Physiology Pharmacology

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)
114 AMD: New Drugs, Delivery Systems and Mechanisms

Sunday, May 6, 2012, 8:30 AM - 10:15 AM
Hall B/C Poster Session
Program #/Board # Range: 433-457/D1110-D1134
Organizing Section: Physiology/Pharmacology
Program Number: 433 Poster Board Number: D1110
Presentation Time: 8:30 AM - 10:15 AM
In-vitro Activity Of Transgenic Anti- VEGF Molecules
Tobias Wimmer, Nina Wagner, Eva Senger, Birgit Lorenz, Knut Stieger.
Department of Ophthalmology, Justus-Liebig University Giessen, Giessen,
Germany.
Purpose: Upregulation of VEGF (vascular endothelial growth factor) in the eye
leads to uncontrolled retinal vessel growth in diseases like AMD (age related
macula degeneration) or DR (diabetic retinopathy). Repeated injections of antiangiogenic molecules like Lucentis (Ranibizumab) or Avastin (Bevacizumab)
are the state of the art treatment for these disorders. The aim of this study was to
develop a gene addition therapy, with which anti-VEGF molecules are produced
over long time periods in the eye. To achieve this, we expressed the soluble
isoform of the VEGF receptor 1 (sFlt1) under the control of the Tetracyclininducible TetOn-promotor and the F(ab)-fragment Ranibizumab (Ra01) under the
control of a CMV promoter in two different eukaryotic cell lines and analyzed the
biological activity.
Methods: The sequences of both chains of Ranibizumab were synthesized and
cloned into the same, IRES (internal ribosomal entry site) containing, expression
vector. The sFlt1 cDNA was amplified from corneal extracts and cloned into a
TetOn expression vector. HEK293 and HeLa cell lines were transfected with these
constructs and the expression of sFlt1 was induced with the Tetracyclin-derivate
Doxycyclin. The gene-product containing supernatand was collected after 24 hours
of expression. HUVEC (human umbilical vein endothelial cells) migration assays
and HUVEC tube formation assays were performed to determine the biological
activity of the expressed molecules compared to Lucentis and Avastin. The
concentration of the expressed molecules was determined by ELISA.
Results: In the HUVEC tube formation assay, Ra01 showed a reduction in tube
formation (inhibition of VEGF) of 67%, the induced sFlt1 57% and the noninduced sFlt1 a reduction of 6%, compared to Lucentis (100ng) of 45% and
Avastin (100ng) of 51%. The migration assay showed an inhibition of VEGFinduced HUVEC migration of 15% with expressed sFlt1 and a decrease of 40%
with Ra01, compared to Lucentis (100ng) with 47%.
Conclusions: The expression of sFlt1 can be controlled using the TetOn system,
which produces sufficient amounts of sFlt1 to inhibit VEGF in the tube formation
assay compared to Lucentis or Avastin. Ra01 can be assembled into a
heterodimer in eukaryotic cells and shows a VEGF inhibiting activity comparable
or slightly better than Lucentis or Avastin. These results provide the basis for a
gene-addition therapy to inhibit VEGF in pathologies like AMD or DR.
Commercial Relationships: Tobias Wimmer, None; Nina Wagner, None; Eva
Senger, None; Birgit Lorenz, None; Knut Stieger, None
Support: DFG Sti 597/2-1
The Olmesartan Effect in the Choroid-scleral Leukocyte Recruitment in
Hypercholesterolemia Model
Rogil J. Torres1, Dalton Precoma1, Lucia Noronha1, Mario Sturzeneker1, Regiane
Torres1, Andrea Luchini2, Antonio M. Casella3, Caroline Torres4, Lucas Torres4,
Robson Torres4. 1Retina Vitreous, Pontificia Universidade Catolica do Parana,
Curitiba, Brazil; 2Centro Oftalmologico de Curitiba, Curitiba, Brazil;
3
Ophthalmology, University of East London, Londrina, Brazil; 4Universidade
Positivo, Curitiba, Brazil.
Purpose: Demonstrate that the blockade of angiotensin II AT-1 receptors, through
the systemic administration of olmesartan, can reduce the MCP-1 expression and
the resulting macrophage accumulation in the choroid and sclera of
hypercholesterolemic rabbits.
Methods: 32 New Zealand rabbits were divided into 3 groups: group 1(NG), was
fed a standard rabbit diet; group 2 (HG) was fed a hypercholesterolemic diet; and
group 3 (OG) was fed a hypercholesterolemic diet enriched with olmesartan. The
rabbits underwent serum dosages of total cholesterol, triglyceride, HDL cholesterol
and fasting blood glucose at the beginning of the experiment and at the euthanasia
day. Sclera and choroid underwent immunohistochemical analysis with MCP-1 and
RAM-11 markers.
Results: No abnormality was detected in NG. HG showed a significant increase in
immunoreactivity for MCP-1 in relation to NG (p= 0.001) and OG (p=0.004). HG
showed a significant increase in immunoreactivity for RAM-11 of the choroidscleral complex in relation to (p<0.001). A significant decrease in
immunoreactivity for RAM-11 was observed in OG in relation to HG (p= 0.034)
and a significant increase in immunoreactivity for RAM-11 was observed in OG in
relation to NG (p=0.008).
Conclusions: Olmesartan reduced the MCP-1 expression and the resultant

macrophage accumulation in the choroid-scleral complex of hypercholesterolemic
rabbits.
Commercial Relationships: Rogil J. Torres, None; Dalton Precoma,
None; Lucia Noronha, None; Mario Sturzeneker, None; Regiane Torres,
None; Andrea Luchini, None; Antonio M. Casella, None; Caroline Torres,
None; Lucas Torres, None; Robson Torres, None
Support: None
Biocompatibility Of A Novel Ocular Drug Delivery System
Nathan Gooch1A, Michael Burr1, Bruce Gale1B, Balamurali Ambati1C.
A
Bioengineering, BMechanical Engineering, COphthalmology, 1University of Utah,
South Jordan, UT.
Purpose: To determine ocular biocompatibility of a novel, sustained release,
refillable intraocular, drug delivery device.
Methods: The intraocular drug delivery device is a reservoir and delivery agent
which is designed to be placed within the capsular bag during cataract surgery. The
capsule drug ring (CDR) prototypes were manufactured by hot melt extrusion of
Bionate II (DSM), a polycarbonate urethane. As the Bionate II tubing was
extruded from the dye, it was wrapped around a 8mm pipe to incorporate the
correct inner and outer diameters into the polymer before fully setting. A filter
composed of polyether sulfone was fitted to one end of the devices for controlled
drug release. The other end was sealed. The devices have been optimized using
Avastin as the drug of interest. In vitro biocompatibility was assessed with human
lens epithelial cell (B-3), mouse macrophage (J774A.1), and mouse fibroblast (L929) cell lines. Cell migration and proliferation were assessed after in vitro culture.
Pro-inflammatory cytokines (i.e. MIP-1, MIP-1, MCP-1, IL-1, TNF, TGF-1)
were quantified using cytometric bead array (CBA). Preliminary in vivo
biocompatibility and pharmacokinetics testing has been performed in rabbits.
Results: The use of hot melt extrusion for CDR manufacture in place of previous
design methodologies has dramatically improved the performance and
reproducibility of drug delivery and pharmacokinetics. The devices have been
designed to be a circular ring shape so as to fit in the capsular bag without
impeding vision and have a drug reservoir of 50L. In vitro cell migration and
proliferation experiments show that the CDR components and devices had no
measurable cytotoxic impact on B-3, J774A.1, and L-929 cell lines. Proinflammatory cytokine concentrations were also unchanged by the use of CDR
device materials when compared to the gold standard tissue culture polystyrene
cultured cells. Manufacturing methods were shown to be sterile as LPS was
detected at levels below 0.0303 EU/mL. Preliminary in vivo histology shows
Avastin concentrations in the retina out to >90 days with very little host foreign
body or inflammatory response.
Conclusions: The results show the successful manufacture of the CDR, a
potentially refillable drug delivery device. In vitro results show the devices and
their individual components to be highly biocompatible with cells showing no
difference in migration, proliferation, and pro-inflammatory cytokine generating
behaviors. Avastin was used as the primary drug of interest to test
pharmacokinetics in vivo. Histology showed that the CDR devices performed as
designed with very good biocompatibility. The CDR shows great potential as an
implantable ocular device for drug delivery.
Commercial Relationships: Nathan Gooch, None; Michael Burr, None; Bruce
Gale, None; Balamurali Ambati, None
Support: NIH Grant EY017185-01A2
Hyaluronic Acid-Containing Silicone Hydrogels: Their Use as Extended Drug
Delivery Systems of Hydrophobic Ocular Drugs
Myrto Korogiannaki, Heather Sheardown. Chemical Engineering, McMaster
University, Hamilton, ON, Canada.
Purpose: While eyedrops are a well-accepted method of delivering drugs to the
eye, they suffer from significant limitations including significant losses, low
residence times, pulsatile concentration profiles in the tear fluid and the need for
patient compliance. The high oxygen permeability of silicone hydrogel (SH) based
materials makes them attractive for extended release in front of the eye
applications. Based on the hypothesis that the drug release can be controlled by
controlling the hydrophilicity of the materials, we have developed a series of
hyaluronic acid (HA) modified SH materials that have utility for extended release
of hydrophobic ocular drugs.
Methods: The modified SH that were used consist of a hydrophilic monomer,
either hydroxyethyl methacrylate (HEMA) or N,N-dimethylacrylamide (DMAA), a
hydrophobic silicone monomer of methacryloxypropyltris (trimethylsiloxy) silane
(TRIS) and hyaluronic acid (HA) (7.5 kDa). Ethylene glycol dimethacrylate
(EGDMA) was used as the cross-linker. The reaction was performed by UV
induced free-radical polymerization. Atropine was used as a model hydrophobic
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.

drug. HA and atropine were incorporated into the SH during synthesis with direct
entrapment. The hydrogels were analyzed for surface hydrophilicity and
equilibrium water content (EWC) in order to determine the effect of HA on these
materials. Drug release was monitored and quantified by UV spectroscopy
technique.
Results: The swelling study showed that the incorporation of HA significantly
increased the EWC of drug loaded-DMAA/TRIS hydrogels. Based on the results of
the captive air bubble study, it was found that the surface of the model SH were
more hydrophobic than our previous HA containing materials presumably due to
the polyester sheets used for materials casting. Moreover, the presence of HA into
the drug loaded-DMAA/TRIS materials improved the surface hydrophilicity as the
advancing contact angles were decreased. The contact angles were also examined at
the end of the release experiment and it was found that the silicone hydrogel
surfaces were significantly more hydrophilic. Atropine was released for more than
two weeks and the incorporation of HA during synthesis led to an increase in the
amount and the duration of the drug released.
Conclusions: The HA-containing SH materials have the potential to be used as
extended drug delivery systems for hydrophobic drugs with potential application in
the treatment of front of the eye diseases as a replacement of eyedrops.
Commercial Relationships: Myrto Korogiannaki, None; Heather Sheardown,
None
Support: NSERC
The Combination of Bevacizumab and 3,4 Dihydroxyphenyl Ethanol Reduces
Angiogenin in Retinal Pigment Epithelial Cells
Tamara J. Granner, Shawn C. Maloney, Sebastian Di Cesare, Tiago Briccoli,
Cristina Miyamoto, Miguel N. Burnier, Jr.. Ophthalmology, McGill University,
Montreal, QC, Canada.
Purpose: Choroidal neovascularization is the hallmark of the wet form of AgeRelated Macular Degeneration (AMD) and is currently the target of multiple antiangiogenic pharmacotherapies. The current study evaluated if 3,4 Dihydroxyphenyl
Ethanol (DPE) reduces secretion of pro-angiogenic cytokines from a human retinal
pigment epithelial cell line (ARPE-19). Moreover, additional anti-angiogenic
effects of DPE in combination with bevacizumab were evaluated.
Methods: ARPE-19 cells were cultured for 24 hours under normoxic conditions or
with a hypoxia-mimicking agent (100M cobalt chloride [CoCl2]). After 24 hours,
all media was removed and replaced with one of the following serum-free
conditions: Control media, DPE (100M), bevacizumab (0.25mg/ml), or
combination of DPE (100M) and bevacizumab (0.25mg/ml). Media was harvested
after 24 hours for sandwich ELISA-based angiogenesis arrays. The secretion of the
following ten pro-angiogenic cytokines was measured: Angiogenin, ANG2, EGF,
bFGF, HB-EGF, PDGF-BB, Leptin, PIGF, HGF, and VEGF-A. Statistical
significance was evaluated using a Students t-test with p<0.05 as a cutoff for
significance.
Results: Of the ten cytokines assayed, three (Angiogenin, ANG2, and VEGF-A)
were secreted by ARPE-19 cells under normoxia and all three were significantly
increased under CoCl2-simulated hypoxia. HB-EGF and PIGF were not secreted
under normoxia, but secretion was induced under simulated hypoxia. Following
treatment with DPE, levels of Angiogenin and VEGF-A significantly decreased
under normoxia while all five detectable cytokines significantly decreased under
simulated hypoxia compared to the control. The combination of bevacizumab with
DPE significantly reduced secretion of Angiogenin and ANG2 under normoxia.
Angiogenin was significantly down-regulated by the combination under simulated
hypoxia compared to bevacizumab alone.
Conclusions: This study demonstrated that DPE significantly reduced the secretion
of multiple pro-angiogenic cytokines to varying degrees under normoxia and
simulated hypoxia. Further, the combination of DPE and bevacizumab proved to be
a more effective approach to reduce Angiogenin than bevacizumab alone.
Therefore the combination of DPE and bevacizumab may represent a valuable
therapeutic option for the wet form of AMD.
Commercial Relationships: Tamara J. Granner, None; Shawn C. Maloney,
None; Sebastian Di Cesare, None; Tiago Briccoli, None; Cristina Miyamoto,
None; Miguel N. Burnier, Jr., None
Support: None
Three Year Results of Visual Outcome with Disease-Activity-Guided
Ranibizumab Algorithm for the Treatment of Exudative Age-Related Macular
Degeneration
Lala Ceklic1,2, Carsten Framme2, Ute E. Schnurrbusch-Wolf2, Sebastian Wolf2.
1
Eye Clinic "Kasindo", Clinical Center of Eastern Sarajevo, Eastern Sarajevo,
Bosnia and Herzegovina; 2University Of Bern, Universitatsklinik fur
Augenheilkunde, Bern, Switzerland.
Purpose: To evaluate three year follow up treatment outcomes with ranibizumab

(Lucentis) 0.5 mg administered either monthly or quarterly on a pro re nata
(PRN) basis according to a disease-activity-guided monitoring and treatment
algorithm.
Methods: A total of 316 treatment-naive eyes of 316 patients with exudative agerelated macular degeneration met the criteria for inclusion in this retrospective,
interventional case series. Patients were treated with ranibizumab 0.5 mg according
to a disease-activity-guided algorithm with monthly monitoring following the
standard loading phase with 3 injections. Spectral Optical coherence tomography
was routinely used to assess disease activity by qualitatively judging subretinal
fluid: active lesions showing subretinal fluid were treated with a series of 3
monthly injections, whereas inactive lesions (dry retinal conditions) were treated
with quarterly injections.
Results: Mean best-corrected ETDRS visual acuity improved from 52 letters at
baseline to 59 letters at 12 months, achieved with a mean of 7.4 injections, 60
letters at 24 months with a mean of 12.1 injections administered up to the second
year and again 60 letters at 36 months with a total mean number of 16 injections.
Conclusions: A morphology driven Pro re nata regimen with ranibizumab
administered either monthly or quarterly in long term follow up (up to 36 months)
resulted in BCVA gain of 8 letters which is in favorable correlation to the expected
visual gain derived from the MARINA- and ANCHOR studies; however, using
significant less injections.
Commercial Relationships: Lala Ceklic, None; Carsten Framme, None; Ute E.
Schnurrbusch-Wolf, None; Sebastian Wolf, None
Support: None
Tissue Kallikrein Suppresses Laser-Induced Choroidal Neovascularization in
Mice
Junichi Fukuhara1A,1B, Kousuke Noda1A,1B, Chikako Yoshizawa1A, Satoshi
Kinoshita1A,1B, Zhenyu Dong1A,1B, Ryo Ando1A,1B, Anton Lennikov1A,1B, Atsuhiro
Kanda1A,1B, Susumu Ishida1A,1B. ADepartment of Ophthalmology, BLaboratory of
Ocular Cell Biology and Visual Science, 1Hokkaido University Graduate School of
Medicine, Sapporo, Japan.
Purpose: Tissue kallikrein is a serine protease that contributes to a flow-dependent
arterial dilation through activation of bradykinin B2 receptors coupled with
endothelial nitric oxide release. Recently, it has been reported that tissue kallikrein
also possesses the antiangiogenic effects through the cleavage of vascular
endothelial growth factor (VEGF) 165 isoform and thereby reduces the
pathological vascular changes in the murine model of oxygen-induced retinopathy.
Here, we investigate the impact of tissue kallikrein in laser-induced choroidal
neovascularization (CNV).
Methods: Male C57Bl/6 mice (7-8 weeks old) were treated in accordance with the
ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
CNV was induced by laser photocoagulation (200mW, 75m, 100msec). The
animals received daily subcutaneous injections of tissue kallikrein (50g/kg), or
vehicle control (PBS) for 2 days before laser photocoagulation, and the treatment
was continued until the end of the study. Seven days after laser injury, choroidal
flat mounts were prepared and the size of the CNV lesions was quantified. The
RPE-choroid complex was harvested 3 days after laser injury and the levels of
inflammation-associated molecules, monocyte chemoattractant protein (CP)-1
and intercellular adhesion molecule (ICAM)-1, were assessed by enzyme-linked
immunosorbent assay. Immunoblotting was performed using the RPE-choroid
complex with anti-VEGF164 antibody.
Results: The animals treated with tissue kallikrein showed a significant decrease in
CNV size (27168.32432.2m2), compared with vehicle-treated controls
(36374.63204.1m2, p<0.05). Tissue kallikrein treatment significantly reduced the
levels of CP-1 (p<0.05) and ICAM-1 (p<0.05). Furthermore, immunoblotting
showed the approximately 16kDa-band, presumably corresponding to fragmented
VEGF164 protein, in the RPE-choroid complex samples obtained from the animals
treated with tissue kallikrein.
Conclusions: The current data indicate an antiangiogenic property of tissue
kallikrein through VEGF164 cleavage in CNV.
Commercial Relationships: Junichi Fukuhara, Sanwa Kagaku Kenkyusho Co.,
Ltd. (F); Kousuke Noda, Sanwa Kagaku Kenkyusho Co., Ltd. (F); Chikako
Yoshizawa, None; Satoshi Kinoshita, None; Zhenyu Dong, None; Ryo Ando,
None; Anton Lennikov, None; Atsuhiro Kanda, None; Susumu Ishida, None
Support: None
VEGF Trap Exhibits VEGF Binding Stoichiometry Distinct From Antibodies,
and Does Not Support Platelet Aggregation
Douglas A. MacDonald1, Jiann-Kae Luo1, Nicholas Papadopoulos1, Erica Pyles1,
Stanley Wiegand1, Franoise Bono2, Nathalie Delesque2, Pierre Savi2, John

Francis3, Todd Meyer3. 1Regeneron Pharmaceuticals, Tarrytown, NY; 2Sanofi,
France, Toulouse, France; 3Center for Thrombosis Research, Orlando, FL.
Purpose: VEGF Trap (aflibercept) is a novel, soluble decoy receptor that binds
VEGF-A, VEGF-B and placental growth factor. Like anti-VEGF antibodies, VEGF
Trap contains an Fc domain. However, given its otherwise distinct structure, VEGF
Trap may bind VEGF with different stoichiometry than antibodies. The present
studies were conducted to compare binding stoichiometries of bevacizumab (Bev,
an anti-VEGF antibody) and VEGF Trap to VEGF, as well as FcRIIA -mediated
platelet activating capability of Bev:VEGF and VEGF Trap:VEGF complexes.
Methods: Stoichiometry of binding for Bev and VEGF Trap to VEGF165 was
measured using multi-angle laser light scattering. Platelet activation was measured
using platelet aggregometry & 14C-serotonin release assays (SRA). Animal studies
used human FcRIIA transgenic mice. Pre-formed 1:1 molar mixtures of Bev or
VEGF Trap with VEGF165 plus heparin were injected into the tail vein
(n=10/group). After 10 min, blood drawn by cardiac puncture was used to measure
platelet number. Lungs were dissected en bloc, PBS washed and formalin-fixed.
H&E sections were analyzed for evidence of thrombosis.
Results: Bev:VEGF165 complexes were heterogeneous and predominantly
multimeric (~400kDa to >2,000 kDa). In contrast, VEGF Trap formed a
homogeneous 1:1 binding complex with VEGF165, with a M.W. of 155 kDa.
Platelet aggregometry and SRA showed that preformed equal molar Bev:VEGF165
complexes (100-500 nM) plus heparin (0.5-50 ug/ml) activated platelets, while
VEGF Trap:VEGF165 complexes did not. Increasing the molar excess of Bev ( 4
fold relative to VEGF) resulted in smaller complexes that did not trigger platelet
activation. Injection of pre-formed VEGF Trap:VEGF165 complexes with heparin
into FcRIIA transgenic mice did not significantly affect platelet count (966168)
vs saline-injected controls (1173179). Animals receiving Bev+VEGF165
complexes with heparin exhibited behavior consistent with thrombotic shock and
experienced profound thrombocytopenia (platelet count of 331217; P < 0.001).
Lung sections from these animals showed patterns of occlusive thrombi.
Conclusions: Bev when mixed at near equal molar ratios with VEGF165, in the
presence of heparin, can support platelet aggregation. This is not the case for VEGF
Trap. The clinical relevance of these observations remains to be determined.
Commercial Relationships: Douglas A. MacDonald, Regeneron
Pharmaceuticals (E); Jiann-Kae Luo, Regeneron Pharmaceuticals (E); Nicholas
Papadopoulos, Regeneron Pharmaceuticals (E); Erica Pyles, Regeneron
Pharmaceuticals (E); Stanley Wiegand, Regeneron Pharmaceuticals (E);
Franoise Bono, Sanofi France (E); Nathalie Delesque, Sanofi France (E); Pierre
Savi, Sanofi France (E); John Francis, None; Todd Meyer, Funding grant from
Regeneron (F)
Support: None
Nanostructured Biopolymer Films for Retinal Drug Delivery
Kevin D. Lance, Daniel A. Bernards, Natalie A. Ciaccio, Robert B. Bhisitkul, Tejal
A. Desai. UCSF, San Francisco, CA.
Purpose: The goal of this research is to develop a sustained drug delivery device
for intraocular release of therapeutics, with a focus on the treatment of age-related
macular degeneration (AMD). We investigate the ocular tolerance, structural
durability, and functionality of our nanoporous biopolymer devices.
Methods: We utilized a modular fabrication approach with polycaprolactone
(PCL) that combines template-based fabrication to produce nanopores and a
polymer mixture to generate a mechanically robust supporting layer. PCL-based
devices were characterized in vitro to assess physical degradation and the
characteristic release of model therapeutics. Parallel experiments in microporous
PCL devices were used to characterize drug payload stability for a model antibody.
New Zealand White rabbits were used for our in vivo studies. Structured
poly(caprolactone) (PCL) thin films were implanted in rabbit eyes for survival
studies and surveillance of ocular tolerability up to 9 months. Histology of
enucleated post-mortem eyes was used to evaluate morphologic abnormalities and
adverse reactions; scanning electron microscopy was used to examine the durability
and stability of extracted thin films. Complete devices loaded with IgG were
implanted for a 6 week feasibility study, and vitreous and device samples were
analyzed to gauge device performance and payload stability.
Results: Devices utilizing nanoporous thin films for controlled release exhibit
constant rates of release in vitro for greater than 6 months. Model protein drug
payloads maintained stability at least 10 weeks. Nanostructured thin films lacked
an observable immune response through 9 months of implantation. Structural
integrity of implanted films was maintained throughout this time course in vivo.
Equivalent films tested in vitro became increasingly fragile after 1 year and had
noticeable structural breakdown occurring in excess of 1 year. A short duration in
vivo trial with completed devices demonstrated stable activity for the IgG payload
over the course of 6 weeks.
Conclusions: The fabrication procedures we have developed are capable of
generating robust nanoporous biopolymer films, and preliminary studies have
established several important benchmarks of device function, including sufficient
drug loading, controlled release of therapeutic over extended times, materials

biocompatibility, and maintenance of drug activity.
Commercial Relationships: Kevin D. Lance, Santen, Inc. (F); Daniel A.
Bernards, Santen, Inc. (F); Natalie A. Ciaccio, Santen, Inc. (F); Robert B.
Bhisitkul, Santen, Inc. (C); Tejal A. Desai, Santen, Inc. (F)
Support: NIH Grant 2 T32 GM 8155-27
Resveratrol inhibits Choroidal Endothelial Cell Proliferation through the
induction of SAPK/JNK Pathway
Vijay Khetpal, S Balaiya, K V. Chalam. Ophthalmology, University of Florida
College of Medicine, Jacksonville, FL.
Purpose: Exudative AMD results from hypoxia induced choroidal
neovascularization. Resveratrol, a common antioxidant present in red wine and
natural plants is anti-angiogenic in carcinoma and pro-angiogenic in experimental
models of vascular endothelial cells. It decelerate the aging process and modulate
vascular endothelial cell function in diverse angiogenic beds. In this study, we
investigated the effects of resveratrol on cell viability and its effects on apoptosis in
hypoxic choroidal endothelial cells.
Methods: Choroidal endothelial cells (RF/6A) where exposed to escalating doses
of resveratrol 4 and 12 g/ml and cell viability was analysed by WST-1 assay.
Hypoxia was induced through cobalt chloride (200 M) and the effect of
resveratrol (4 and 12g/ml) was evaluated by assessing Stress Activated
ProteinKinase/c-Jun N-terminal Kinase (SAPK/JNK) pathway using immunoblot
and densitometric analysis.
Results: In the presence of hypoxia (200M of cobalt chloride), cell proliferation
increased to 129.31.13% whereas it decreased to 125.61.02% after the addition
of resveratrol at 4g/ml and 86.12.16% at 12g/ml of concentration. In
comparison to basal level (0.3% ODU), phosphorylation state of SAPK/JNK
increased after hypoxic insult to 5.3% at 200 M concentration of cobalt chloride
which decreased to 3.8 and 2.5% after the addition of 4 and 12 g/mL of
resveratrol, respectively. (P<0.01)
Conclusions: Resveratrol inhibits proliferation of hypoxic choroidal endothelial
cells and its apoptotic effects may be relayed through the SAPK/JNK pathway.
Commercial Relationships: Vijay Khetpal, None; S. Balaiya, None; K. V.
Chalam, None
Support: None
Anti-phosphatidylserine Antibodies As A Potential New Therapy Against
Choroidal Neovascularization
Rafael Ufret-Vincenty1, Bogale Aredo1, Kaiyan Zhang1, Cynthia X. Wang1,
Shusheng Wang1, Jose Pulido2, Philip E. Thorpe1. 1Ophthalmology, UT
Southwestern Medical Center, Dallas, TX; 2Ophthalmology, Mayo Clinic,
Rochester, MN.
Purpose: Despite the dramatic changes in clinical outcomes brought by anti-VEGF
agents, additional drugs directed against different targets in the neovascular process
are needed. This may allow for combination therapies that could potentially
eradicate choroidal neovascularization (CNV). In normal cells the
aminophospholipid phosphatidylserine (PS) is almost exclusively localized to the
inner leaflet of the cell membranes lipid bilayer. Endothelial cells of tumor
neovasculature lose their capacity to maintain PS asymmetry. Anti-PS antibodies
bind to the newly exposed phosphatidylserine in the outer leaflet of the tumor
vascular endothelium, mediating antibody-dependent cell-mediated cytotoxicity,
and causing the collapse of the tumor neovasculature. Radiotherapy increases the
exposure of PS in tumor vasculature, enhancing the antitumor effects of anti-PS
antibodies. We propose to evaluate if there is also exposure of PS in CNV, and if
anti-PS antibodies can treat CNV.
Methods: We induced CNV in B6 mice using the laser model. Immunostaining for
PS was done after perfusing mice with an anti-PS antibody and paraffin-embedding
the eyes. We tested the effect of intravitreal anti-PS antibodies alone, or in
combination with eye radiation, on the CNV size as measured with ICAM-2
staining.
Results: Paraffin sections of eyes perfused with an anti-PS antibody stained
positive for PS. The staining co-localized with ICAM-2-stained CNV,
demonstrating that PS was exposed on the choroidal neovascular membranes. The
anti-PS antibody (11.31) led to a 52% reduction in the laser-induced CNV size
(p=0.02) when injected intravitreally. We have established a system for irradiation
of the eyes. We will show data on the effect of radiation alone or in combination
with anti-PS antibodies on the neovascular complex.
Conclusions: Anti-phosphatidylserine antibodies may have therapeutic value in
wet AMD alone or in combination with radiation or anti-VEGF agents.
Commercial Relationships: Rafael Ufret-Vincenty, None; Bogale Aredo,
None; Kaiyan Zhang, None; Cynthia X. Wang, None; Shusheng Wang,
None; Jose Pulido, None; Philip E. Thorpe, Peregrine (P)

Support: Unrestricted RPB Department Grant, Core grant to Department of
Ophthalmology (EY020799), Disease Oriented Clinical Scholars Grant to RUV
Biological Activity of Different Transgenic Ranibizumab Compositions
Knut Stieger, Tobias Wimmer, Birgit Lorenz. Department of Ophthalmology,
Justus-Liebig-University Giessen, Giessen, Germany.
Purpose: Ocular neovascularisation due to uncontrolled growth of new vessels into
the retina following overexpression of vascular endothelial growth factor (VEGF)
is the main cause of visual impairment in retinal neovascular diseases such as agerelated macular degeneration (AMD) or diabetic retinopathy (DR). The intraocular
expression of anti-VEGF molecules potentially represents a therapeutic strategy to
block neovascularization in these pathologies. The aim of this study was to
characterize the optimal composition of the expression cassette for the production
of these molecules in standard cell lines in vitro.
Methods: Both antibody chains of Ranibizumab, the light and part of the heavy
chain were designed containing secretory leader sequences or restriction sites for
subsequent subcloning. The fragments were either expressed separately using an
IRES containing expression cassette (Ra01), or were cloned together into one
reading frame containing either a glycine or glycine-proline anchor in between
(Ra02-Ra06). Plasmids were transfected into HEK293 and Hela cell lines and the
expression of the molecules verified by Western blot analysis. A Ranibizumab
specific ELISA was developed in order to measure the concentration of the antiVEGF molecules. The biological activity was tested using HUVEC (human
umbilical vein endothelial cell) tube formation assays and HUVEC migration
assays.
Results: All Ra compositions were detected in the supernatant of transfected Hela
and HEK293 cells. Generation of long cellular tubes in the HUVEC tube formation
assay, which is predominantly due to VEGF activity, was reduced to 50% with
Ra01 and similar levels were achieved with Ra02-Ra06. VEGF activity in the
HUVEC migration assay was reduced by about 50% for all Ra compositions.
Similar VEGF inhibition results were obtained using commercially available
recombinant Ranibizumab (Lucentis) at equal concentrations.
Conclusions: Transgenic Ranibizumab, either expressed separately or as one
molecule have similar inhibitory effects on VEGF activity as commercially
available Ranibizumab in vitro. These results lay the foundation for the
development of an alternative treatment strategy for patients with AMD or DR, in
which Ranibizumab is produced at low doses directly in retinal cells.
Commercial Relationships: Knut Stieger, None; Tobias Wimmer, None; Birgit
Lorenz, None
Support: DFG STI 597/2-1
RGD-targeted Nanoparticles Expressing Flt23k Inhibit CNV In a Murine
CNV Model
Xiaohui Zhang, Ling Luo, Hironori Uehara, Tadashi Miya, Christina Mamalis,
Alex Jones, Bonnie Archer, Balamurali K. Ambati. John Moran Eye Center,
University of Utah, Salt Lake City, UT.
Purpose: Choroidal neovascularization (CNV) is a leading cause of blindness in
age-related macular degeneration (AMD) patients in the developed world.
Currently, the most effective therapies are monthly intravitreal injections of antiVEGF agents such as bevacizumab or ranibizumab. However there are significant
risks associated with repeated intravitreal injections. The purpose of this study was
to determine whether a single intravenous administration of RGD-coated
nanoparticles delivering plasmids expressing Flt23k intraceptors could suppress
CNV in a murine model.
Methods: We prepared nile-red labeled nanoparticles which were blank, loaded
with pCMV.Flt23k, or loaded with pCMV.Flt23k conjugated with RGD
oligopeptides (which home to alpha-v-beta-3 integrin). All three nanoparticles were
dissolved in MES buffer. The total volume delivered was 4 l (plasmid
concentration is 0.1g/l) in each mouse, and similar volumes of MES buffer
served as blank control. Mice CNV was induced by 532 nm laser or subretinal
injecton of adeno-associated virus mediated small hairpin ribonucleic acid
(shRNA) sFlt-1. Tail vein injection was performed 2 weeks after induction of
CNV. CNV regression was evaluated 2 weeks after tail vein injection in
histological sections and CNV volume quantified using newly developed software,
Seg3D in vivo image. RGD.Flt23k.NR.NP was detected by immunostaining in
CNV.
Results: 5 expression in CNV area was confirmed by immunostaining. H&E
stained sections show the CNV size was dramatically decreased in
RGD.Flt23k.NR.NP injected mice (treatment group) compare to the other three
control groups (Flt23k.NR.NPs, blank NR.NPs or MES buffer). The treatment
group mice CNV volume was decreased by 23%, which showed significantly more
reduction than observed with unlabeled nanoparticles, blank nanoparticles, or MES
control (all ps <0.05), As a positive control, CNV lesions treated with anti-mouse
VEGF antibody intravitreal injections were decreased by 11% (p=0.1).

Conclusions: This study exploits a new method to treat CNV by providing a novel
therapeutic, a safer route of delivery, and also a nonviral delivery system for
extended gene delivery.
Commercial Relationships: Xiaohui Zhang, None; Ling Luo, None; Hironori
Uehara, None; Tadashi Miya, None; Christina Mamalis, None; Alex Jones,
None; Bonnie Archer, None; Balamurali K. Ambati, None
Support: 5R01EY017182-04
Microbial and Fungal Contamination Following A Day Use Of Multiple Use
Bottles Of Fluoresceine Sodium 0.25% And Benoxinate Hydrochloride 0.4%
In An Outpatient Ophthalmology Clinic
Olivier Lasnier, Anne Faucher. Ophthalmology, Sherbrooke University,
Sherbrooke, QC, Canada.
Purpose: Fluorescein and benoxinate solution in a multiple use bottle is an
important clinical tool for diagnostics and tonometry measurement in
ophthalmology clinics. However, this solution may also be the source for
dissemination of ocular infections. Contamination of ocular solutions may occur
due to rapid administration, poor patient cooperation and clinician distraction. We
aimed to determine the rate of bacterial and fungal contamination of 5 mL multiple
usage bottles of fluorescein 0.25% and benoxinate 0.4% solution after a single day
of use in an ophthalmology outpatient clinic. We also calculated the average
duration of a 5 mL solution.
Methods: This project is a prospective blinded study. One unopened bottle of
fluorescein sodium 0.25% and benoxinate hydrochloride 0.4% solution was placed
in every exam room of the University of Sherbrooke ophthalmology clinics at the
beginning of the day. All staff members working in the clinics were unaware of the
ongoing project to prevent any change in their usual practice. At the end of the
clinical day, all bottles were collected, left at room temperature for less than 24
hours and sent for culture. Three drops from each bottle were put on each of five
culture media: blood agar, chocolate agar, Brucella agar, Sabouraud agar and
Thioglycolate broth. All media where immediately stored in an air oven at 35
degrees Celsius until being delivered to the microbiology laboratory for further
analysis. All cultures were kept for at least one week of observation before being
discarded.
Results: A total of 27 bottles were collected from nine ophthalmologists. All of the
bottles had negative culture after one week of incubation. All physicians were
unaware of the project at the time of sample collection and reported to have used
this solution, on average, for 80% of their patients. Considering the average
frequency of usage and the average number of patients seen per day, a 5 ml of this
solution would last two and a half days.
Conclusions: The multiple use of a single bottle of Fluoresceine sodium 0.25% and
Benoxinate hydrochloride 0.4% is safe on a 24 hours basis in regard of the bacterial
and fungal contamination risk. To our knowledge, this is the largest in-use
contamination experimentation for this solution. The safety profile over a longer
period of utilization warrants further investigation.
Commercial Relationships: Olivier Lasnier, None; Anne Faucher, None
Support: None
Treatment For Persistent Uveitic Macular Edema With intravitreal
dexamethasone 0.7 mg implant (Ozurdex)
Alfredo Adan Civera1, Laura Pelegrin1, Amanda Rey Torrente1, Victor Llorens1,
Marina Mesquida1, Blanca Molins2. 1Ophthalmology, Hospital Clinic, Barcelona,
Spain; 2Ophthalmology, IDIBAPS, Barcelona, Spain.
Purpose: To evaluate the effects of intravitreal dexamethasone (DEX) 0.7 mg
implant (Ozurdex; Allergan, Inc., Irvine, CA) for the treatment of persistent
uveitic macular edema (ME)
Methods: Medical records of 16 patients (23 eyes) with persistent (>or= 90 days)
uveitic ME treated with intravitreal DEX 0.7 mg implant were reviewed. Complete
ophthalmic examination including visual acuity, fundus biomicroscopy, fundus
photography, and spectral domain optical coherence tomography (Cirrus HDOCT,Carl Zeiss Meditec, Dublin, CA) was performed at baseline and follow-up
.Main outcome measures were improvement of central retinal thickness (CRT)
measured with optical coherence tomography and changes in best corrected visual
acuity (BCVA). Tolerability of the implant was assessed.
Results: At a mean postoperative follow-up of 5.3 months,central retinal thickness
on optical coherence tomography exams decreased significatively (p < 0.001) at
one month and three month examination.At day 60, a 10-letter or more BCVA
improvement was seen in 60.86% (14/23) of eyes.A 15-letter or more mprovement
was achieved in 34,78% (8/23) of eyes.ME relapsed in 21.7% (5/23) and an
additional intravitreal injection of DEX 0.7 mg implant was performed. The
implant was well tolerated. Throughout the study, an increase in intraocular
pressure of 10 mm Hg or more was seen in 26,08 % (6/23) of eyes. There were no

cases of endophthalmitis. At the time of intravitreal injection, 17 eyes (73.9%) were
vitrectomized and 6 were non-vitrectomized (26.1%)
Conclusions: Intravitreal DEX 0.7 mg implant seems to be a safe and effective for
treatment of persistent uveitic ME .Our results suggest that efficacy of the implant
in vitrectomized eyes with uveitic ME.
Commercial Relationships: Alfredo Adan Civera, None; Laura Pelegrin,
None; Amanda Rey Torrente, None; Victor Llorens, None; Marina Mesquida,
None; Blanca Molins, None
Support: None
JUNCTION Study: SD-OCT Shows Shrinking And Disappearing Of Drusen
In Nonexudative AMD Eyes Treated With AREDS 2 Supplements Plus A
Complex Containing The Natural Compounds Curcumin, Omega 3 DHA/EPA
and Phospholipids
Andreas U. Bayer. "The Life100 Concept" Study Group, Weilheim, Germany.
Purpose: To evaluate safety and efficacy of Ayurvedas anti-inflammatory and
anti-oxidative natural compound Curcumin in the treatment of nonexudative AMD.
Preliminary results (6-months follow-up) of the Justification for the Use of
Natuaral Compounds in the Treatment of Inflammatory, Ophthalmic and
Neurodegenerative diseases Study (JUNCTION Study) are presented here.
Methods: 126 patients with nonexudative AMD with large drusen in both eyes
(AREDS Category 3) were randomized to the AREDS2 supplements 1000mg
Omega 3 and 10mg Lutein / 2mg Zeaxanthin alone or to these supplements in
addition to 1000mg CuromTM (complex of Curcumin, Omega 3 DHA/EPA and
phospholipids). SD-OCT (Heidelberg Spectralis OCT) and fundus photography
(Zeiss Visucam NMpro) were performed at baseline and after 6 months. Before the
beginning of this study, the bioavailability of different Curcumin products have
been studied.
Results: The treatment with CuromTM shows very good 24-hours bioavailabilities.
In the 66 patients treated with AREDS2 plus CuromTM, there was a highly
significant reduction of drusen size between baseline and after 6 months
(p<0.0001). In the 132 eyes of these 66 patients, we found a disappearing of one or
more drusen in 109 eyes. There was only a non-significant change of drusen size in
the eyes of the 60 patients who were treated with AREDS2 supplements alone.
Tolerance of AREDS2 supplements was slightly better than of AREDS2
supplements plus CuromTM. There were 5 out of the 66 patients who were treated
with AREDS2 supplements plus CuromTM, who complained about diarrhea and/or
a dull stomach. Biomarkers of atherosclerosis improved significantly in patients
who were treated with AREDS2 supplements plus CuromTM (p<0.05).
Conclusions: CuromTM is a powerful natural compound (complex) to treat
nonexudative AMD (high-risk dry AMD). The anti-inflammatory agent Curcumin
should be part of any treatment of nonexudative AMD. Thereby, bioavailability is
of major concern. Follow-up of patients in the JUNCTION Study will test the
hypothesis, that (whether) CuromTM is able to reduce the risk of progression of
high-risk dry AMD (AREDS Category 3) to exudative AMD and/or Geographic
Atrophy (AREDS Category 4). In addition, following the guidelines for assessment
of cardiovascular risk in asymptomatic adults of the American College of
Cardiology Foundation/American Heart Association, the large number of patients
recruited until April 30, 2012, will give insights into a possible prevention of stroke
and/or congestive heart failure.
Commercial Relationships: Andreas U. Bayer, Dr. Andreas Bayer (P)
Support: None
Clinical Trial: Bavarian Medical Association, Munich, Germany, 11130
Design and Optimization of a Transscleral Iontophoresis Applicator for
Delivering Biologics to the Anterior and Posterior Segments
Michael Manzo1, Peyman Moslemy1, Begona Ruiz-Perez1, Fengqui Fan1, Lydia
Mbocha1, Will Schubert1, Phil Isom1, Tracy Dowie1, Pammy Subramony1, Michael
A. Patane2. 1Eyegate Pharmaceuticals Inc, Waltham, MA; 2EyeGate
Pharmaceuticals, Inc, Waltham, MA.
Purpose: To develop a transscleral iontophoresis applicator capable of delivering
biologics to the anterior and posterior segments.
Methods: The engineering designs and testing focused on optimizing conductivity,
buffering capacity, product stability, and minimizing drug volume requirements.
Multiple biocompatible conductive matrices were prepared and evaluated in the
ocular iontophoresis applicator drug reservoir. The devices were loaded with
Cytochrome C (CytC, 12.4 kDa globular protein) in H2O and tested ex vivo via
anodal iontophoresis in a Franz cell apparatus with phosphate buffered saline
solution in the receptor chamber.
For in vivo testing, New Zealand albino rabbits were dosed with CytC using the
test devices impregnated with different biocompatible conductive matrices.
Results: Following iontophoretic transport experiments in rabbits, bioanalytical
results revealed that CytC was present in all ocular tissues harvested. The levels of
CytC in conjunctiva, sclera, choroid, retina, vitreous, iris and ciliary body, and
aqueous humor were quantified by triple-quad MS. The iontophoretic transport
efficiency (percent ratio of total delivery to the initial amount of protein in dosing
solution) increased from < 0.7% for passive delivery of 40 and 80 mg/mL CytC
solutions to > 7% for iontophoresis. The concentration of CytC in the ocular tissues
increased directly with the increase in dosing solution concentration from 10 - 40
mg/mL but remained at comparable levels for 80 mg/mL. Initial pH of dosing
solution (7.4 vs. 5.0) had no considerable effect on the amount of protein delivered
to the ocular tissues. Varying the current intensity (1 - 8 mA) with a fixed 5 min
application time enhanced the concentration of CytC in the tested ocular tissues.
Likewise, increasing the iontophoretic dose at a fixed current intensity resulted in
enhanced delivery of CytC to the ocular tissues.
Conclusions: The experimental results demonstrate that significant amounts of
CytC can be delivered non-invasively into all rabbit ocular tissues using a novel
transscleral iontophoresis ocular applicator.
Commercial Relationships: Michael Manzo, Eyegate Pharmaceuticals Inc (E);
Peyman Moslemy, Eyegate Pharmaceuticals (E); Begona Ruiz-Perez, Eyegate
Pharmaceuticals (E); Fengqui Fan, Eyegate Pharmaceuticals (E); Lydia Mbocha,
Eyegate Pharmaceuticals (E); Will Schubert, Eyegate Pharmaceuticals (E); Phil
Isom, Eyegate Pharmaceuticals (E); Tracy Dowie, Eyegate Pharmaceuticals (E);
Pammy Subramony, Eyegate Pharmaceuticals (E); Michael A. Patane, Eyegate
Pharmaceuticals (E)
Support: None
Multicenter Phase 1 Clinical Trial Targeting Tissue Factor for the Treatment
of Neovascular AMD
John A. Wells, III1, Brian B. Berger2, Christine Gonzales3, Victor H. Gonzales4,
David L. Johnson1, Brian D. Sippy5, Manju Soni6. 1Ophthalmology, Palmetto
Retina Center, West Columbia, SC; 2Retina Research Center, Austin, TX;
3
Ophthalmology, Retina and Vitreous Center of Southern Oregon, Ashland, OR;
4
Valley Retina Institute, McAllen, TX; 5Ophthalmology, Rocky Mountain Eye Ctr,
PC, Missoula, MT; 6Numa LLC, Mystic, CT.
Purpose: To evaluate the safety and tolerability of binding tissue factor with hIcon1, alone or in combination with anti-VEGF therapy, in eyes with active
neovascular AMD.
Methods: This prospective, multi-center, dose-escalating clinical study evaluated
the safety and tolerability of a single, intravitreal injection of 60g, 150g and
300g of hI-con1 in 18 patients (6 per cohort). Ocular inclusion criteria included
active CNV with at least a 30% classic component on angiography and VA 20/63 CF. Eyes with end-stage CNV, eyes on chronic anti-VEGF therapy and treatment
nave eyes were enrolled. Anti-VEGF therapy was allowed 2 weeks after the hIcon1 injection at the investigators discretion.
Results: No ocular or systemic dose limiting toxicities were identified. No retinal
or choroidal vascular, inflammatory or hemorrhagic toxicities were identified.
Patients reported no drug related adverse events. hI-con1, administered alone or
adjunctively with an anti-VEGF agent, showed multiple, dose-related biologic
signals across the broad spectrum of treated eyes. An interim analysis of 6 eyes in
the high-dose 300g cohort at Day 57 showed:
67% showed reduced OCT thickness, some CNV regression on angiography and
3 lines improved BCVA.
100% of the 3 treatment nave eyes showed reduced OCT thickness, some CNV
regression on angiography and 3 lines improved BCVA.
An interim analysis of all dose cohorts additionally showed:
The mean BCVA of 7 eyes on chronic anti-VEGF therapy (mean number of
previous anti-VEGF injections = 6; all with persistent sub-retinal fluid; VA = 20/80
or worse) at Day 29 was +9 letters compared to baseline; at Day 57, +7 letters
compared to baseline.
The mean BCVA of 9 patients receiving one hI-con1 plus one anti-VEGF
injection was +11 letters 29 days after the anti-VEGF injection.
67% of all 18 eyes went 57 days without resuming anti-VEGF therapy; 50% of all
eyes went 85 days or longer without additional anti-VEGF therapy.
Conclusions: A single injection of hI-con1, alone or in combination with antiVEGF agents, showed no ocular or systemic safety signals. Evidence of biologic
activity with reduced OCT thickness, evidence of CNV regression, and gains in
BCVA was observed in many of the treated eyes. Further studies are planned.
Commercial Relationships: John A. Wells, III, Eyetech (C, R), Genentech (F),
LPath Inc (F), Novartis (F), Ophthotech (F), Pfizer Inc (F), Steba (F); Brian B.
Berger, Genentech (F), Iconic Therapeutics (F), Lpath Inc (F), NeoVista
Pharmaceuticals (F), Pfizer Inc (F); Christine Gonzales, Eyetech (C), Iconic
Therapeutics (F), Ophthotech (F); Victor H. Gonzales, Eyetech (C, R), Genentech
(F, C), Iconic Therapeutics (F), Ophthotech (F), Pfizer (C, R), Pfizer Inc (F),
Regeneron (F), Steba (F); David L. Johnson, Genentech (F), Iconic Therapeutics
(F), Lpath Inc (F), Ophthotech (F), Regeneron (F); Brian D. Sippy, Iconic
Therapeutics (F), Regeneron (F); Manju Soni, Iconic Therapeutics (C)
Support: Iconic Therapeutics, Inc
Clinical Trial: http://www.clinicaltrials.gov, NCT01485588

Trasferrin-functionalized PLGA Nanopartilces Sustain Diclofenac Delivery to
Choroid-RPE
Uday B. Kompella1A, Arun K. Upadhyay1B. APharmaceutical Sciences,
Ophthlamology and Bioengineering, BPharmaceutical Sciences, 1University of
Colorado Denver, Aurora, CO.
Purpose: To develop topically applied transferrin-functionalized, biodegradable,
polymeric nanoparticles to enhance and sustain diclofenac delivery for treating
choroidal neovascularization.
Methods: Diclofenac and Nile red loaded poly(lactide-co-glycolide) (PLGA)
nanoparticles were prepared using double emulsion and solvent evaporation
method. Nanoparticles were chemically conjugated to amino groups on transferrin
through 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl (EDAC) activation
of carboxyl group present on PLGA. In vitro release of diclofenac from
nanoparticles was assessed under sink condition at 37 C. Diclofenac and Nile red
were quantified using UV absorbance at 276 nm and 520 nm (Nile red absorbance
maxima in 1:1 acetonitrile:water mixture), respectively. In vitro uptake of
nanoparticles was evaluated in ARPE-19 and MDCK1 cells. Briefly, cells at 80 %
confluence were incubated with 100 g/ml nanoparticles in serum free medium for
5 min, the medium was removed, cell monolayer was washed - 2 times each with
physiological PBS (7.4) and acidic PBS (pH 5.2), cells were lysed using 2% Triton
X-100 and Nile red was extracted from particles using dichloromethane, and Nile
red content was estimated using UV absorbance at 554 nm (absorption maxima in
dichloromethane). Following single topical application of two drops of 10 l
nanoparticle suspension (0.35% w/v diclofenac acid) NP or equivalent dose of
plain diclofenac sodium solution in Brown Norway rats, drug delivery was
determined by LC-MS method. Nanoparticles were assessed for size using Malvern
nanosizer.
Results: Transferrin-functionalized diclofenac PLGA nanoparticles had a mean
diameter of 230-260 nm and a negative zeta-potential. Drug release was sustained
during the one week in vitro study. ARPE-19 and MDCK1 cells showed 3-4 times
higher uptake of transferrin-functionalized diclofenac-PLGA nanoparticles when
compared to non-functionalized nanoparticles. At the end of one week following
single dose, diclofenac levels in rat choroid-RPE were significantly higher with
functionalized PLGA nanoparticles when compared to non-functionalized
nanoparticles and plain drug solution.
Conclusions: Transferrin functionalization enhances cellular delivery of
nanoparticles and allows sustained and enhanced delivery of diclofenac to choroidRPE for one week following eye drop administration.
Commercial Relationships: Uday B. Kompella, International Patent
Application. 12/09/2007. WO/2008/033924 (P); Arun K. Upadhyay, None
Support: NIH Grant R41 EY020097
Planar SU-8/PEGDMA Microdevices for Retinal Drug Delivery of Lucentis
Jennifer S. Wade, Tejal A. Desai Ph.D.. Bioengineering and Therapeutic Sciences,
University of California - San Francisco, San Francisco, CA.
Purpose: Due to the complications associated with intravitreal injection novel drug
delivery technologies are desired. We have developed planar SU-8/PEGDMA
microdevices, coated with the permeation enhancer Chitosan. These devices
maximize contact surface area and provide consistent drug volume. Using the
retinal epithelial cell line ARPE19, the influence of microdevice geometry and
surface coating on paracellular drug delivery has been investigated.
Methods: Device fabrication was achieved by a three-mask photolithography
process. SU-8 and PEGDMA hydrogel solutions of FITC-Dextran (FD) or Lucentis
were spun onto a silicon wafer. UV-light was used to crosslink the hydrogel in the
device reservoir. Surface modification was conducted by deposition of a 1.6% w/v
Chitosan solution onto a drug-loaded wafer until dry film formation. Devices were
placed in the apical chamber of an ARPE19 coated transwell insert. Aliquots were
removed from the basolateral chamber and analyzed for released drug
concentration using a fluorimeter or spectrophotometer.
Results: Microdevices with payloads of FD and Lucentis were succesfully
fabricated. Consistent elution of FD and Lucentis from devices was achieved. The
quantity of FD transported across the ARPE19 monolayer of cells using a
microdevice was greater than the amount transported using a bolus administration.
The effect Chitosan coating has on the amount of drug transported is still being
investigated and further optimization of the coating process will clarify if the
mucoadhesive inhibits drug elution.
Conclusions: A planar microdevice capable of housing therapeutics of varying
molecular weight was developed. Preliminary data suggests this device enhances
the transport of large molecules across an ARPE19 in vitro retina model in
comparison to bolus administration alone.
Commercial Relationships: Jennifer S. Wade, Genentech Inc (F); Tejal A.

Desai Ph.D., Genentech Inc (F)
Support: Genentech Grant 71868-01
Therapeutic Effect of Fenofibrate Eyedrops on Diabetic Retinopathy
Ying Chen1, Boyu Lu1,2, Qingjiong Zhang2, Jianxing Ma1. 1Physiology, OUHSC,
Oklahoma City, OK; 2Sun Yat-sen University, Zhongshan Ophthalmic Center,
Guangzhou, China.
Purpose: The new studies from The Fenofibrate Intervention and Event Lowering
in Diabetes (FIELD) reported that oral administration of fenofibrate, a relativlye
safe and low cost lipid-lowering drug, prevented the progression of diabetic
retinopathy (DR) in type 2 diabetic patients. The purpose of this study is to evaluate
if fenofibrate has therapeutic effects on DR in type 1 diabetes and if topical
administration of fenofibrate is able to prevent DR in type 1 diabetes.
Methods: Human retinal endothelial cells (HRECs) were cultured on Matrigel or
Transwell inserts in the presence or absence of various concentrations of
fenofibrate, the effects of fenofibrate on tube formation and cell migration were
evaluated. Oxygen-induced retinopathy (OIR) was generated by exposing newborn
rats to 75% oxygen. Diabetes was induced in adult rats by injection of
streptozotocin (STZ). Eyedrop containing 3% fenofibrate or control vehicle were
topically administered to the cornea of OIR rats or diabetic rats 5 and 3 times a day,
respectively. Levels of vascular endothelial growth factor (VEGF) and intercellular
adhesion molecule 1 (ICAM-1) were measured by Western blot and enzyme-linked
immunosorbent assay (ELISA). Retinal vascular leakage was evaluated by retinal
vascular permeability assay, and retinal neovascularization (NV) was evaluated by
fluorescein angiography.
Results: Fenofibrate inhibited HREC tube formation and cell migration. Topical
application of fenofibrate significantly decreased retinal vascular permeability and
reduced levels of VEGF and ICAM-1 in the retina from OIR rats and from STZinduced diabetic rats, and arrested pre-retinal NV in the OIR rat model.
Conclusions: Fenofibrate attenuates retinal angiogenesis and inflammation, and
topical administration of fenofibrate has therapeutic effects on DR in type 1
diabetes.
Commercial Relationships: Ying Chen, None; Boyu Lu, None; Qingjiong
Zhang, None; Jianxing Ma, None
Support: 711JF10-Junior Faculty Award from ADA , P20RR024215 from the
National Center for Research Resources.
Development of an Optimized Culture Media to Improve VEGF Antagonist
Secretion and Stability by Encapsulated Cell Technology Implants

Alline M. Lelis1A, Michael Rivera1A, Sue Elliott1A, Brenda Dean1A, Pam
Heatherton1A, Bruce Bouchard1A, Melissa Stiles1A, Vicent Ling1A, Konrad Kauper1B,
Weng Tao1. AResearch and Development, BCore Technology Development,
1
Neurotech USA, Lincoln, RI.
Purpose: Encapsulated Cell Technology (ECT) is designed to deliver therapeutic
factors directly to the retina through a semi-permeable, hollow fiber membrane
implant that encapsulates genetically modified cells. The goal of this study was to
optimize ECT pre-implant culture conditions to maximize and stabilize secretion of
a VEGF-antagonist targeting microgram per day daily production levels and to
remove all animal and human components from the culture media. Effects of
culture media and additional supplementation on encapsulated cell protein secretion
levels and encapsulated cell health were evaluated.
Methods: ECT devices were manufactured and encapsulated using a platform cell
line engineered to secrete a VEGF antagonist molecule. Media supplements,
including lipids, vitamins, amino acids and growth factors were added to the
platform culture media in an effort to increase protein expression and optimize
encapsulated cell line performance. Multiple commercial media designed for
enhanced recombinant protein expression, differentiation, and viability were
compared to the current platform media. Protein production of the encapsulated
cells was evaluated by ELISA. Device health and cell viability was analyzed
qualitatively by the preparation of histology slides. Media analyte and metabolite
concentration were evaluated using a chemistry analyzer.
Results: The addition of GlutaMAXTM and removal of L-glutamine from all media
led to decreased ammonia accumulation and improved encapsulated cell stability.
One animal and human component free media formulation increased protein
production of the encapsulated cell line three to four fold, maintaining steady-state
expression levels in micrograms per day. Most media supplements evaluated did
not improve protein expression, with the exception of cholesterol. However, longterm exposure to high concentrations of cholesterol was detrimental to cell health.
While select stem cell factors improved protein production, changes to cell
morphology and growth rate occurred as well.
Conclusions: Optimization of media used to support ECT function resulted in
identification of an improved formulation capable of achieving sustained ECT
production of a VEGF antagonist at a rate of micrograms daily secretion. In
addition, the absence of animal and human components enables this media to be
used for clinical development and commercialization.
Commercial Relationships: Alline M. Lelis, Neurotech USA (E); Michael
Rivera, Neurotech USA (E); Sue Elliott, Neurotech USA (E); Brenda Dean,
Neurotech USA (E); Pam Heatherton, Neurotech USA (E); Bruce Bouchard,
Neurotech USA (E); Melissa Stiles, Neurotech USA (E); Vicent Ling, Neurotech
USA (E); Konrad Kauper, Neurotech USA (E); Weng Tao, Neurotech USA (E)
Support: None
Long-Term, Sustained Intraocular Delivery of a VEGF Antagonist Using
Encapsulated Cell Technology Implant for the Treatment of Choroidal
Neovascular Diseases
Konrad Kauper, Vincent Ling, Sue Elliot, Cahil McGovern, Sandy Sherman,
Brenda Dean, Lisa Orecchio, Mike Rivera, Pam Heatherton, Weng Tao. Core
Technology Development, Neurotech USA, Lincoln, RI.
Purpose: To develop Encapsulated Cell Technology (ECT) intraocular implants
capable of microgram daily intraocular delivery of a VEGF-antagonist over a
sustained period. Long-term delivery of a VEGF antagonist will potentially
improve the standard-of-care treatment modality of monthly injections by
providing a consistent dose, eliminating patient compliance issues and minimizing
safety concerns associated with repeated injections in the eye.
Methods: A human derived cell line with a clinically tested history of safety and
long-term implant viability was genetically engineered to produce a VEGFantagonist in doses ranging from nanogram to microgram daily sustained delivery
by ECT intraocular implants. The ability to neutralize VEGF and to block VEGFinduced endothelial cell proliferation was evaluated for the VEGF-antagonist.
Studies were conducted to determine the steady state concentrations of VEGFantagonist in the rabbit eye over a one year period. Maximum vitreous
concentrations from several encapsulated cell lines producing escalating doses of
VEGF-antagonist were quantified. Sustained intraocular levels of VEGF-antagonist
delivered by ECT implants were compared to modeled data of standard-of-care
injections for LucentisTM, AvastinTM and EyleaTM to determine the projected
efficacy requirements of steady-state concentration of a VEGF antagonist.
Results: All doses of VEGF-antagonist produced by ECT implants were
determined to be potent and bioactive. Controlled delivery by ECT ranged from
nanogram per day to greater than 5 microgram per day sustained delivery in the
rabbit eye. While improvements to the production rate of the ECT implant
continue, the current steady state concentration of VEGF antagonist exceeds 25
micrograms in the rabbit eye. Potential levels of VEGF-antagonist in the human
eye, adjusted for the half-life of the ECT produced protein, conservatively
extrapolate to concentrations greater than 50 ug, exceed the modeled levels for
steady-state, standard-of-care treatments by monthly injections.
Conclusions: ECT intraocular implant is capable of sustained delivery of a VEGF

antagonist for periods greater than one year in the rabbit. The ability of ECT
implant to deliver an efficacious dose over sustained periods, suggest that the ECT
implant would have unique advantages compared to traditional standard-of-care
treatment for choroidal neovascular diseases, including improved patient
compliance and safety.
Commercial Relationships: Konrad Kauper, Neurotech USA (E); Vincent
Ling, Neurotech USA (E); Sue Elliot, Neurotech USA (E); Cahil McGovern,
Neurotech USA (E); Sandy Sherman, Neurotech USA (E); Brenda Dean,
Neurotech USA (E); Lisa Orecchio, Neurotech USA (E); Mike Rivera, Neurotech
USA (E); Pam Heatherton, Neurotech USA (E); Weng Tao, Neurotech USA (E)
Support: None
Nanostructured Porous Silicon Dioxide Microparticles as an Intravitreal
Injectable Drug Delivery System for Avastin (Bevacizumab) Lasting Six
Months
William R. Freeman1, Michael Sailor2, Michelle Chen3, Lingyun Cheng1.
1
Ophthalmology, UCSD Jacobs Retina Center, La Jolla, CA; 2Chemistry and
Biochemistry, UCSD, La Jolla, CA; 3Spinnaker Biosciences, La Jolla, CA.
Purpose: Intravitreal anti-VEGF therapy has become the standard of care in
treatment of CNV, diabetic macular edema and other conditions. Most studies
suggest that injections of Avastin (bevacizumab) every 4 weeks may be the optimal
treatment although some interruption in therapy may be possible. The goal of our
study was to develop a long-acting intravitreally injectable form of Avastin bound
in a nanostructured porous silicon dioxide microparticles and to show the ability of
these Avastin-loaded microparticles to release drug over many months.
Methods: Porous silicon dioxide was prepared by electrochemical etch of a single
crystal silicon wafer in hydrofluoric acid and then oxidized. Microparticles were
prepared by ultrasonic fracture. Commercial Avastin was loaded into the
nanopores, which had mean diameters of ~ 100 nm. The porous silicon dioxide
carrier prepared in this fashion had previosuly been shown to be non-toxic after
intravitreal injection. Elution of drug into phosphate buffered saline (PBS) solution
at pH 7.4 was determined by placing 5 mg of drug loaded microparticles into
tightly capped glass vials containing 1.5 mL PBS and gently reciprocating the vials
at 37C. Free Avastin released from the microparticles was measured using a micro
BCA protein test (Pierce).
Results: The mass loading efficiency of Avastin in the porous silicon dioxide
microparticles was 137 ug Avastin/mg porous silicon dioxide. Elution experiments
were initially performed with a drug load of 700 ug. Avastin release was nearly
linear with a steady-state free (released) drug concentration between 10 and 30
ug/mL (therapeutic is >> 0.06 ug/mL). The free drug concentration remained > the
therapeutic concentration for 5.5 months.
Conclusions: Commercial Avastin can be loaded into nanoporous silicon dioxide.
We loaded a total of 1 mg of Avastin into a 0.1 cc injection volume (50% particles/
50% dextrose). Drug releases in a linear manner maintaining a therapeutic
concentration for 5.5 months (165 days). Optimized Avastin loading can increase
the load to 4.7 mg per 0.1 cc injection and would result in a therapeutic effect for
over six months. A clinical trial with an Avastin-loaded porous silicon dioxide
formulation is anticipated with in the next 12 months.
Commercial Relationships: William R. Freeman, Spinnaker Biosciences (F, I,
C); Michael Sailor, Spinnaker Biosciences (F, I, C); Michelle Chen, Spinnaker
Biosciences (E); Lingyun Cheng, Spinnaker Biosciences (F, I, C)
Support: NIH EY020617-01A1 and Research to Prevent Blindness, Inc.
A Novel Eye Drop Formulation of Squalamine For Exudative AMD:
Evaluation Of Ocular Distribution And Ocular Safety In Rabbits
Irach B. Taraporewala1, Michael J. Elman2, Shalom Z. Hirschman1, Samuel I.
Backenroth1. 1Ohr Pharmaceutical Inc, New York, NY; 2Elman Retina Group,
Baltimore, MD.
Purpose: To evaluate the ocular safety and ocular tissue distribution of a novel eye
drop formulation of Squalamine, a potent antiangiogenic small molecule inhibitor
of multiple growth factors (VEGF, PDGF, bFGF) with previously demonstrated
systemic activity in vivo in ocular pathologies and in clinical trials for exudative
macular degeneration.
Methods: Male Dutch belted rabbits (n=24) were administered Squalamine eye
drops bilaterally, either QD (every 24 hours) or BID (every 12 hours) for 1, 7, and
14 days (n=4/group/dose). Ocular tissues were harvested 24 (2) or 12(1) hours
post last dosing in the QD or BID groups, respectively. Posterior sclera/choroid,
aqueous and vitreous humors, and plasma were assayed for Squalamine
concentrations using a validated LC-MS/MS method with a lower limit of
quantification (LLOQ) of 10ng/g of tissue. Ocular toxicity and irritation were also
evaluated.
Results: Squalamine eye drops, given QD or BID were well tolerated with no

adverse clinical effects. Given QD, mean concentrations of Squalamine in
posterior sclera/choroid were 9.5, 21.9, and 39.8ng/g in the 1, 7, and 14 day
groups, respectively. Given BID, mean concentrations of Squalamine in posterior
sclera/choroid were 21.7, 62.6, and 68ng/g in the 1, 7, and 14 day groups,
respectively. Values represent levels at one full dosing interval after last
administration (QD 24(+2) hours, BID 12(+1) hours). Squalamine concentrations in
aqueous and vitreous humors were <LLOQ in all animals and <LLOQ in plasma in
23/24 animals.
Conclusions: Squalamine eye drops were well tolerated, consistent with previous
longer term preclinical studies in which there were no adverse clinical findings or
changes in ocular histopathology. Mean posterior segment tissue concentrations of
Squalamine given QD and BID exceeded the threshold at which Squalamine is
known to inhibit neovascularization in a cell-based model. Importantly, as
evidenced by concentrations in posterior sclera/choroid above the anti-angiogenic
threshold level, sustained therapeutically relevant posterior ocular exposure levels
were maintained for the duration of a full dosing interval (QD 24h, BID 12h).
Squalamine has prolonged residence time and slow tissue clearance when
administered QD and BID up to 14 days. Minimal systemic uptake reduces
potential systemic safety concerns. The absence of Squalamine concentrations in
aqueous humor suggests a passive diffusion mechanism from anterior to posterior
sclera and subsequently into the choroid. These results, consonant with previous
preclinical topical data and intravenous clinical studies, warrant further clinical
investigation of Squalamine eye drops to treat neovascular ophthalmic disorders.
Commercial Relationships: Irach B. Taraporewala, Ohr Pharmaceutical (I, E,
P); Michael J. Elman, Genentech (C), Ohr Pharmaceutical (C); Shalom Z.
Hirschman, Ohr Pharmaceutical (C); Samuel I. Backenroth, Ohr Pharmaceutical
(I, E, P)
Support: None
115 Drug Delivery I
Sunday, May 6, 2012, 8:30 AM - 10:15 AM
Molecularly Imprinted Hydrogels with Hyaluronic Acid for Ocular Drug
Delivery
Giuliano Guidi, Andrea K. Weeks, Heather Sheardown. Chemical Engineering,
McMaster University, Hamilton, ON, Canada.
Purpose: The use of eye drops for ocular drug delivery while convenient is
inefficient and poses a risk of systemic side effects. Contact lenses with a welldocumented history of use in the eye represent a promising alternative vehicle. The
potential of a contact lens delivery system for the prostaglandin analogue, timolol
maleate was investigated and the effect of factors such as material polarity,
molecular imprinting and HA were analyzed to further understand the drughydrogel interactions which govern the release kinetics.
Methods: Two model lens materials were created based on combinations of
dimethylacrylamide (DMAA), hydroxyethyl methacrylate (HEMA) and
methacryloxypropyltris(trimethylsiloxy)silane (TRIS). The materials were prepared
with and without the direct addition of 7.5 kDa hyaluronic acid at 0.1 wt% and
timolol maleate at 0.3 wt% and cured under UV light. Molecular imprinting was
accomplished and a subsequent uptake study was performed for two days using two
solutions, one containing timolol in PBS at a concentration of 0.2mg/mL and
another containing both timolol and hyaluronic acid in PBS at 0.2mg/mL. Release
studies were then performed on the molecular imprinted materials using UVspectroscopy to quantify release. Materials were also characterized by swelling and
contact angles.
Results: Release studies show that 90% of the release for both pHEMA/TRIS and
DMAA/TRIS occurs within approximately 3 days and 2 days respectively. The
pHEMA/TRIS materials show an average 25% increase in total drug release over
the duration of the study in comparison to the DMAA/TRIS materials. The effect of
imprinting yields a consistent trend with both timolol and HA imprinted materials
independently showing increasing release and a compounded effect when materials
are imprinted with both compounds simultaneously. The increase in uptake affinity
associated with timolol imprinted materials is consistent with established literature,
however, the effect of HA increasing drug release is a novel result.
Conclusions: It is evident the polarity of monomers, the ratio of their contribution
and the nature of the therapeutic are all determining factor in uptake and release for
these systems. Hyaluronic acid imprinting appears to affect the uptake and release
of therapeutics regardless of its similarity to the target compound and this
interaction requires further characterization.
Commercial Relationships: Giuliano Guidi, None; Andrea K. Weeks,
None; Heather Sheardown, None
Support: NSERC 20/20 Ophthalmic Network

Preparation And Characterization Of Fk-506 Micelles For Potential
Application In The Treatment Of Uveoretinitis
li xingyi, chen hao. Institute of Biomedical Engineering, wenzhou medical college,
wenzhou, China.
Purpose: To develop a novel injectable FK-506 micelles and the potential
application in the treatment of uveoretinitis was primarily evaluated in rat model.
Methods: Briefly, PEG-PCL diblock copolymer was prepared by ring-opening
copolymerization of e-CL initiated by MPEG at 130oC using Sn(Oct)2 as
catalyst.To improve the water solubility of FK506, thin-film method was employed
to develop a novel FK506 nanoformulation based on MPEG-PCL
copolymer.Briefly, 0.2g of FK506 and 0.8g of MPEG-PCL copolymer were codissolved into 10ml acetone solution, and then evaporated by a rotary evaporation
at 37oC. Finally,the product was re-suspended into 40ml water solution at 55oC to
obtain FK506 nanoformulation. The obtained FK 506 nanoformulation was
lyophilized and storage at 4oC for further usage. In the rate model, rats were
intravitreally injected with saline, FK506, FK506 nanoformulation and its
efficiency on the inflammation was evaluated.
Results: Due to the solubilization of MPEG-PCL co-polymer, the water solubility
of FK506 was greatly improved,and the obtained FK506 nanoformulation could be
freeze-dried into a power state while could re-dissolved into the water solution. As
presented in Fig.1, the obtained FK506 nanoformulation were uniform particle size
(about 50nm) and monodisperse with no aggregation. Meanwhile, the preliminary
study on the rate model revealed that the application of FK506 nanoformulation
could significantly reduce the intraocular inflammation and markedly inhibite the
development of uveoretinitis .
Conclusions: In the presented paper, a novel FK 506 micelles was developed and
its potential application in the treatment of uveoretinitis was investigated. The
results suggested that the application of FK506 nanoformulation could significantly
reduce the intraocular inflammation and markedly inhibite the development of
uveoretinitis .
Commercial Relationships: li xingyi, None; chen hao, None

Support: Zhejiang Provincial Program for the Cultivation of High-level Innovative
Health talents
Corticosteroid Delivery from a Punctum Plug in a Canine Model Over 21Days
Ankita Desai, Michael Bassett, Art Driscoll, Peter Jarrett, Amar Sawhney, Leslie
Jost, Abbe Miller, Charles Blizzard. R&D, Ocular Therapeutix, Inc., Bedford, MA.
Purpose: To examine the influence of aqueous solubility on the in vivo release rate
of three corticosteroids from a hydrogel punctum plug in a canine model.
Methods: Micronized prednisolone (Pred), prednisolone acetate (PA) and
dexamethasone (Dex) having a water solubility of 223, 89 and 17 g/mL,
respectively were suspended in a multi-arm PEG solution and injected into small
bore tubing prior to cross-linking. The steroid in hydrogel matrix was subsequently
dried and cut into punctum plugs. The plugs were inserted into the inferior
canaliculus of beagles and a subset was removed each week for photographic
imaging and to analyze drug content following extraction. Percent drug release was
plotted over time relative to content prior to insertion.
Results: As shown in Figure 1, the steroids release in vivo from the plug at a rate

relative to their aqueous solubility. The most soluble Pred is > 95% released in 14
days, whereas the lesser soluble Dex and PA has released 54 and 41%, respectively
over 21 days. Images of a steroid loaded plug demonstrating weekly drug release
are seen in the bottom of Figure 1.
Conclusions: Topical corticosteroids, such as Dex, Pred and PA are used to treat
inflammation in a variety of ophthalmic conditions. Many therapies require
multiple daily administrations (hourly for severe conditions) for a prolonged period
making a single dose therapy a more convenient option that may help ensure
patient compliance and better provide necessary treatment. Aside from drug
potency and ocular penetration, aqueous solubility must be considered when
developing an ophthalmic sustained drug delivery system, as it can greatly
influence the in vivo release rate as demonstrated in the canine model. A more
water soluble drug may be preferred if short term therapy is required, whereas a
less soluble drug may be preferred if long term therapy is
needed.
Commercial Relationships: Ankita Desai, Ocular Therapeutix, Inc. (E);

Michael Bassett, Ocular Therapeutix, Inc. (E); Art Driscoll, Ocular Therapeutix,
Inc. (E); Peter Jarrett, Ocular Therapeutix, Inc. (E); Amar Sawhney, Ocular
Therapeutix, Inc. (E); Leslie Jost, Ocular Therapeutix, Inc. (E); Abbe Miller,
Ocular Thjerapeutix, Inc. (E); Charles Blizzard, Ocular Therapeutix, Inc. (E)
Support: None
Presentation Time: 8:30 AM 10:15 AM
Comparison Of Ocular Effects After Use Of Topical Eye Drops Versus Use Of
The WhisperTM Topical Drug Applicator
Jacklyn H. Salmon1, Sydney Cartiff1, Skip Ballou2, Corey Ballou2, Brian C. Gilger1.
1
Clinical Sciences, North Carolina State University, Raleigh, NC; 2Corinthian
Ophthalmic, Inc., Raleigh, NC.
Purpose: To compare the mydriatic, mitotic, and intraocular pressure (IOP) effects
of topical tropicamide and latanoprost when delivered as an eye drop versus
delivery of the same medication via the WhisperTM device.
Methods: Six adult, female beagles were used in this study. With at least 1 week
washout between treatments, each dog received topical 1% tropicamide HCl
(Bausch & Lomb Inc., Tampa, FL) or 0.005% latanoprost (Greenstone LLC,
Peapack, NJ) delivered as a single eye drop (via the commercial bottle) or in a
separate study, via a single application using the WhisperTM device (Corinthian
Ophthalmic, Inc.) in the right eye while receiving balanced salt solution (BSS,
Alcon Laboratories, Fort Worth, TX) in the left eye using the same application
method. Pupil diameter (PD) (tropicamide and latanoprost) using a digital
pupilometer (VIPTM 200, Neuroptics, Irvine, CA) and IOP (latanoprost only)
using a TonoVet tonometer (Icare, Espoo, Finland) were measured in both eyes at 1, 0, 0.5, 1, 2, 3, 4, 6, and 8 hours after topical application.
Results: Eyes receiving topical tropicamide via eye drops or WhisperTM device
had significantly greater PD (P<0.01) than those receiving BSS from 30 minutes to
6 hours after application. Eyes receiving topical latanoprost via eye drops or
WhisperTM device had significantly smaller (P<0.0001) PD from 30 minutes to 8
hours after application and significantly lower (P<0.001) IOP from 1 to 6 hours
after application compared to eyes receiving BSS. There were no significant
differences in PD in eyes treated with tropicamide by eye drops or by the

WhisperTM device through 6 hours after treatment. Eyes treated with latanoprost
via the WhisperTM device had significantly lower IOP 1 hour (P=0.049) after
treatment compared to IOP of eye drop latanoprost. IOP was not significantly
different in eyes treated with latanoprost by either method at times 2, 3, and 4 hours
after treatment, but IOP was significantly lower (P=0.048) in eyes treated with
latanoprost eye drops compared to the WhisperTM latanoprost at 6 and 8 hours
after treatment.
Conclusions: Drugs delivered via the WhisperTM device, an innovative approach
to application of topical ocular medications, induce similar ocular effects compared
to traditional eye drops when using 2 common ocular medications in the dog.
Furthermore, the latanoprost IOP data suggests that the WhisperTM device may
induce a more rapid onset of effect compared to the same drug administered via an
eye drop. These results strongly support the further development of this innovative
device for application of topical ocular medications.
Commercial Relationships: Jacklyn H. Salmon, Corinthian Ophthalmic, Inc.;
Sydney Cartiff, Corinthian Ophthalmic, Inc.; Skip Ballou, Corinthian Ophthalmic,
Inc.; Corey Ballou, Corinthian Ophthalmic, Inc., Brian C. Gilger, Corinthian
Ophthalmic, Inc.
Support: None
Effect Of Melatonin On Prednisolone Eye Disposition In Cats
Maria J. Del Sole1, Paula Schaiquevich2, Marcelo A. Aba1, Carlos E. Lanusse1,
Laura Moreno1. 1Fisiopatologia, Facultad Ciencias Veterinarias, UNCPBA, Tandil,
Argentina; 2Unit of Clinical Pharmacokinetics, CONICET-Hosp de Ped JP
Garrahan, Ciudad de Buenos Aires, Argentina.
Purpose: To characterize prednisolone (PRED) ocular and systemic
pharmacokinetics in cats after oral administration and to study the effect of
concomitant administration of melatonin (MEL) on PRED disposition.
Methods: Six (6) castrated young physically and ophthalmologically healthy male
European Short Hair cats were orally administrated with a single dose (10 mg) of
PRED or a single dose of PRED (10 mg) and MEL (3 mg) in tablet. In anesthetized
cats 2 mL of blood and 450 L of aqueous humor (AH) were obtained from
preplaced cephalic antebrachial intravenous catheters and from one eye by direct
puncture, respectively, at: 0.25, 0.5, 1, 1.5, 2, 3, 4 and 5 h after administration.
Plasma and AH samples were assayed for PRED by HPLC. A two-compartment
model was used to simultaneously fit PRED plasma and aqueous concentration vs.
time data using ADAPT 5. The estimated pharmacokinetic (PK) parameters
included the absorption rate constant (ka), elimination rate constant from the central
compartment (kc), intercompartment rate constants between plasma and aqueous
humor (kpa), apparent volume of distribution of the central compartment (Vc/F).
PK parameters were compared between groups (PRED vs. PRED+MEL) by means
of Wilcoxon matched pairs test (p<0.05).
Results: The model adequately fitted the data and the estimated median (interval)
PK parameters are shown in Table 1. No significant difference was observed in the
PK parameters when comparing between groups of treatments (p>0.05).
Conclusions: These results indicated that MEL does not modify PRED systemic or
ocular disposition in cats when both are administrated simultaneously. A possible
synergic pharmacological effect may be account for different mechanisms of
action, but not for pharmacokinetic synergism and will be further
studied.
Commercial Relationships: Maria J. Del Sole, None; Paula Schaiquevich,

None; Marcelo A. Aba, None; Carlos E. Lanusse, None; Laura Moreno, None
Support: None

Vitreous Humor Buffering Capacity Of Rabbit, Bovine, And Porcine
Mohannad Shawer, Martin J. Coffey, Eric Phillips. Bausch and Lomb, Rochester,
NY.
Purpose: To compare the vitreous buffering capacity of three species: rabbit,
bovine, and porcine. This study examines the ability of the vitreous to
accommodate formulations with wide ranges of pH and its effect on the local and
whole vitreous pH, and clarity of the vitreous as result of such changes in its pH.
Methods: The vitreous from three species (bovine, porcine, and rabbit) were used
for pH titration. For each measurement, a 5-mL sample of vitreous was placed in a
scintillation vial and stirred. After the initial pH stabilization, the pH was recorded
and aliquots of either 0.1M HCl or 0.1M NaOH were added every minute for 30
minutes. Prior to the performing the titrations, vitreous samples were equilibrated
with a 5% CO2 in air mixture and the pH was adjusted to 6.5 - 7.5. Equilibration
with the 5% CO2 in air mixture was maintained during the HCl titrations, but not
during the NaOH titrations because doing so was found to artificially increase the
observed buffer capacity as the CO2 solubility increased at higher pH. Vitreous
clarity was evaluated by optical density measurements at low pH after incubation
with HCl aliquots for 20 minutes.
Results: Although the titration curves for the three species had some notable
differences, all three species exhibited similar buffering capacity toward both acidic
and basic titration. Titration toward both acidic and basic sides (over about 3 pH
units) showed a buffer capacity of 6.3-8 mmol L-1 pH-1. Using published
information about the composition of the vitreous in various species, we can
identify which vitreous components are contributing to the observed buffer
capacity. The clarity of the vitreous was found to diminish at low pH. The vitreous
maintained its clarity at pH 3 and 2, but significant turbidity was observed at pH 1.
Conclusions: All three animal species are predicted to react similarly with regard
to vitreous pH when injected with a non-neutral pH formulation. The observed
buffer capacity for the vitreous in all three species examined here is in good
agreement with what would be predicted based on published information on the
vitreous composition.
Commercial Relationships: Mohannad Shawer, Bausch and Lomb (E); Martin
J. Coffey, Bausch and Lomb (E); Eric Phillips, Bausch and Lomb (E)
Support: None
Transscleral Iontophoretic Delivery of a Macromolecule into the Rabbit Eye
John Higuchi1, Kongnara Papangkorn1,2, Sarah Molokhia2, Donald Mix1,
Charlotte Butler1, Prasoona Karra1, Balbir Brar1, S. Kevin Li1,3, William I.
Higuchi1,2. 1Aciont Inc, Salt Lake City, UT; 2University of Utah, Salt Lake City,
UT; 3University of Cincinnati, Cincinnati, OH.
Purpose: To study the in vivo transscleral anodal iontophoresis (AI) delivery of
Immunoglobulin G (IgG, MW~150 kDa and a surrogate for bevacizumab) and to
evaluate key iontophoretic conditions (i.e., current, current density, and drug
solution volume) on the amount and distribution of IgG delivered into the eye.
Methods: AI was performed on New Zealand White rabbits using a Visulex
system containing IgG (2.5%). There were 4 groups of rabbits (n=3 for each group)
under 4 different conditions of AI. After 20 min of AI delivery, the rabbits were
sacrificed and the eyes were enucleated. The eyes were dissected and the cornea,
aqueous humor, vitreous, conjunctiva, sclera, choroid and retina analyzed for IgG
content by ELISA.
Results: At 4 mA (current density = 1.8 mA/cm2), the total amount IgG delivered
was 438 63 and 364 27 g with 400 L and 200 L IgG solution volumes in
Visulex system, respectively. At 8 mA (current density = 3.6 mA/cm2), the total
amount IgG delivered was 727 38 and 457 94 g with 400 L and 200 L
solution volumes, respectively. With regard to tissue distributions, at both current
densities, most of the IgG was found in the conjunctiva and sclera at amounts
roughly in proportion to their total amounts at the two current densities. There were
significant amounts of IgG found in the deeper tissues, including the retina/choroid.
It was interesting to find that the average amounts in the deeper tissues (e.g.,
retina/choroid and vitreous) were much higher at the higher current density relative
to the total amounts (e.g., 8 vs 42 g for the retina/choroid).
Conclusions: This study might be the first to demonstrate successful noninvasive
macromolecule delivery in vivo to the posterior eye tissues with the amounts of
IgG delivered being of the same order of magnitude of that used in the intravitreal
injection of Avastin. It was estimated that iontophoretic delivery of IgG is
approximately 1000-fold superior over passive. The reproducibility was found to be
quite satisfactory (5-20%). The volume of drug solution used as well as current
density appears to be significant in AI drug delivery efficiency for both amounts
and depth of penetration.
Commercial Relationships: John Higuchi, Aciont Inc (E); Kongnara
Papangkorn, Aciont Inc (E); Sarah Molokhia, Aciont Inc (C); Donald Mix,
Aciont Inc (E); Charlotte Butler, Aciont Inc (E); Prasoona Karra, Aciont Inc
(E); Balbir Brar, Aciont Inc (E); S. Kevin Li, Aciont Inc (C); William I. Higuchi,
Aciont Inc (E)
Evaluation of a Candidate Cell Line Employed to Deliver an Antiangiogenic
Factor Using Encapsulated Cell Technology
Cahil McGovern1A, Sandy Sherman1A, Crystal Cortellessa1A, Suzanna Borges1A,
Melissa Stiles1B, Karen Ahola1A, Alice Lee1B, Konrad Kauper1C, Bruce Bouchard1D,
Weng Tao1E. AProcess Development, BQuality Control, CEngineering, DHistology,
E
Reasearch and Development, 1Neurotech USA, Lincoln, RI.
Purpose: To investigate the cell stability and in vitro performance of Encapsulated
Cell Technology (ECT) devices using a cell line to deliver an antiangiogenic factor.
Methods: The cell line secreting the antiangiogenic factor was constructed using
NTC-200 cells. The candidate cell line was cultured for 40 passages. At
predetermined passages, cells were seeded at a defined density in 24 well plates and
allowed to attach. Cells were washed twice with a balanced salt solution and
incubated with 1 ml of Endo-SFM for 2 hours. The pulsate was then analyzed for
antiangiogenic factor release. Once cell stability had been established out to 40
passages, the cell line was encapsulated in a hollow fiber membrane. Each device
was manufactured using standard protocols and held at 37C in closed packages
containing 37 mls of Endo-SFM. ECT devices were pulsed for factor production in
1 ml of Endo-SFM for 24 hours at days 2, 7, and 14 post manufacture. The devices
were subsequently analyzed for metabolic activity and then subjected to either total
DNA or histological analysis. Unencapsulated cells and device performance were
quantified by Elisa. Device metabolic activity was determined using the CCK-8
assay (Dojindo). Total DNA was determined using the Hoefer DyNA Quant 200
fluorometer (Pharmacia). Histological examination of the devices was performed
using standard hematoxylin and eosin staining techniques.
Results: Cell stability: The candidate cell line released the desired factor for 40
passages which is favorable for manufacturing purposes. During this time, the cells
maintained a typical and consistent morphology as well as exhibited a consistent
doubling rate. Device stability: The ECT devices released the desired factors and
maintained viable cells during the evaluation period. Total DNA analysis of
devices showed a consistent number of cells was maintained between 1 and 2
weeks and histological analysis of device sections showed a high density of healthy
cells distributed throughout the device.
Conclusions: The data suggested that the Encapsulated Cell Technology platform
was able to achieve sustained delivery of antiangiogenic factors under in vitro
conditions. This technology can deliver other factors for a broad range of
indications where long-term treatment is required.
Commercial Relationships: Cahil McGovern, Neurotech (E); Sandy Sherman,
Neurotech (E); Crystal Cortellessa, Neurotech (E); Suzanna Borges, Neurotech
(E); Melissa Stiles, Neurotech (E); Karen Ahola, Neurotech (E); Alice Lee,
Neurotech (E); Konrad Kauper, Neurotech (E); Bruce Bouchard, Neurotech (E);
Weng Tao, Neurotech (E)
Support: None
Rediscovering An Old Drug: Topical Application Of Acetazolamide Using A
Ternary Complex With Hp--cd And Tea
Luis I. Tartara, Santiago D. Palma, Daniela A. Quinteros, Marcela R. Longhi,
Daniel A. Allemandi, Gladys E. Granero. Departamento de Farmacia, Universidad
Nacional de Cordoba, Cordoba, Argentina.
Purpose: In order to enhance the ocular bioavailability of acetazolamide (ACZ), a
novel liquid formulation based on a multicomponent complex with hydroxypropyl-cyclodextrin (HP--CD) and triethanolamine (TEA) was prepared. In vitro e In
vivo performance of this formulation was assayed in rabbits.
Methods: The background of the design of this formulation was the interaction
between the components of the ternary complex. 1H- and 13C- NMR experiments
were undertaken to verify the real inclusion of ACZ in the
ACZ-HP--CD-TEA complex. The biopharmaceutical performance of the
formulation was evaluated by mean of In vitro corneal permeation and the In vivo
effect on the intraocular pressure (IOP). The marketed ophthalmic solution
AZOPT (1% w/v brinzolamide) was also included in the assays for comparison.
Results: The ternary system ACZ-HP--CD-TEA seemed to be able to reduce IOP
in about 30% after two hours. This effect was sustained for four hours after
instillation. In vitro corneal permeation studies demonstrated that the ACZ
permeation was increased as consequence of the multicomponent complex
formation. RMN experiments indicated that TEA can weaken the association
between ACZ and HP--CD increasing the drug ocular hypotensive effect by
increasing rate and extent of drug dissolution; due to the relative stability of the
ternary ACZ-HP--CD-TEA system. All formulations, including the commercial
product, were considered practically non-irritant.
Conclusions: These results indicate that this new strategy for ACZ formulation
could improve the treatment of IOP. The new formulation thus obtained was to be

able to facilitate ACZ corneal permeation and showed
appreciable pharmacological activity.
Commercial Relationships: Luis I. Tartara, None; Santiago D. Palma,
None; Daniela A. Quinteros, None; Marcela R. Longhi, None; Daniel A.
Allemandi, None; Gladys E. Granero, None
Support: FONCYT PICT 2010-0380
Moxifloxacin: A Valuable New Addition To Chemotherapeutic
Armamentarium For The Treatment Of Retinoblstoma
Megha Barot, Mitan R. Gokulgandhi, Dhananjay Pal, Ashim K. Mitra.
Pharmaceutical Science, University of Missouri, Kansas City, Kansas City, MO.
Purpose: Multidrug resistance (MDR) proteins (P-gp and MRPs) mediated
chemoresistance have been considered as a major cause of treatment failure in the
management of retinoblastoma (RB) with systemic chemotherapy. Here, we have
examined an interaction of fluoroquinolone moxifloxacin (MFX) with three
anticancer drugs (Topotecan, Etoposide, Vinblastine) for the treatment of RB. We
hypothesized that in such interactions, MFX will not only modulate the
bioavailability of anticancer agent at the ocular site such as retina (due to
competitive inhibition at efflux sites) but it will also synergize antiproliferative
activity of anticancer agent.
Methods: Time dependent uptake and transport of three anticancer drugs was
performed in presence of MFX across model cell lines (MDCK-MDR1 and
MDCK-MRP2). Modulation of time dependent cell cytotoxicity and caspase-3
enzyme activity of anticancer drugs in presence of MFX was evaluated using
retinoblastoma (Y-79) cells. IC-50 value of anticancer drugs alone and in presence
of MFX was also determined.
Results: 2-2.5 fold increased uptake of all three anticancer drugs was observed in
presence of MFX across MDCK-MDR1 and MDCK-MRP2 cells suggesting MFX
mediated evasion of efflux pumps. Significant reduction in efflux ratio (B-A/A-B
permeability) of three anticancer drugs was also observed in presence of MFX
indicating MFX mediated improved transport. Following cytotoxicity study,
tenfold reduction in IC-50 value of Topotecan and Etoposide and twofold reduction
in IC-50 value of Vinblastine was observed in combination with MFX. Significant
enhancement in caspase-3 enzyme activity of three anticancer drugs was observed
in combination with MFX on Y-79 cells.
Conclusions: Strategy of utilizing efflux pump inhibitors (which is yet not
clinically approved) for improving ocular bioavailability of anticancer agent has its
own limitations in terms of little or no improvement in toxicity of chemotoxic
agents. However, ocular cells have shown good tolerability against MFX, which is
a clinically well accepted drug even at higher dose level. Our results suggest that
MFX may be a valuable new addition to chemotherapeutic armamentarium,
concurrently improving cytotoxic activity while evading MDR mediated
chemoresistance of various anticancer drugs currently used for the treatment of RB.
These novel drug interactions will provide dual benefit in terms of overcoming
chemoresistance and synergistic cytotoxic effect will help reducing
chemotherapeutic dose which eventually reduces probability of dose-limiting
toxicity.
Commercial Relationships: Megha Barot, None; Mitan R. Gokulgandhi,
None; Dhananjay Pal, None; Ashim K. Mitra, None
Support: NIH grants RO1 EY 09171-16 and RO1 EY 10659-14
Lc-ms/ms Quantification Of Melphalan Plasma Levels In Children
Undergoing Selective Intra-arterial Infusion Of Chemotherapy For
Retinoblastoma
Jonathan W. Kim, Ludmila Alexandrova, Emilia DeMarchis, Diana Lee, Allis
Chien. Ophthalmology, Stanford University, Palo Alto, CA.
Purpose: Melphalan is an alkylating agent with effective tumoricidal properties but
also severe systemic side effects. A recent clinical trial has demonstrated promising
results in retinoblastoma patients when melphalan is infused selectively into the
ophthalmic artery. A minority of subjects developed neutropenia, which suggests
that systemic diffusion of the drug does occur. Developing a reliable systemic assay
to determine plasma levels of melphalan following ophthalmic artery infusion is
critical in optimizing the benefits of this treatment.
Methods: We utilized high performance liquid chromatography tandem mass
spectrometry (LC-MS/MS) to determine the melphalan levels in human plasma.
Melphalan and the internal standard (N-phenyldiethanolamine (N-PEA)) were
purchased from Sigma (Steinhein, Germany) and drug-free normal human plasma
was obtained from a regional blood bank. Stock solutions of Melphalan 2 mg/mL
solution in dimethyl sulfoxide (DMSO) and the internal standard (IS) 1 mg/mL in
ethanol were both stored at -80C. Two concentration ranges (range 1: 2 - 400
ng/mL and range 2: 20 - 4000 ng/mL) were tested in order to develop a calibration
curve. The extraction procedure for the concentration ranges 1 and 2 utilized 100
uL and 50 uL plasma respectively. Blank human plasma was spiked with
appropriate calibration solutions of melphalan and internal standard. A

methanol/acetonitrile protein precipitation was performed and the samples were
analyzed with an LC-MS/MS assay.
Results: Mass chromatograms for the range 1 provided eight calibration points:
2ng/mL; 4; 10; 20; 50; 100; 200; 400 ng/mL. A calculated ratio of the peak
intensities for both melphalan and the internal standard were then used to generate
a calibration curve. Similarly, mass chromatograms for range 2 generated eight
calibration points: 20 ng/mL; 40; 80; 200; 400; 1000; 2000; 4000 ng/mL. A
calculated ratio of the peak intensities for both melphalan and the internal standard
were then used to generate a second calibration curve (figure 2). Weighting of 1/x 2
was used to fit the data to a linear least-squared regression curve, where x
represents concentration (ng/mL). The linear detection response was defined for
concentrations within the range of 2 to 400 ng/mL and within the range of 20 to
4000 ng/mL.
Conclusions: A LC-MS/MS method for determination of melphalan levels in
human plasma has been developed, and calibration curves for range 1 and range 2
were generated by using a 100L plasma aliquot and a 50L plasma aliquot,
respectively. Ongoing aspects of the project include assay validation to demonstrate
accuracy, reproducibility and stability of melphalan in human plasma.
Commercial Relationships: Jonathan W. Kim, None; Ludmila Alexandrova,
None; Emilia DeMarchis, None; Diana Lee, None; Allis Chien, None
Support: None
3D Computational Fluid Dynamic Model Comparing Rabbit and Human
Intravitreal Pharmacokinetics Using Fluorescein Dyes
Julie E. Whitcomb1, Susan S. Lee1, Marc Horner2, Mohammad R. Kazemi3, Michael
R. Robinson1A, Jie Shen1. AOphthalmology, 1Allergan, Irvine, CA; 2ANSYS,
Evanston, IL; 3ANSYS, San Jose, CA.
Purpose: Ocular pharmacokinetic experiments are commonly performed in rabbits
to predict the intraocular distribution of drug in humans. However, results may not
translate directly to humans because of anatomical and physiological differences.
Computational fluid dynamic (CFD) models were developed in an effort to discern
the intravitreal drug distribution differences between the two species.
Methods: 3D half-globe symmetric CFD models of rabbit and human eyes were
developed to simulate diffusion and convection of drug in the vitreous. The
geometries were constructed using SpaceClaim Direct Modeler and anatomical
dimensions were based on average values for each species. ANSYS Fluent was
used to simulate the flow of vitreous humor and drug delivery from a centrallyplaced bolus of fluorescent dye. Fluorescein and fluorescein glucuronide were
selected as model compounds based on available experimental data from Araie and
Maurice, 1991.
Results: Anatomical differences, such as a larger lens and smaller vitreous volume
in the rabbit, altered the aqueous humor flow in the rabbit relative to the human.
The average drug concentration in the rabbit eye was found to be higher than the
human due to the smaller vitreous volume.
Conclusions: While the rabbit is an excellent animal model for studying ocular
pharmacokinetics, anatomical and physiological differences should be considered
when extrapolating rabbit data to human. CFD modeling can be effective to use
rabbit ocular pharmacokinetic data to simulate human drug
distribution.
Commercial Relationships: Julie E. Whitcomb, Allergan (E); Susan S. Lee,

Allergan (E); Marc Horner, ANSYS (E); Mohammad R. Kazemi, ANSYS (E);
Michael R. Robinson, Allergan (E); Jie Shen, Allergan (E)
Support: None
Protective Effects of Transscleral Drug Delivery Device Against Light-induced
Retinal Damage in Rats
Nobuhiro Nagai1A, Hideyuki Onami1A, Hirokazu Kaji1B, Takuya Yamada1B, Yuki
Katsukura1A, Machiko Sato1A, Yumi Ishikawa1A, Toru Nakazawa1A, Matsuhiko

Nishizawa1B, Toshiaki Abe1A. AGraduate School of Medicine, BGraduate School of
Engineering, 1Tohoku University, Sendai, Japan.
Purpose: To evaluate the protective effects of a transscleral drug delivery device
that can release geranylgeranylacetone (GGA) in a controlled release manner
against light-induced retinal damage in rats.
Methods: The device consists of a reservoir, controlled-release cover, and drug
formulations, which were made of photopolymeized poly(ethyleneglycol)
dimethacrylate that partially contains tri(ethyleneglycol) dimethacrylate. These
parts were fabricated via a microfabrication technique that used an AutoCAD
design. GGA, a heat shock protein (HSP) inducer, was loaded in the device. Highperformance liquid chromatography was used to evaluate the release amount of
GGA. After the devices were placed onto the sclera of rat eyes, HSP inductions of
retinal tissues were evaluated by real-time RT-PCR and western blot analyses.
Flash electroretinograms were recorded 4 days after white light exposure (8000 lux
for 18h). Histological examinations were perfomred to evaluate the thickness of the
outer nuclear layer.
Results: GGA was released with zero-ordered kinetics from the device. One or
four weeks after implanation, gene and protein expression of HSP70 were
upregulated in the sclera-choroid-retinal pigment epithelium fraction of the eyes
treated with GGA-loaded devices compared with those treated with saline-loaded
devices or non-treated rats. Electroretinographic amplitudes of the a- and b-waves
increased significantly in rats treated with GGA-loaded devices compared with
saline-loaded devices. The outer nuclear layer thickness was thinned in the group
treated with saline-loaded devices, but the group treated with GGA-loaded devices
suppressed the photic damage.
Conclusions: Transscleral GGA delivery device protected against light-induced
retinal damage in rats. The device may offer a less-invasive method of drug
delivery to achieve sustained release of medications for intravitreal drug delivery
and the treatment of various retinal diseases.
Commercial Relationships: Nobuhiro Nagai, None; Hideyuki Onami,
None; Hirokazu Kaji, None; Takuya Yamada, None; Yuki Katsukura,
None; Machiko Sato, None; Yumi Ishikawa, None; Toru Nakazawa,
None; Matsuhiko Nishizawa, None; Toshiaki Abe, None
Support: Grant-in-Aid for Young Scientists (A) from the MEXT, Japan, Health
Labour Sciences Research Grant from the MHLW, Japan
Delivery Of Dexamethasone To The Posterior Segment Of The Eye Using An
Eye Gel Formulation
Vincenzo Papa1A, Elena Solfato1B, Claudine Civiale1B. AMedical Marketing-BU
Pharma, BPharma Tech & Anal Chem-BU Pharma, 1SIFI SPA, Lavinaio, Catania,
Italy.
Purpose: Corticosteroids are valuable drugs in the management of macular edema
(ME) but therapeutic concentrations in the retina can be usually achieved only after
invasive injections, making the use of topical corticosteroids not suitable for the
treatment of ME. We describe herein the PK results obtained in rabbits after topical
administration of a gel-based delivery system containing 1% xanthan gum and
0.15% dexamethasone phosphate (DP), a water soluble prodrug of dexamethasone
(D).
Methods: A single dose of 50 l of 0.15% DP gel (Etacortilen Gel) was applied to
one eye of pigmented rabbits (n=24). D levels were measured by LC/MS-MS in
aqueous humour (AH), vitreous humour (VH) and retina/choroid (R/C) of both
eyes after 15, 30, 60, 120, 180 and 240 min. CMAX and AUCALL were calculated for
each tissue. Data obtained from control eyes were considered expression of
systemic absorption.
Results: CMAX (mean SE): 83.72 10.64 (study eye) vs. 0 (control eye) ng/ml in
the AH, 1.76 0.59 vs. 4.24 1.89 ng/g in the VH and 67.79 22.73 vs. 39.98
9.97 ng/g in the R/C. AUCALL (mean SE): 178.92 17.08 (study eye) vs. 0
(control eye) hr*ng/ml in the AH, 1.88 0.38 vs. 6.19 1.25 hr*ng/g in the VH
and 129.92 16.81 vs. 77.14 9.02 hr*ng/g in the R/C. In the R/C the AUCALL in
study eyes was statistically significant higher (p<0.01, Student t test) than that in
control eyes.
Conclusions: A single topical administration of DP gel is able to deliver effective
concentrations of D to the posterior segment of the eye through both systemic and
topical absorption. Topical absorption across the conjunctiva and the sclera
accounted for about 40% of the D reaching the posterior part of the eye. Since D
levels found in the R/C are sufficient to reduce central macular thickness in patients
with diabetic ME (Tanito IOVS 2011), this eye gel formulation may be useful as
adjuvant topical treatment of ME, obviating any concerns associated with more
invasive routes.
Commercial Relationships: Vincenzo Papa, SIFI SpA (E); Elena Solfato, SIFI
SpA (E); Claudine Civiale, SIFI SpA (E)
Support: None
SMVT Targeted Lipid Prodrug Of Cidofovir: Novel Treatment Strategy For

CMV Retinitis
Mitan R. Gokulgandhi, Megha Barot, Mahuya Bagui, Dhananjay Pal, Ashim K.
Mitra. Pharmaceutical Science, University of Missouri Kansas City, Kansas City,
MO.
Purpose: Cidofovir (CDF) has shown potential antiviral activity and currently
indicated for the treatment of AIDS-related cytomegalovirus (CMV) retinitis. The
major drawback with CDF is its low bioavailability (attributable to low lipid
solubility) and hence poor passive transport into virus infected tissue such as retina
which leads to limited therapeutic efficacy. Therefore, with a strategy to
simultaneously enhancing lipid mediated and transporter targeted cellular uptake of
CDF, we have evaluated novel transporter targeted lipid prodrugs of CDF for the
treatment of CMV retinitis.
Methods: We have successfully synthesized sodium dependent multivitamin
transporter (SMVT/Biotin) targeted lipid prodrugs with lipid moiety of carbon
chain length C6 or C12 as a linker between biotin (B) and CDF (B-C6-CDF and BC12-CDF). All synthesized prodrugs were characterized and evaluated for their
physicochemical properties and cytotoxicity. We have also performed in vitro
uptake and transport of [3H]Biotin in presence of these prodrugs on a model cell
line MDCK-MDR1 and human retinal cell line ARPE-19. To elucidate the affinity
interaction of these prodrugs with SMVT transporter, uptake of [3H]Biotin in
presence of different concentrations of prodrugs has been performed and IC50
value of individual prodrugs has been calculated.
Results: Inhibition of [3H]Biotin uptake in presence of all prodrugs shows strong
interaction with SMVT transporter. B-C12-CDF (0.900.07M, IC50) shows
higher affinity towards SMVT transporter than B-C6-CDF (11.251.48 M, IC50)
and B-CDF (31.42 3.05M, IC50) suggesting that increasing in lipid chain length
will improve cellular uptake via SMVT transporter. Cellular uptake of B-C12-CDF
(18 fold), B-C6-CDF (5 fold) and B-CDF (3.5 fold) prodrugs was found to be
higher relative to CDF, shows our novel conjugates have higher cellular
accumulation in retina.
Conclusions: Above studies demonstrated that transporter targeted lipid prodrugs
of CDF pose superior affinity for SMVT transporter and hence will have higher
bioavailability into human retinal cells owing to higher expression of SMVT on the
human retina. This strategy will augment antiviral and therapeutic efficacy of CDF
into retina due to synergistic effect of lipid and transporter targeting moiety.
Finally, these novel prodrugs appear to be potential clinical candidates for the
treatment of CMV retinitis.
Commercial Relationships: Mitan R. Gokulgandhi, None; Megha Barot,
None; Mahuya Bagui, None; Dhananjay Pal, None; Ashim K. Mitra, None
Support: NIH grants RO1 EY 09171-16 and RO1 EY 10659-14
Three Methods of Topical Lidocaine-Based Anesthesia for Intravitreal
Injections
Anthony F. Kokx, Gary J. Miller. Ophthalmology, West Virginia University,
Morgantown, WV.
Purpose: The purpose of this study is to compare 3 different methods of topical
anesthesia for intravitreal injections. This study will compare effectiveness of each
method at achieving pre-injection anesthesia and each method's cost effectiveness.
Methods: Patients already requiring an intravitreal (0.05mL) injection with either
Bevacizumab or Ranibizumab were enrolled in this study. Three different methods
of topical anesthesia were utilized; 3.5% lidocaine gel (LG), 4% lidocaine soaked
pledget (LP), and 4% lidocaine soaked cotton tipped applicator (CTA). Twentyfour patients were enrolled in the study (power of 80%), with all patients scheduled
to receive each treatment arm. After each injection, a pain survey (100 point Visual
Analog Scale) was administered, recording pain associated with both numbing and
injection. The following day, the patients used the same scale to retrospectively rate
their pain from the day of the injection as well as rate their residual pain (RP).
Patients had no reference to the prior survey.
Results: The average number of injections prior to enrollment in the study was 4.7.
Average post-injection IOP was 30mm Hg. Subconjunctival hemorrhage (SCH)
was noted in 1 out of 7 patients. The average pain score for each patient (all
methods same day) associated with the numbing method was 6.5/100 (n=7); for the
injection was 5.43/100 (n=7). The average pain score for each patient (all methods
next day) associated with the numbing method was 6.8/100 (n=6); for the injection
was 8.4/100 (n=6). The pain score in the LP group (n=5) was 5.8/100 for numbing
and 3.5/100 injection (same day). The pain score in the LP group was 5.3/100 for
numbing and 7.9/100 for injection (next day). The RP was 1.0/100. There was one
SCH in this group that did not have higher retrospective or residual pain scores.
The pain score in the LG group (n=1) was 0 for both numbing and injection (same
and next day) without any RP. The pain score in the CTA group (n=1) was
16.5/100 for numbing and 18.5/100 injection (same day). The pain score in the
CTA group was 7.5/100 for numbing and 6.0/100 injection (next day). RP was
1.5/100. There were no cases of endophthalmitis. The average unit cost for each
method is as follows: LG $1.13 per injection; LP $0.80 per injection; CTA $0.24
per injection. Average staffing time for each numbing protocol; LG group - 10

seconds (for technician); LP group - 30 seconds (for technician); CTA group - 60
seconds (for physician).
Data collection will finish March 2012.
Conclusions: All three anesthetic methods appear to be capable of achieving an
effective level of anesthesia. Currently, there is not a link between SCH and higher
RP or retrospective pain scores. There is a range of cost and time of staffing
required with each method.
Commercial Relationships: Anthony F. Kokx, None; Gary J. Miller, None
Support: WVU Research to Prevent Blindness
Pharmacokinetics of a VEGF Antagonist Delivered by an Intraocular
Encapsulated Cell Technology Implant
Michael Rivera1A, Alline Lelis1A, Bruce Bouchard1A, Melissa Stiles1A, Vincent
Ling1A, Konrad Kauper1B, Weng Tao1. ADevelopment, BCore Technology
Development, 1Neurotech USA, Lincoln, RI.
Purpose: Agents that neutralize VEGF isoforms have been shown to be effective
for treating the wet form of Age-Related Macular Degeneration (AMD), a leading
cause of blindness. Neurotechs encapsulated cell technology (ECT) platform
utilizes genetically engineered cells to secrete therapeutic proteins from an
implantable hollow fiber capsule. The purpose of the current study was to
investigate the pharmacokinetics (PK) of a VEGF antagonist delivered by an
intraocular ECT implant in rabbits over a 3 months implantation period.
Additionally, an arm evaluating the PK of Bevacizumab injection was included for
comparison.
Methods: ECT implants were manufactured using polymer membrane
encapsulated cells genetically engineered to continuously secrete a VEGF
antagonist molecule, designated NT-503. Standard-of-care injections of 1.25 mg
Bevacizumab were given at Day 0 and Day 42. Plasma and vitreous were harvested
from 4 eyes/two animals per time point following implantation or injection.
Implant secretion rates, vitreous and serum concentrations of the VEGF antagonist
were quantified by ELISA. Samples were collected at days 1, 3, 7, 14, 28, 40, 56
and 84 for both ECT implanted and injected eyes.
Results: The NT-503 implants have demonstrated stable expression of the VEGF
antagonist molecule through the time period evaluated. Data suggests that the
levels of VEGF antagonist are delivered at up to 5 g/day, maintaining a vitreous
steady state concentration of approximately 25 g/eye. Bevacizumab injections
have been shown to maintain vitreous steady state concentrations of > 5 g/mL for
up to 30 days.
Conclusions: Pharmacokinetics data demonstrates that ECT delivery of a VEGF
antagonist can maintain microgram per eye concentrations over an extended period
and the steady state level established by the ECT implant exceeds the maintenance
dose established by Bevacizumab monthly intraocular injections.
Commercial Relationships: Michael Rivera, Neurotech USA (E); Alline Lelis,
Neurotech USA (E); Bruce Bouchard, Neurotech USA (E); Melissa Stiles,
Neurotech USA (E); Vincent Ling, Neurotech USA (E); Konrad Kauper,
Neurotech USA (E); Weng Tao, Neurotech USA (E)
Support: None
Suppression of Rat Choroidal Neovascularization by Transscleral Vasohibin-1
Delivery Device
Hideyuki Onami1A, Nobuhiro Nagai1A, Ryosuke Wakusawa1A, Hirokazu Kaji1B,
Takuya Yamada1B, Yumi Ishikawa1A, Matauhiko Nishizawa1B, Yasufumi Sato1A,
Toru Nakazawa1A, Toshiaki Abe1A. AGraduate school of medicine, BGraduate school
of engineering, 1Tohoku univercity, Sendai, Japan.
Purpose: To evaluate the effects of transscleral sustained vasohibin-1(VASH)
delivery for rat laser-induced choroidal neovascularization (CNV) by a novel drug
delivery device.
Methods: Transscleral VASH delivery device (VASH-DD) consists of a drug
reservoir covered with a controlled-release membrane. The controlled release
membrane is made of photopolymerised polyethylene glycol dimethacrylate
(PEGDM) that contains interconnected collagen microparticle. VASH is pelletised
with PEGDM and loaded in the reservoir. The amount of released VASH from
VASH-DD was measured by ELISA. The release was also confirmed in vivo by
immunostaining 2 weeks after the device transplantation. Rat laser-induced CNV
model was used to evaluate the effect of VASH-DD. Fluorescein angiography and
choroidal flat mounts were used to evaluate the CNV. The results of not only
transplantation different amount of VASH loaded device (0, 1, and 10M,
respectively) and just pelletised VASH (VASH-pellet) but also VASH and vehicle
itravitreal injection were compared each other.
Results: The sustained release of VASH from VASH-DD was confirmed by
ELISA. VASH was detected in the retina, especially RPE and ganglion cells aroud
the transplanted region and optic nerve. Statisitically significant less fluorescein
leakage was observed in eyes with VASH-DD than eyes with 0 M VASH-DD 2
weeks after transplantation (p=0.038). Statistically significant less CNV size was
also observed in eyes transplanted with VASH-DD or intravitreal VASH injection
than those of others by choroidal flat mounts.
Conclusions: Our device showed sustained protein release and might offer a lessinvasive method than those previously reported for treatment, such as age-related
macular degenelation.
Commercial Relationships: Hideyuki Onami, None; Nobuhiro Nagai,
None; Ryosuke Wakusawa, None; Hirokazu Kaji, None; Takuya Yamada,
None; Yumi Ishikawa, None; Matauhiko Nishizawa, None; Yasufumi Sato,
None; Toru Nakazawa, None; Toshiaki Abe, None
Support: Health Labour Sciences Research Grant from the MHLW,Japan
Polyesteramide Microparticles For Ophthalmic Drug Delivery
Vanessa Andres-Guerrero1A, Beatriz de las Heras1B, George Mihov2, Aylvin Dias2,
Roco Herrero-Vanrell1A. APharmaceutical Technology, BPharmacology, 1Faculty
of Pharmacy/Complutense University, Madrid, Spain; 2DSM Ahead & DSM
Biomedical, Geleen, The Netherlands.
Purpose: Polyesteramides (PEAs) are a new family of polymeric materials. PEAs
combine good mechanical, thermal and processing properties and are also
biodegradable. The purpose of the current study was to evaluate PEA-II
microparticles to be used as carriers for controlled drug delivery in the eye.
Methods: Microparticles (MPs) were prepared using an emulsion-solvent
evaporation technique. Particle size and morphology of MPs were characterized by
dynamic light scattering and scanning electron microscopy (SEM), respectively. To
study the in vitro degradation behavior, MPs were incubated in a phosphate
buffered solution isotonized with NaCl (PBS, pH 7.4, 37C) at a constant agitation
speed of 100 rpm. At different time points (1 hour, 24 hours, 48 hours and 5 days)
MPs morphology was studied by SEM. In vitro tolerance studies were performed
by the MTT technique in human corneal limbal epithelial cells and macrophage
cells. Cells were exposed to MPs suspensions (5mg and 10mg MPs/ml in PBS) for
15 minutes (short term exposure), 1 hour and 4 hours (long term exposure).
Dexamethasone (DX) was used as a lipophilic drug model to determine the
encapsulation efficiency of PEA-II MPs (0.5:10 DX:PEAII).
Results: Unloaded PEA-II MPs were spherical and had smooth surface. MPs size
ranged between 10-30m (mean particle size 21.30.2 m). MPs started to lose
their shape after being incubated in PBS for 1 hour. 5 days later, MPS had turned
into an unshaped depot of polymer. The cytotoxicity assays demonstrated good
tolerance in the two cell lines in all cases after short- and long-term exposures (cell
viability>90%) at the assayed concentrations. DX-loaded MPs (9.620.56 g
DX/mg MPs and mean particle size 21.31.8 m) did not show any morphological
difference with unloaded MPs.
Conclusions: Biodegradable PEA-II MPs are potentially useful to develop new
controlled drug delivery systems for treating ophthalmic diseases.
Commercial Relationships: Vanessa Andres-Guerrero, None; Beatriz de las
Heras, None; George Mihov, DSM Biomedical (E); Aylvin Dias, DSM
Biomedical (E); Roco Herrero-Vanrell, None
Support: PANOPTES (project number 246180) under the 7th Research
Framework Programme of the European Union and Spanish Ministry of Health
(RETICS RD07/0062).
Sustained Back Of The Eye Delivery Following Sub-tenon Administration Of
Dexamethasone-loaded PLGA Microspheres In Rabbits
Rocio Herrero-Vanrell1, Deyanira Barbosa-Alfaro1, Irene Bravo-Osuna1, RS
Kadam2, I. Fernandez-Bueno3, MT Garca-Gutierrez3, Jose-Carlos Pastor3, Uday
B. Kompella2, Irene Teresa Molina Martnez1. 1Pharmaceutical Techn Sch of
Pharm, Complutense University, Madrid, Spain; 2Pharmaceutical Sci & Ophthal,
University of Colorado Denver, Aurora, CO; 3IOBA-Campus Miguel Delibes,
University of Valladolid, Valladolid, Spain.
Purpose: Quantification and pharmacokinetics evaluation of dexamethasone in
ocular tissues after sub-tenon administration of dexamethasone-loaded PLGA
microspheres in rabbits. Dexamethasone is widely used in ophthalmology to treat
several ocular diseases affecting the posterior segment. Due to short half-live of
dexamethasone, repeated intraocular injections are employed in the treatment of
most of these diseases. Controlled drug delivery systems, such as as microspheres
(MPh) are being designed to avoid frequent injections. MPhs based on a
biodegradable polymer (PLGA) have been developed to release dexamethasone in
vitro for 65 days. MPhs can be periocularly administered as a conventional
injection with less risk of adverse effects than intravitreal administration.
Methods: Sterilized dexamethasone-loaded PLGA microspheres (5 mg of
microspheres, dose: 828 g of Dexamethasone) were administered by sub-tenon
injection to rabbits. Dexamethasone concentrations in rabbit ocular tissues
(choroid-RPE, retina and vitreous) were evaluated at different time points (1, 2, 4
and 6 weeks after injection). Samples were treated and the drug was quantified by

means of electrospray ionization MS/MS analysis after separation by HPLC.
Results: After administration in rabbits, dexamethasone reached measurable
concentrations in choroid-RPE, retina and vitreous during the six-weeks study. The
tmax was observed at 2 weeks post-administration in the three tissues evaluated,
with Cmax values of 579.02 ng/g, 1,749.65 ng/g and 69.19 ng/mL for choroidRPE, retina and vitreous respectively. Calculations of area under the concentrationtime curve (AUC0,42d) in the target tissue (retina) were performed as measurement
of drug bioavailability, with a value of 21,337.52 ng day/g.
Conclusions: According to the results obtained in this work, the microsphere
formulation developed is suitable for sustained transscleral delivery of
dexamethasone. Periocular microspheres can be considered as a potential
alternative to dexamethasone intravitreal administrations indicated for the treatment
of diseases affecting the posterior segment of the eye.
Commercial Relationships: Rocio Herrero-Vanrell, None; Deyanira BarbosaAlfaro, None; Irene Bravo-Osuna, None; RS Kadam, None; I. FernandezBueno, None; MT Garca-Gutierrez, None; Jose-Carlos Pastor, None; Uday B.
Kompella, None; Irene Teresa Molina Martnez, None
Support: Research Group UCM 920415 (GR35/10-A). MAT-2010-18242.
A Topical Ocular Toxicity Study of Dexamethasone Sodium Phosphate
Formulations Administered with Visulex-p in Rabbits
Kongnara Papangkorn1,2, Donald Mix1, Charlotte Butler1, David J. Knauss2, John
W. Higuchi1, Balbir Brar1, William I. Higuchi1,2. 1Aciont Inc, Salt Lake City, UT;
2
Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City,
UT.
Purpose: To assess the ocular tolerability of Visulex-p (Vp) containing
dexamethasone sodium phosphate (DSP) following daily dosing for 7 days and
weekly dosing for 12 weeks in New Zealand white rabbits.
Methods: Twenty-one 5-6 months old rabbits were assigned to seven groups
according to Table 1. Vp loaded with 250 ul of the test formulation was applied to
the right eye. Vp was then removed at the end of the application. The ocular
examinations, with an indirect ophthalmoscope, modified McDonald-Shadduck
scoring, and fluorescein staining were completed at pre-, post-, and at selected
time-points post-dose. The body weight was recorded at baseline and during the
study. After the final observation, animals were sacrificed and the eyes were
collected for histological examination for any signs of inflammation, including
edema/congestion (conjunctiva, ciliary body, cornea), inflammatory cell infiltration
(conjunctiva, cornea, anterior chamber, trabecular meshwork, iris, ciliary body),
and neovascularization of cornea.
Results: Ocular findings are summarized in Table 1. Conjunctival injection and
chemosis were observed right after dosing in all groups, with severity increasing
with dosing concentrations. Resolution was observed within the 1-7 days after each
application. At doses of 8% and higher, there was a longer time to resolution with
continued weekly treatment. Weight loss occurred with 15% and 25% DSP. No
corneal damage was seen at any of the dose groups. No ocular findings were
observed in histopathology.
Conclusions: Vp containing 4% and 8% DSP formulation is well tolerated in
rabbit for 7 days of daily dosing and over a 3 month period of weekly dosing.
Although Vp containing 8% and 15% DSP formulation treatments showed an
accumulation of irritation scores over 3 months, there were no signs of irreversible
ocular tissue damage in any of the rabbits.
Commercial Relationships: Kongnara Papangkorn, Aciont Inc (E); Donald

Mix, Aciont Inc (E); Charlotte Butler, Aciont Inc (E); David J. Knauss, Aciont
Inc (F); John W. Higuchi, Aciont Inc (E); Balbir Brar, Aciont Inc (E); William I.
Higuchi, Aciont Inc (E)
Support: NEI Grant 2R44EY014772-02A1
Drug Eluting Contact Lenses For The Treatment Of Glaucoma
Joseph B. Ciolino1, Cristina F. Stefanescu2,3, Katherine A. Wymbs2, Sarah L.
Spraque2, Daniel R. Mascoop2, Shireen S. Rudina2, Fabiano Cade1, Daniel S.
Kohane3,2. 1Ophthalmology, Massachusetts Eye and Ear Infirmary/ Harvard
Medical School, Boston, MA; 2Massachusetts Institute of Technology, Cambridge,
MA; 3Anesthesiology, Children's Hospital Boston/Harvard Medical School,
Boston, MA.
Purpose: To report the in vivo and in vitro testing of a latanoprost-eluting contact
lens designed for the treatment of glaucoma.
Methods: Drug-eluting therapeutic contact lenses (TCL) were created by
encapsulating latanoprost-Poly(lactic-co-glycolic acid) films in methafilcon A by
ultraviolet light polymerization. TCLs (n=4) were placed in 5 mL of phosphate
buffered saline at 37C with continuous rotation (64 RPM). The release media was
sampled and changed daily. Drug concentrations were measured with an enzymelinked immunosorbent assay (ELISA). TCLs were inserted on the left eye of New
Zealand white rabbits (n=3) for 10 days. The eyes were examined under an
operating microscope and the anterior chamber (AC) fluid was collected during 1,
3, 5, 7, 10, and 14 days of continuous wear. Latanoprost 0.005% solution (drops)
was topically applied to rabbit eyes and the AC fluid was sampled at the following
times after drop administration (1, 3, 6, 12, and 24 hrs). Each sample was collected
on a different day and the AC drug concentration was measured by ELISA. The 24hr area under the curve (AUC) for latanoprost drops was calculated and compared
to the AC concentration measured during 14 days of TCL wear.
Results: In vitro, TCLs demonstrated an initial burst and then eluted a sustained
and therapeutic daily amount of latanoprost for 4 weeks. In vivo, the TCLs
demonstrated no signs of toxicity. Through 14 days of continuous wear, TCLs
delivered more drug to the anterior chamber each day than latanoprost drops.
Conclusions: This contact lens design can potentially be used as a treatment for
glaucoma and as a platform for ocular drug delivery with widespread applications.
Commercial Relationships: Joseph B. Ciolino, M0925.70250US00
(P); Cristina F. Stefanescu, None; Katherine A. Wymbs, None; Sarah L.
Spraque, None; Daniel R. Mascoop, None; Shireen S. Rudina, None; Fabiano
Cade, None; Daniel S. Kohane, M0925.70250US00 (P)
Support: 1K08EY019686
Thermoresponsive and Biodegradable Star-Branched Dendritic Polymers for
Drug Delivery across the Blood-Ocular Barriers
Xiaoxun Li, Sibo Jiang, Tao L. Lowe. Department of Pharmaceutical Sciences,
University of Tennessee Health Science Center, Memphis, TN.
Purpose: The objective of this study was to develop thermoresponsive and
biodegradable star-branched dendritic polymers as drug carriers for drug delivery
across the blood-ocular barriers.
Methods: The thermo-responsive and biodegradable star-branched dendritic
polymers were synthesized using a combination of solid phase peptide synthesis,
ring-opening polymerization and atom transfer radical polymerization techniques.
These dendritic polymers contained biodegradable poly-L-lactic acid (PLLA)
branches, thermoresponsive poly (N-isopropyl acrylamide) segmants, and poly-Llysine (PLL) dendrons. Their chemical structure was characterized using 1H-NMR
and FT-IR. Their molecule weights were determined using MALDI-TOF. Their
thermoresponsive property was measured using both UV-vis spectroscopy (to
measure the transmittance) and dynamic light scattering (to measure the
hydrodynamic size of the dendritic polymers). In the degradation studies, the
viscosity, molecular weight, and chemical structure of the dendritic polymers were
monitored using rheometer, MALDI-TOF and FTIR, respectively. Their
cytotoxicity to retinal pigment epithelium (RPE) cells was evaluated using the
MTT assay. The permeabilities of dendritic polymers across the porcine sclerachoroid-RPE, sclera, and cornea were determined using side-by-side diffusion
cells. The DTAF-labeled nanoparticle solutions were added to the donor cell while
equal volume of transport buffer was put in the receiver cell. The fluorescence
intensity in the receiver cell was assayed for 4 h.
Results: 1H-NMR, FT-IR and MALDI-TOF confirmed the successful synthesis of
the star-branched dendritic polymers. UV-vis spectroscopy and dynamic light
scattering measurements showed that they were thermoresponsive with LCSTs
around of 30-40 C at different concentrations (0.05-1mgmL). Degradation studies
showed that both viscosity and molecular weight decreased with time during the
degradation course. MTT data indicated that the dendritic polymers were not toxic
the retinal pigment epithelium (RPE) cells. The ex-vivo data demonstrated that
these dendritic polymers were more permeable to the porcine sclera-choroid-RPE
than the control, dextran with a molecular weight of 70,000.

Conclusions: The developed thermo-responsive and biodegradable star-branched
dendritic polymers showed great potential to be used as drug carriers for deliver
drug across the blood-ocular barriers.
Commercial Relationships: Xiaoxun Li, None; Sibo Jiang, None; Tao L. Lowe,
None
Support: NIH Grant
Molecular Parameters Associated With Uptake Of Multi-functional
Antioxidants For The Lens, Retina, And Brain
Hiroyoshi Kawada, Zifeng Zhang, Karen A. Blessing, Peter F. Kador.
Pharmaceutical Sciences, College of Pharmacy, Univ of Nebraska Medical Center,
Omaha, NE.
Purpose: Our laboratory has reported the synthesis of multi-functional antioxidant
analogs of N,N-dimethylsulfamoyl-4-(2-pyrimidyl)piperazine, which can
independently chelate redox-active metals and scavenge reactive oxygen species
(ROS). While they readily accumulate in the lens and retina after oral
administration, they fail to significantly reach the brain. To increase the ability of
these multi-functionals to cross the blood brain barrier (BBB), a new series of
analogs retaining the 2-amino-4-hydroxypyrimidine ring system have been
synthesized (HK 1-16). Here, the molecular parameters required to target these
analogs to the lens, retina, or brain are defined.
Methods: Molecular parameters such as log P and Clog P, that are associated with
lipophilicity; dipole moment, which implies an electric moment apart from the total
net charge on the molecule; kappa shape index, which is associated with steric bulk
of the molecule; molar refractivity, which is related to polarizability of the
molecule; and hydrophilic volume of the molecule were calculated by MOE. The
new descriptor fMF, which is associated with topology, was calculated as described
in J. Med. Chem. 1996, 39, 2887-289. All compounds examined were administered
to C57BL/6 mice for 14 days by individually incorporating each compound into
chow (0.05%). After 14 days of feeding, all mice were perfused with PBS and drug
levels in the brain, lens, and retina were quantified by HPLC-ESI-MS.
Results: Significant negative correlations between the accumulation of drugs in the
brain and either the kappa shape index, dipole moment, molar refractivity, or
hydrophilic volume were observed (linear fit analysis, R2 = 0.66-0.95, P< 0.05). In
contrast, significant positive correlations were observed between the lenticular
accumulation of drugs and either the kappa shape index or dipole moment (linear
fit analysis, R2 = 0.63-0.87, P< 0.05). No significant correlations between any
molecular parameter and the uptake of drugs into the neural retina were observed.
Conclusions: Inverse correlations between the uptake of drugs and their molecule
parameters related to steric bulk, refractivity, polarity, and hydrophilic volume,
were observed in the brain versus the lens. Although the retina is considered an
extention of the brain, no correlations between the examined molecular parameters
and accumulation of compound into the retina were observed.
Commercial Relationships: Hiroyoshi Kawada, None; Zifeng Zhang,
None; Karen A. Blessing, None; Peter F. Kador, None
Support: R21EY016460
Fluid Infusion Into the Corneal Stroma Using a Microneedle
Samirkumar R. Patel1, Eric Powers1, Mark R. Prausnitz1, Henry F. Edelhauser2.
1
Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta,
GA; 2Ophthalmology, Emory Univ Eye Center, Atlanta, GA.
Purpose: Corneal infections, especially deep stromal infections, can require an
aggressive therapy regimen using topically applied agents. It would be ideal to have
a delivery method that can administer drug directly to the stroma to stop spread of
the infection quickly without relying on patient compliance. Here, we investigate
the capability of a microneedle to infuse a fluid into the corneal stroma and study
the volume that can be delivered, the pressures needed for injection and the spread
of injected material.
Methods: Enucleated porcine eyes were placed in a holder and microneedles (700800 m in length) were inserted perpendicularly into the cornea. The needles were
attached to tubing with an in-line pressure transducer connected to a computer for
continuous pressure monitoring. The tubing was attached to a 1 mL syringe that
was mounted onto a syringe pump. The needle was inserted near the central cornea
and the pump was used to inject a target volume of sulforhodamine B into the
stroma at a flow rate of 100 L/min. Images of the cornea were taken to measure
the spread of the colored dye.
Results: Intrastromal injections of up to 300 L were achieved in all attempts using
a microneedle. The spread of the injection was radial from the site of injection and
the injection of 50, 80, 100, 200 and 300 L resulted in a spread diameter of
6.30.5, 6.91.0, 8.20.1, 10.00.5 and 11.40.7 mm, respectively. The pressure
profile during injection reached a peak within 25-35 s after the injection was started
and then dropped off. The peak pressure during all injections was between 27-32
psi. During injection of volumes between 100-300 L, the pressure leveled off
before injection was stopped. In these cases the pressure averaged between 15-20
psi. All volumes caused the cornea to expand and injection of 200-300 L resulted
in a 3-4 mm increase in thickness.
Conclusions: This study demonstrated that a microneedle can inject up to 300 L
of fluid directly into the corneal stroma of porcine eyes. The injected fluid spread
radially and a volume as small as 50 L was able to spread significantly. Large
volumes, however, caused significant corneal thickening. As a result, if minimal
corneal thickening is desired, small volumes, < 50 L should be used. This
approach could be used to deliver drugs to the cornea for the treatment of corneal
infections.
Commercial Relationships: Samirkumar R. Patel, 12/767,768 (P), Clearside
Biomedical (E); Eric Powers, None; Mark R. Prausnitz, 12/767,768 (P),
Clearside Biomedical (I); Henry F. Edelhauser, 12/767,768 (P), Clearside
Biomedical (I)
Support: R24 EYE017404, P30 06360, RPB
Pharmacokinetics of intravitreal bevacizumab (avastin) In vitrectomized eyes
Se Joon Woo1, Jeeyun Ahn2, Ji Hyun Park1, Sunyoung Park3, Kyu Hyung Park1,
Hyuncheol Kim3. 1Ophthalmology, Seoul National University Bundang Hospital,
Seongnam, Republic of Korea; 2Department of Ophthalmology, Seoul Metropolitan
Boramae Medical Center, Seoul, Republic of Korea; 3Chemical & Biomolecular
Engineering, Sogang University, Seoul, Republic of Korea.
Purpose: To perform comparative analysis of pharmacokinetics of intravitreally
injected bevacizumab in vitrectomized versus non-vitrectomized rabbit eyes.
Methods: Among the 35 eyes of 35 rabbits included in the study, 25-gauge pars
plana vitrectomy was performed in 18 right eyes (vitrectomized eyes), and the
remaining 17 right eyes served as control (non-vitrectomized eyes). Both groups
received 1.25mg/0.05mL bevacizumab intravitreally. Eyes were enucleated at 1
hour, 1, 2, 5, 14 and 30 days after the intravitreal injection and were immediately
frozen at -70C. Bevacizumab concentrations were determined after separation of
frozen vitreous and aqueous humor compartments using direct enzyme-linked
immunosorbent assay. Bevacizumab concentration-time data were fit by twocompartmental analysis to determine half-life.
Results: The vitreous concentration of bevacizumab in both groups showed two
phases of clearance which are the fast distribution phase and the slow elimination
phase. Bevacizumab vitreous concentration in vitrectomized eye showed 94.7% (1
hour), 70.5% (1 day), 89.2% (2 days), 94.2% (5 days), 99.2% (14 days), and 79.1%
(30 days) of that of non-vitrectomized eyes. The calculated overall half-lives of
intravitreal bevacizumab were 6.99 days for vitrectomized eyes and 7.06 days for
non-vitrectomized eyes (1.6 hours difference). The clearance of intravitreal
bevacizumab in vitrectomized eyes was accelerated only in the first phase but not
in the second phase.
Conclusions: The increase of intravitreal bevacizumab clearance after vitrectomy
is not substantial in rabbit eyes. This experimental data suggests that the therapeutic
efficacy and duration of intravitreal bevacizumab in patients who previously
underwent vitrectomy may be comparable to those without vitrectomy history.
Commercial Relationships: Se Joon Woo, None; Jeeyun Ahn, None; Ji Hyun
Park, None; Sunyoung Park, None; Kyu Hyung Park, None; Hyuncheol Kim,
None
Support: SNUBH research grant 11-2011-016
In Vivo Verification of the Bioresponsive Potential of an Intelligent
Intraocular Implant
Lisa C. du Toit1A, Viness Pillay1A, Trevor R. Carmichael1B, Thirumala Govender2,
Yahya E. Choonara1A. APharmacy and Pharmacology, BOphthalmology,
1
University of the Witwatersrand, Johannesburg, South Africa; 2Pharmaceutics,
University of KwaZulu-Natal, Kwazulu-Natal, South Africa.
Purpose: An autofeedback polymeric platform was used in the design of an
intelligent intraocular implant - the I3 - using stimuli-responsive polymers,
producing a smart release system capable of delivering therapeutic levels of antiinflammatory agent and antibiotic for posterior segment disorders of the eye in
response to inflammation. Here the I3 was assessed for its ability to respond to the
inflammatory state created in a suitable animal model.
Methods: In vivo design and surgical technique in the healthy rabbit eye: Fifty
New Zealand Albino rabbits were used, randomly assigned to the experimental (25
rabbits) and control (25 rabbits) groups (n=5). A placebo device was implanted into
one eye of the control group at sub-Tenons space and the drug-loaded implant
(containing indomethacin and ciprofloxacin) into one eye of the experimental
group. All procedures were undertaken in accordance with ARVO. For each study,
one animal in each group was euthanized at each sampling point (3, 7, 14, 21, 28
days) with consequent device removal, enucleation and vitreous humor aspiration.
In vivo design and surgical technique in the inflamed rabbit eye: Rabbits were
assigned to experimental (5 rabbits) and control (5 rabbits) groups (n=5 at the

sampling point on day 7). Device implantation in the control and experimental
groups was on day 0. Intraocular inflammation was induced via intravitreal
injection of Salmonella typhimurium lipopolysaccharide (LPS) (100-200 ng/10L
of phosphate-buffered saline) into the I3-implanted eye of each rabbit. One animal
in each group was euthanized on day 7 with device removal, enucleation and
vitreous aspiration. Analysis of implant, tissue and fluid samples: Device
biocompatibility and the degree of inflammation present were histologically
established. The disparity in drug release behaviour in the healthy versus the
inflamed rabbit eye was ascertained via analysis of vitreous samples.
Results: Histological evaluation indicated that sub-Tenon device implantation did
not induce a notable degree of inflammation. In vivo results demonstrated good
penetration of drug from sub-Tenons space, through the ocular membranes, into
the vitreous humour. There was enhanced release of both drugs in the inflamed
rabbit eye even after 7 days with indomethacin levels of 0.7490.126g/mL and
1.1680.186g/mL, and ciprofloxacin levels of 1.1810.150g/mL and
6.6530.605g/mL being attained in the normal and inflamed eye, respectively.
Conclusions: The I3 displayed exemplary inflammation-responsive behaviour
following assessment in a rabbit eye model.
Commercial Relationships: Lisa C. du Toit, None; Viness Pillay, None; Trevor
R. Carmichael, None; Thirumala Govender, None; Yahya E. Choonara, None
Support: None
Generation Of Dual Secreting CNTF / VEGF-antagonist Cell Lines And ECT
Devices
Vincent Ling, Susan Elliott, Brenda Dean, Lisa Orecchio, Konrad Kauper, Michael
Rivera, Weng Tao. Biological Sciences, Neurotech USA, Lincoln, RI.
Purpose: Anti-angiogenic molecules have been successfully used in the treatment
of wet AMD. However, only about 30% of treated patients gain 3 lines of vision.
The presence of 70% afflicted patients suggests a separate mechanism in wet AMD
pathology not addressed by standard anti-VEGF therapies. The current study was
carried out to explore the concept of a combination therapy - the simultaneous
delivery of two biotherapeutics, each with different bioactivities, from one device.
An example of a clinically relevant dual-secreting biologic device would be one
that produces an anti-angiogenic VEGF antagonist and also the neuroprotective
cytokine, CNTF, as dual mode therapy for Wet AMD with underlying
photoreceptor lesions.
Methods: An iterative transfection strategy was used to create combination VEGF
antagonist / CNTF cell lines. VEGF antagonist expression vectors were cloned,
then transfected into NTC-200 cells followed by screening for highest productivity.
Top producing VEGF antagonist cell lines were subsequently expanded,
transfected with CNTF expression vectors and screened for top CNTF producers.
Dual producing VEGF antagonist / CNTF cell lines were obtained and
subsequently encapsulated to create Neurotech ocular ECT implants. The rate of
protein secretion was determined by ELISA.
Results: Dual biologic producing clonal cell lines exhibited robust recombinant
protein secretion, with levels of some cell lines approaching 15 pcd for VEGF
antagonist, and 0.5 pcd for CNTF cytokine. Cell lines were then encapsulated, and
production of recombinant proteins from individual ECT devices were detected.
Conclusions: Proof-of-concept, dual-secreting cell line studies suggest that
Neurotech ECT devices may be an effective method of delivering biologics in a
combinatorial approach where such therapies may be preferable, such as a case for
Wet / Dry AMD.
Commercial Relationships: Vincent Ling, Neurotech (E); Susan Elliott,
Neurotech (E); Brenda Dean, Neurotech (E); Lisa Orecchio, Neurotech (E);
Konrad Kauper, Neurotech (E); Michael Rivera, Neurotech (E); Weng Tao,
Neurotech (E)
Support: None
Microneedle-Based Suprachoroidal Injections in Rabbits: Histological Study
Using Triamcinolone Acetonide
Damian E. Berezovsky1, Samirkumar R. Patel2, Hans E. Grossniklaus1, Henry F.
Edelhauser1. 1Ophthalmology, Emory University, Atlanta, GA; 2Chem &
Biomolecular Eng, Georgia Institute of Technology, Atlanta, GA.
Purpose:
To assess the histopathological appearance of retina, choroid and sclera following
suprachoroidal injection of Triamcinolone Acetonide (TA, Triesence, Alcon
Laboratories) and balanced salt solution in live pigmented rabbits.
Methods:
Ten Dutch-belted rabbits weighing 4-6 lbs. were used in accordance with ARVOs
Statement for the Use of Animals in Ophthalmic and Visual Research. Eyes
received single 100uL injections of 2mg TA (n=9) or balanced salt solution (n=9)
using a single hollow metal microneedle in the superior temporal quadrant.
Injection location was 5mm posterior to the limbus. Eyes were enucleated
immediately (n=6), after 1 day (n=6), or after 7 days (n=6). Two eyes were used as
negative controls. After enucleation, eyes were fixed in 2.5% Glutaraldehyde.
Circular sections (4mm in diameter) were cut from the area between injection site
and optic nerve, embedded in resin and stained using toluidine blue.
Results:
Sections from all eyes were examined for inflammation, atrophy, necrosis, edema,
enlargement of the suprachoroidal space, choroidal hemorrhage or any other
abnormality. No inflammation, atrophy, necrosis, edema, hemorrhage or scarring
was found in any of the injected eyes, or in the normal controls. In about one-third
of tissue samples, an abnormal separation was observed in the area of the
suprachoroidal space. Normal-appearing parallel thin septae were visible within
this enlarged suprachoroidal space, without cellular infiltration or evidence of
hemorrhage. Two TA-injected samples contained an amorphous, acellular,
nonstaining material within the enlarged suprachoroidal space, believed to
represent triamcinolone collections. The retina retained normal appearance in all
tissue samples.
Conclusions:
This histological study of in vivo suprachoroidal injections shows that
triamcinolone acetonide or balanced salt solution can be injected into living rabbit
eyes without causing an immediate or short-term inflammatory response or
choroidal hemorrhage. These two materials, injected using a hollow metal
microneedle, did not cause any significant disruptions in tissue organization or
appearance through seven days post-injection.
Commercial Relationships: Damian E. Berezovsky, None; Samirkumar R.
Patel, 12/767,768 (P), Clearside Biomedical Inc (I, E); Hans E. Grossniklaus,
None; Henry F. Edelhauser, 12/767,768 (P), Clearside Biomedical Inc (I)
Support: NIH grants R24 EY017404, P30 EY06360, and RPB
Superior Delivery to Choroid-Retina following Suprachoroidal Injections in
Rats: Assessment using Fluorophotometry
Puneet Tyagi, Rajendra S. Kadam, Uday B. Kompella. Pharmaceutical Sciences,
University of Colorado Anschutz Medical Campus, Aurora, CO.
Purpose: The objective of this study was to evaluate the delivery and
pharmacokinetics of sodium fluorescein after suprachoroidal, intravitreal, and
posterior subconjunctival injections in Sprague Dawley rats using noninvasive
fluorophotometry.
Methods: Delivery and pharmacokinetics of sodium fluorescein was evaluated in
Sprague Dawley rats (n=4) after suprachoroidal (500 ng in 5l), intravitreal (50 ng
in 5l), or posterior subconjunctival injection (500 ng in 5l). Sodium fluorescein
levels were monitored noninvasively using Fluorotron Master up to 6 hours.
Pharmacokinetic parameters were estimated by noncompartmental analysis using
WinNonlin.
Results: Sodium fluorescein delivery to choroid-retina was in the order:
suprachoroidal > intravitreal > posterior subconjunctival injection. Peak
concentration (Cmax; ng/ml) in the choroid-retina was the highest after
suprachoroidal (1673 363) injection when compared to intravitreal (103 44) and
posterior subconjunctival (76 6) injections. The extent of delivery (AUC0-t) to the
choroid-retina after suprachoroidal injection was 2- and 6- fold higher than
intravitreal and posterior subconjunctival injections, respectively.
Conclusions: Suprachoroidal injections resulted in the highest delivery of sodium
fluorescein to choroid-retina when compared to intravitreal and posterior
subconjunctival injections. Noninvasive monitoring of sodium fluorescein is
possible in rats using Fluorotron Master ocular fluorophotometry.
Commercial Relationships: Puneet Tyagi, None; Rajendra S. Kadam,
None; Uday B. Kompella, None
Support: This work was supported by the NIH grants EY017533, EY018940, and
EY017045.
Nanoemulsion As A Vehicle To Enhance The Ocular Absorption After
Topically Applied Cyclosporine A In The Rabbit Eye
Junjie Zhang, Tianyang Zhou, Liya Wang, Jijun He, Huiyun Xia. Dpt of
Pharmaceutical Science, Henan Eye Institute, Henan Key Laboratory of
Keratopathy, Zhengzhou, China.
Purpose: To investigate the effect of vehicles on ocular absorption of topically
applied Cyclosporin A (CSA) in the rabbit eye.
Methods: 0.05% Cyclosporine nanoemulsion (CSA-NE) was prepared. A single
dose of 50l CSA was applied using CSA-NE or oil dissolved drug (CSA-OD), the
loading dose (50l5 times with an interval of 5min) of CSA-NE or CSA-OD was
also performed. CSA concentrations were measured at intervals of 5, 15, 30, 6, 120,
180, 240min, 6, 8, 24 and 48 hours for the single dose and 30, 120, 240min, 6, 24,
48 and 72 hours for the loading dose by high performance liquid chromatography
with tandem mass spectrometry (HPLC-MS/MS).
Results: The size of nanoemulsion was 29.00.5nm and zeta potential was -

0.0360.135mv. After single application of CSA-NE, the highest concentrations of
CSA in cornea and conjunctiva were 1433.1340.1ng/g and 1445.5410.2ng/g,
respectively at 30min and remained 236.1595.03ng/g in cornea at 48h while CSA
in cornea and conjunctiva could not be detected after 8 hours for CSA-OD group.
The concentrations in cornea at all the time points were significantly higher than
the CSA-OD group at the corresponding time points (p<0.01), respectively. The
concentrations in conjunctiva were at all the time points except 3h, 4h and 8h were
significantly higher than the CSA-OD group at the corresponding time points
(p<0.05) , respectively. Relatively low concentrations were measured in the
aqueous for both CSA-NE and CSA-OD group 7.609.81 and 6.225.68ng/ml) and
no significant difference were found between the two groups (p>0.05). After
loading dose application of CSA-NE, The highest concentrations in cornea and
conjunctiva were 2790.8409.2 and 3365.0806.1ng/g at 30min, respectively. CSA
levels in cornea and conjunctiva were significantly higher than the corresponding
time points (p<0.0001 and <0.05),respectively while the drug in both cornea and
conjunctiva could not be detected after 8 hours for CSA-OD group. Relatively low
concentrations were measured in the aqueous for both CSA-NE and CSA-OD
group (7.024.36 ng/ml and 4.295.35 ng/ml) and no significant difference were
found between the two groups (p>0.05). The concentrations in blood for single and
loading dose all groups were below the detection limit (5ng/ml).
Conclusions: These findings indicate that nanoemulsion may be a promising
delivery system for topical use of CSA in ocular immune-mediated diseases.
Commercial Relationships: Junjie Zhang, None; Tianyang Zhou, None; Liya
Wang, None; Jijun He, None; Huiyun Xia, None
Support: 072103810606
Ex Vivo Permeation of Cystine across Bovine Corneas - A Step towards the
Development of an Antioxidant Eye Drop for Cataract Prevention
Ilva D. Rupenthal1A, Simon Raesch1A,2, Bo Li1B, Ulrich F. Schaefer2, Claus-Michael
Lehr2,3, Julie C. Lim1B. ADepartment of Ophthalmology, BDepartment of Optometry
and Vision Science, 1The University of Auckland, Auckland, New Zealand;
2
Department of Biopharmaceutics and Pharmaceutical Technology, Saarland
University, Saarbruecken, Germany; 3Department of Drug Delivery, HelmholtzInstitute for Pharmaceutical Research Saarland, Saarbruecken, Germany.
Purpose: Age-related nuclear (ARN) cataract is the leading cause of blindness
worldwide and is estimated to affect more than 40 million people by 2020. Even
though surgical removal of the clouded lens is highly effective, the current demand
exceeds the supply. Since ARN cataract is associated with a depletion of
antioxidants, mainly glutathione, in the lens core, our research has concentrated on
restoring antioxidant levels in this region in order to prevent or delay the onset of
cataract formation. This study investigated the permeation of cystine, the limiting
factor for glutathione synthesis, across bovine corneas.
Methods: Freshly excised bovine corneas were placed between donor and acceptor
chambers of standard Franz diffusion cells. A 0.15 mg/ml cystine solution (1 ml)
was placed in the donor compartment, while modified Ringer solution, kept at
37C, served as acceptor medium. Samples were taken at predetermined time
points over 24 h and cystine concentrations were analyzed by derivatization with
monobromobimane and subsequent fluorescence HPLC analysis. In addition to a
simple cystine solution, various penetration enhancers were tested for their ability
to improve the cystine permeation across bovine corneas.
Results: The established HPLC method allowed cystine quantification to a lower
limit of <0.1 nmol/ml with high linearity (R2=0.9994) and a reduced sample run
time (17 min). Over the 24 h period 45 nmol of cystine permeated across the
bovine cornea using the simple solution as donor. This amount was almost tripled
(118 nmol over 24 h) when incorporating 0.5% EDTA, a tight junction opener,
while 0.01% BAC only exhibited a slight improvement. Immunolabelling of frozen
tissue sections revealed the presence of the cystine/glutamate (Xc-) exchanger in
the corneal epithelium, which may be responsible for partial active transport of the
antioxidant across the tissue.
Conclusions: Franz diffusion cell experiments combined with HPLC analysis offer
an accurate way to study the permeation of various cystine eye drops across bovine
corneas, with the addition of 0.5% EDTA showing the most promising results so
far. Further penetration enhancers in combination with other delivery approaches,
such as in situ gelling eye drops, will further be evaluated before testing the most
promising formulations in vivo.
Commercial Relationships: Ilva D. Rupenthal, None; Simon Raesch, None; Bo
Li, None; Ulrich F. Schaefer, None; Claus-Michael Lehr, None; Julie C. Lim,
None
Support: NZPERF Grant 219
Differential Flow Rate of Triamcinolone and Preservative-Free Triamcinolone
through Small-Gauge Needles
Jorge A. Fortun1, Alex Gonzalez2, Cornelis Rowaan2, Mariela Aguilar2, William

Lee2, Andrew A. Moshfeghi1, Thomas A. Albini1, Jean-Marie A. Parel2. 1Bascom
Palmer Eye Institute, Univ of Miami Miller Sch of Med, Miami, FL; 2Ophthalmic
Biophysics Center, Bascom Palmer Eye Institute, Miami, FL.
Purpose: The most commonly used commercially available steroid preparations
used for intravitreal injections are triamcinolone acetonide (TA) (Kenalog 40,
Bristol-Myers Squibb, Princeton, NJ) and the preservative-free triamcinolone
acetonide (PFTA) (TRIESENCE, Alcon, Inc., Fort Worth, TX) both in the 40mg/mL formulation. The most commonly used needle for the injection of TA is a
27-gauge needle. Smaller gauge needles are avoided because of the possibility of
clogging, presumably caused by obstructive accumulation of triamcinolone
aggregates in these narrow needles. PFTA has been shown to form more aggregates
of smaller size than TA. We evaluated the differential flow of TA and PFTA
through small gauge needles.
Methods: A hydraulic piston mechanism was utilized to transfer a constant vertical
1kg gravitational force to the horizontally positioned 1cc syringe plunger (see
figure). The 1 kg force was measured during experimental intravitreal injections
given by three clinicians (JAF, AAM, TAA). A piezo electric pressure transducer
was placed between the syringe and needle. Specialized software developed with
Labview, National Instrument platform, was used to digitize and record the
pressure signal over time during an injection of 1cc of TA or PFTA through a 27,
30 or 32 gauge needle. All needles were commercially available and 0.5 inches in
length. Measurements were performed in triplicate. From each set of measurements
we deduced the injection rate (ml/sec). Injections during which flow stopped prior
to complete injection of 1cc, were recorded as having a flow rate of 0.
Results: Using a 27-gague needle the mean flow rate of TA was found to be 0.513
cc/sec (0.064 95% Confidence interval [CI]) and of PFTA 0.620 cc/sec (0.055
95% CI). Using a 30-gague needle the mean flow rate of TA was found to be 0.06
cc/sec (0.1 95% CI) and of PFTA 0.180 cc/sec (0.017 95% CI). Using a 32gague needle no flow was sustained by TA on any of three attempts and the mean
flow rate of PFTA was 0.117 cc/sec (0.020 95% CI).
Conclusions: PFTA can be injected through a 32 gauge needle but TA cannot. The
flow rate of PFTA through a 32-gague is adequate for intravitreal injection, i.e.
about 0.1 cc/ sec. This is consistent with the finding of smaller trimacinolone
aggregates in PFTA than
TA.
Commercial Relationships: Jorge A. Fortun, Alcon (C); Alex Gonzalez,
None; Cornelis Rowaan, None; Mariela Aguilar, None; William Lee, None;
Andrew A. Moshfeghi, Alcon (C); Thomas A. Albini, Alcon (C); Jean-Marie A.
Parel, None
Support: The Morgenstern Foundation (TAA); Florida Lions Eye Bank; NIH
Center Grant P30EY14801; Research to Prevent Blindness; Henri and Flore
Lesieur Foundation (JMP).
Transscleral Iontophoretic Delivery of Avastin In Vivo: Drug Distribution
and Safety Aspects
Sarah A. Molokhia1, Kongnara Papangkorn1,2, Donald Mix2, Charlotte Butler2,
Prasoona Karra1, John Higuchi2, Balbir Brar2, S. Kevin Li3, William I. Higuchi1,2.
1
University of Utah, Salt Lake City, UT; 2Aciont Inc, Salt Lake City, UT;
3
University of Cincinnati, Cincinnati, OH.
Purpose: To study anodal iontophoretic delivery of the commercial formulation
and a new iontophoretic formulation of Avastin in vivo using the Visulex
iontophoresis system.
Methods: All studies were performed under anodal iontophoresis (AI) on New
Zealand white rabbits using a Visulex device system with 2.5% bevacizumab
(Avastin). For pharmacokinetics (PK) studies (n=6) the commercial Avastin
formulation and an enhanced (A0-01) iontophoretic formulation were used in
Group 1 and 2, respectively. After 20 min of AI delivery, the rabbits were

sacrificed and the eyes enucleated. The eyes were dissected and the cornea,
aqueous humor, vitreous, conjunctiva, sclera, choroid and retina were assayed for
drug concentrations with ELISA. For the safety study Group 3 (n=6), the eyes were
examined with an indirect ophthalmoscope before and immediately after
iontophoretic dosing, then again on days 2, 4, and 6. The eyes were graded
according to a modified McDonald-Shaddock scale for conjunctival injection,
chemosis, corneal haze, iritis and other ocular irritation signs. At day 6, the animals
were sacrificed and all eyes were processed for histopathological examination.
Results: The total amount of Avastin delivered into the eye after 20 min of AI
(1.8 mA/cm2) was 223 71 g and 315 123 g for the commercial formulation
and (A0-01) iontophoretic formulation, respectively. High concentrations of
Avastin were distributed in the conjunctiva and sclera tissues. However,
approximately 4 g was delivered to the retina/choroid tissues. For the safety study,
the only adverse effect noted was very minor conjunctival injection, present in four
of the six eyes treated on Day 2 which resolved by Day 6. Histopathological
evaluation showed no effects due to the iontophoretic treatment.
Conclusions: This study demonstrated promising results toward higher Avastin
delivery with the new iontophoretic formulation (AO-01), although this is not
statistically significant. Further modifications are required to the new formulation
to achieve significant enhanced delivery. The amount delivered to the
retina/choroid using the Visulex iontophoretic system is within same order of
magnitude reported in literature using intravitreal injection. This study is among the
first to demonstrate safe delivery of Avastin in vivo to the posterior eye tissues
within the therapeutic range using iontophoresis.
Commercial Relationships: Sarah A. Molokhia, Aciont Inc (C); Kongnara
Papangkorn, Aciont Inc (E); Donald Mix, Aciont Inc (E); Charlotte Butler,
Aciont Inc (E); Prasoona Karra, None; John Higuchi, Aciont Inc (E); Balbir
Brar, Aciont Inc (E); S. Kevin Li, Aciont Inc (C); William I. Higuchi, Aciont Inc
(E)
Ocular Drug Delivery Using A Thermo-responsive Lacritin Fusion Protein
John A. MacKay, Wan Wang, Benjamin Droese, Mihir Shah, Pang-Yu Hsueh,
Guoyong Sun, Sarah F. Hamm-Alvarez. Pharmacology and Pharmaceutical
Sciences, University of Southern California, Los Angeles, CA.
Purpose: A fusion protein between recombinant human lacritin and an elastin-likepolypeptide (ELP) was designed as a therapeutic for dry eye disease(DED). Fusion
of these domains imparts thermo-sensitive assembly to lacritin, which is a
candidate biopharmaceutical. Affecting over 3.2 million Americans, predominantly
women, DED is characterized by decreased tear production, which leads to
discomfort and visual impairment. To develop biopharmaceuticals for DED, better
approaches are required to retain proteins on the eye. Protein polymers, such as
ELPs, are an emerging solution to this problem composed of an amino acid repeat
(VPGXG). ELPs have characteristic inverse phase transition temperatures (Tt),
below which they are soluble and above which they self-associate. By adjusting Tt
between room and body temperature ELP fusion proteins may act as a therapeutic
depot. The hypothesis tested here is that the fusion protein of ELP and lacritin will
undergo thermally-induced phase separation at ocular temperatures without
compromising the mitogenic and secretagogue signaling functions of lacritin.
Methods: Multiple libraries of synthetic ELP genes were engineered with a range
of assembly properties. Genetic engineering enables precise control over their
molecular weight, orientation, and Tt. ELP phase behavior was used to purify these
biopolymers from bacteria. Fusion protein, free lacritin, and free ELP were
characterized for assembly, biodegradation, and induction of secretion from
primary rabbit lacrimal gland acinar cells (LGACs).
Results: ELP lacritin fusion proteins were developed with three properties: 1) a
control that is soluble at body temperature; 2) a thermo-responsive fusion that
forms viscous microparticles at body temperature; and 3) a polypeptide
nanoparticle that assembles ~200 nm diameter particles at body temperature. Phase
behavior and self-assembly was observed over a range of concentrations (5-100
uM) using UV-Vis spectrophotometry and dynamic light scattering.
Conclusions: ELP lacritin fusion proteins were developed that assemble between
25 and 30 C. All fusion constructs induce significant secretion of betahexosaminidase from primary LGACs. These results suggest that ELP-mediated
assembly may be a useful platform to control ocular drug delivery and efficacy.
Commercial Relationships: John A. MacKay, Patent disclosure filed on ELP
fusion proteins for ocular drug delivery (P); Wan Wang, None; Benjamin Droese,
None; Mihir Shah, None; Pang-Yu Hsueh, None; Guoyong Sun, None; Sarah F.
Hamm-Alvarez, None
Support: R21EB012281 to JAM, RO1EY017293 and RO1EY017293S1 to SHA
Ultrasound-enhanced Penetration Of Topical Fluorescein into the Vitreous in
Rabbit Eyes
Jay M. Stewart1A, Chris J. Diederich1B, Elliot Chan1A, Vasant Salgaonkar1B,

Ricardo Lamy1A. AOphthalmology, BRadiation Oncology, 1Univ of California-San
Francisco, San Francisco, CA.
Purpose: To determine whether ultrasound treatment can promote the permeation
of topical fluorescein into the vitreous.
Methods: Fresh cadaveric rabbit eyes were placed in saline solution at 37C. An
eye cup containing 0.1% fluorescein solution was centered on the sclera
approximately 5 mm posterior to the limbus. Continuous wave ultrasound at 1
W/cm2 was applied to the sclera within the eye cup for 10 minutes, and the eyes
were then left in solution for an additional 50 minutes. Control eyes received the
same exposure to fluorescein solution in the eye cup without ultrasound treatment.
Eyes were then washed, and vitreous was collected using an automated vitrectomy
system. Vitreous samples were analyzed using fluorescence spectophotometry.
Fluorescence intensity levels were compared to a standard curve representing
known fluorescein concentrations.
Results: The vitreous concentration of fluorescein was increased in treated eyes
(mean 4.34 mcg/mL, n=10) compared to control eyes (mean 2.88 mcg/mL, n=8), P
< 0.05.
Conclusions: Ultrasound treatment facilitated the entry of topical fluorescein into
the vitreous of intact rabbit eyes. These results suggest that ultrasound-enhanced
drug delivery may offer a non-invasive means of treating posterior segment
diseases.
Commercial Relationships: Jay M. Stewart, None; Chris J. Diederich,
None; Elliot Chan, None; Vasant Salgaonkar, None; Ricardo Lamy, None
Support: None
Drug Loaded Microparticles For Long-term Sustained Release Of Anti-VEGF
Therapies In Age-related Macular Degeneration
Leilei Zhang1A,1B, Cynthia J. Roberts1A,1B, Alan D. Letson1B, Ronald X. Xu1A,1B.
A
Biomedical Engineering, BOphthalmology, 1The Ohio State University,
Columbus, OH.
Purpose: To investigate an alternative microfabrication technique to enhance the
drug encapsulation efficiency and reduce the antibody damage for long-term
sustained release of anti-VEGF drug loaded microparticles in age-related macular
degeneration.
Methods: The co-axial electrohydrodynamic atomization process was used to
overcome the limitations of the traditional double emulsion method for
microfabrication of multifunctional drug-loaded microparticles. The process setup
included a co-axial needle, a ground plate, a droplet collector, and an inline video
camera. The inner and outer channels of the co-axial needle were connected to two
syringe pumps and infused with a phosphate buffered saline (PBS) solution of the
anti-VEGF drugs, and an organic solution of poly (lactic-co-glycolic acid) (PLGA),
respectively. Various fluorescent dyes, such as Nile Red, were added to the PLGA
solution to visualize the shell structure. Different drugs, such as Lucentis, were
encapsulated. A total voltage of 14kV was applied between the co-axial needle and
the ground plate so that a stable cone-jet was formed at the co-axial needle, broken
into droplets, and collected by the droplet collector filled with distilled water. The
core-shell ratio of the fabricated microparticles was controlled by adjusting several
process parameters, such as the ratio of the inner and outer flow rates, the
concentration of PLGA, and the applied voltage.
Results: With the co-axial electrohydrodynamic atomization process, we were able
to fabricate drug-loaded microparticles with the size ranging from 1 to 5um. The
morphology of these microparticles was characterized by scanning electron
microscopy, and the cone-shell structure was confirmed by confocal microscopy.
Conclusions: The co-axial electrohydrodynamic atomization process was
successful in producing drug-loaded mircoparticles. Future studies will investigate
protection of anti-VEGF drugs from process-induced damage with a high
encapsulation efficiency. The core-shell ratio can be easily controlled for
programmed long-term drug release.
Commercial Relationships: Leilei Zhang, None; Cynthia J. Roberts,
None; Alan D. Letson, None; Ronald X. Xu, Genentech, Inc. (R)
Support: The Ohio Lions Eye Research Foundation Fellowship and a kindly
donation through the Muskingum County Community Foundation
Wound Healing Of Human Corneal Epithelial Cells Impacted By
Nanoparticles
Enhua H. Zhou1, Richard Pizzo2, Christa Watson1, Joel Cohen1, Quynh Dang1,
Georgios Pyrgiotakis1, Joseph D. Brain1, Jeffrey J. Fredberg1, Philip Demokritou1.
1
Department of Environmental Health, Harvard School of Public Health, Boston,
MA; 2Department of Physics, Boston Colleague, Boston, MA.
Purpose: As engineered nanoparticles (ENPs) enter products, workers and
consumers are increasingly exposed. Our bodys first line of defense against ENPs
is the epithelium. However, there is a lack of understanding of the effects of ENPs

on the epithelium of the eye, which is a site of environmental exposure and
resulting irritation. Moreover, those effects may be aggravated by preexisting
wounds in the epithelium. Here we wanted to explore whether ENPs can impede
the healing of a wounded epithelium.
Methods: To address this question in human corneal epithelial cells (gift from Dr.
Ilene K. Gipson), we developed a novel wound healing assay. Using this assay we
studied a panel of well characterized, industrially relevant ENPs, including several
commercially available ENPs and metals and metal oxides generated using our
recently developed Versatile Engineered Nanomaterial Generation System
(VENGES).
Results: TiO2 and SiO2 ENPs did not affect wound healing at doses up to 100
micro g/ml. By contrast, copper oxide, zinc oxide, and silver ENPs substantially
impede wound healing in a dose-dependent manner.
Conclusions: Taken together, these studies provide a novel in-vitro model system
for evaluating the physiological impact of nanoparticles in the ocular surface.
Commercial Relationships: Enhua H. Zhou, None; Richard Pizzo,
None; Christa Watson, None; Joel Cohen, None; Quynh Dang, None; Georgios
Pyrgiotakis, None; Joseph D. Brain, None; Jeffrey J. Fredberg, None; Philip
Demokritou, None
Support: Pilot Project funding from the HSPH-NIEHS Center for Environmental
Health (ES000002) and the Center for Nanotechnology and Nanotoxicology
Degradation And The Routes Of Clearance Of Intravitreal Hydrosilylated
Porous Silicon Particles
Lingyun Cheng1A, Elizabeth Wu1B, William R. Freeman1A, Michael J. Sailor1B,
Jennifer Andrew1B, Laura Conner1A, Alejandra Nieto1B. AJacobs Retina Ctr at
Shiley Eye Ctr, BDepartment of Chemistry and Biochemistry, 1Univ of CaliforniaSan Diego, La Jolla, CA.
Purpose: We have reported the degradation and intravitreal safety profiles of
porous silicon particles as vehicles for ocular drug delivery. Though porous silicon
can be degraded in vitreous without toxicity, the routes of clearence of the
degradation product, monomeric silicic acid (Si(OH)4), from the eye environment
is not yet understood.
Methods: Ten pigmented rabbits were used for this study. The right eye of each
animal was intravitreally injected with 3mg of hydrosilylated (1-undecylenic acid)
porous silicon particles in 100 L of 5% dextrose. The particles had an average size
of 33 by 46 M. After the intravitreal injections, two rabbits were sacrificed at each
of these time points: 1 week, 2 weeks, 3 weeks, 5 weeks, and 8 weeks. Before
sacrifice 1 to 2 ml of blood was sampled and the enucleated eye globes were
dissected individually into aqueous, vitreous, and retina for quantitation of
dissolved Si (as orthosilicate) using ICP-OES (Inductively Coupled Plasma-Optical
Emission Spectroscopy). Normal rabbit vitreous and retina were used as controls.
Results: No excess Si was detected in the blood samples. The Si concentration in
the vitreous and aqueous compartments gradually decreased over time and Si
concentration was consistently higher in vitreous than that in aqueous. The peak
concentration in the vitreous was 25.55g/ml and 18.90.05g/ml in aqueous,
while the peak concentration in the retina was only 2.40.8g/g which was similar
to the Si concentration in normal control retina samples (1.90.4g/g). Si
concentration in the vitreous at the last time point was 10.51.7g/ml which was
not significantly different from Si concentration in the normal control vitreous
(11.851.6g/ml). The concentration-time profile suggested a sustained
degradation.
Conclusions: The data suggest that the degraded porous silicon in vitreous was
mainly cleared through aqueous outflow
pathways.
Commercial Relationships: Lingyun Cheng, None; Elizabeth Wu,

None; William R. Freeman, None; Michael J. Sailor, None; Jennifer Andrew,
None; Laura Conner, None; Alejandra Nieto, None
Support: NIH EY020617
Development of Microemulsions for Enhanced Topical Delivery to the Eye
Ronald A. Wassel, Didier Nuno, Alexander Quiambao, Fadee Mondalek, Rafal
Farjo. Charlesson LLC, Oklahoma City, OK.
Purpose: MSH-1001, a novel highly insoluble small molecule, is an ATP-sensitive
K channel opener and has been shown to reduce intraocular pressure. The purpose
of this study was to develop a microemulsion based eye drop that can deliver
therapeutic drug concentrations to the retina better than a micronized suspension.
Methods: Twelve different nano- and micro-emulsions were prepared. There
formulations were made up of oils and surfactants that are commonly used in
ophthalmic formulations. Quantification of MSH-1001 in rabbit aqueous humor at
1 hour following a 60 ul eye drop of the aqueous humor performed using LCMS/MS.
Results: Following topical administration of the various eye drops it was observed
that a 0.5% MSH-1001 microemulsion delivered the same drug levels to the
aqueous humor as a 3% nanoemulsion and the 10% micronized suspension.
Conclusions: MSH-1001 can be formulated into optically transparent and
thermodynamically stable microemulsion eye drops. These eye drops, while having
two orders of magnitude lower concentrations of the active ingredient, have
demonstrated the ability to deliver the same concentration to the aqueous humor as
more traditional eye drop formulations. These results suggest that this eye drop
formulation platform technology could be applied to increase ocular delivery of
other lipophilic active pharmaceutical ingredients.
Commercial Relationships: Ronald A. Wassel, None; Didier Nuno,
None; Alexander Quiambao, None; Fadee Mondalek, None; Rafal Farjo, None
Support: NIH SBIR Grant 1R43EY021074-01A1
Pharmacokinetics and Biodistribution of Bevacizumab Following
Suprachoroidal Injection into the Rabbit Eye Using a Microneedle
Saffar Mansoor1, Samikrumar R. Patel1, Cetin Tas2, Rashmi Pacha-Ravi3, Uday B.
Kompella3, Henry F. Edelhauser4, Mark R. Prausnitz1. 1Chemical & Biomolecular
Engineering, Georgia Institute of Technology, Atlanta, GA; 2Pharmaceutical
Sciences, Gulhane Military Academy, Ankara, Turkey; 3Pharmaceutical Sci &
Ophthal, University of Colorado Denver, Aurora, CO; 4Ophthalmology, Emory
Univ Eye Center, Atlanta, GA.
Purpose: As an alternative to intravitreal injection, this study presents injection of
bevacizumab into the suprachoroidal space (SCS) using a hollow microneedle. This
minimally invasive approach deposits bevacizumab between the sclera and choroid,
which targets drug delivery to its therapeutic site of action. The purpose of this
study is to quantify the pharmacokinetics and biodistribution of bevacizumab in the
rabbit eye. This is the first study to assess bevacizumab delivery in vivo after
injection into the SCS using a microneedle.
Methods: Bevacizumab (Avastin, 1250 g/50 l) was injected into the SCS of
pigmented rabbits using a metal microneedle measuring 700-800 m in length
inserted 5 mm posterior to the limbus. Rabbits were euthanatized and eyes were
enucleated at 15 min, 1 day, and 2 days following SCS injection. Ocular tissues
(sclera, choroid, retina, aqueous humor, vitreous, anterior tissues, lens and optic
nerve) were separated. Bevacizumab was then extracted from these tissues and
quantified using ELISA.
Results: The percent bevacizumab recovered from the eyes was 88.40.9% at 15
min, 4.60.5% at 1 day, and 0.20.1% at 2 days after injection. The distribution of
bevacizum in ocular tissues at 15 min after injection was 76% in choroid, 13% in
sclera and 2.9% in retina, 1.0% in vitreous, 0.5% in aqueous humor, 0.9% in
anterior chamber, 0.6% in lens and 0.1% in optic nerve. After 1 day, drug levels
were 34% in choroid, 27% in sclera and 23% in retina, 11% in vitreous, 0.7% in
aqueous humor, 1.6% in anterior chamber, 3.8% in lens and 0.3% in optic nerve.
After 2 days, the distribution of bavacizum was 0.5% in choroid, 3.3% in sclera,
0.5% in retina, 55% in vitreous, 3% in aqueous humor, 36% in anterior chamber,
1.1% in lens and 0.6% in optic nerve.
Conclusions: A microneedle was able to inject bevacizumab into the SCS of rabbit
eyes using a minimally invasive procedure. There was a quick biodistribution of
bevacizumab into choroid, sclera, and retina, indicating excellent drug targeting.
However, choroidal levels of bevacizumab declined very rapidly. The current
formulation of bevacizumab is therefore not optimal and a sustained-release
formulation of bevacizumab may be needed to achieve sustained delivery to the
posterior segment of the eye.
Commercial Relationships: Saffar Mansoor, None; Samikrumar R. Patel,
12/767/768 (P), Clearsdie Biomedical (I); Cetin Tas, None; Rashmi Pacha-Ravi,

None; Uday B. Kompella, None; Henry F. Edelhauser, None; Mark R.
Prausnitz, 12/767,768 (P), Clearside Biomedical (I)
Support: NEI grant R24 EY017404
Prolongation of Proparacaine Corneal Anesthesia by an In Situ Cross Linked
Hydrogel of Carboxymethylcellulose
Houman D. Hemmati1,2, Miguel Manzano-Garcia2, Daniel S. Kohane3, Robert
Langer2. 1Ophthalmology, Mass. Eye & Ear Infirmary, Harvard Medical School,
Boston, MA; 2Chemical Engineering, Massachusetts Institute of Technology,
Cambridge, MA; 3Anesthesiology, Laboratory for Biomaterials and Drug Delivery,
Children's Hospital Boston, Harvard Medical School, Boston, MA.
Purpose: To formulate and characterize a rapid hydrogel-forming eye drop
delivery excipient designed to provided extended release of a topical anesthetic
drug to the ocular surface.
Methods: Periodate oxidation of carboxymethylcellulose (CMC) was performed to
generate aldehyde-modified CMC (CMC-ALD). Modification of CMC with adipic
dihydrazide (ADH) was performed to generate ADH-modified CMC (CMC-ADH).
Separate 0.5% proparacaine solutions were created in phosphate-buffered saline
(PBS) with CMC-ALD, CMC-ADH, unmodified CMC, and without CMC. 1:1
mixtures of CMC-ALD and CMC-ADH solutions were created to test hydrogel
formation time, adhesiveness, and optical clarity. 25 uL of each of the
aforementioned solutions were applied to the corneas of adult male Sprague
Dawley rats, with one group of animals receiving 25 uL of PBS alone as a negative
control (n=6 rats per group). Hydrogel eyes received 12.5 uL each of CMC-ALD
and CMC-ADH simultaneously. Corneal sensation was measured by CochetBonnet esthesiometry 1-2 times a day for 10 days.
Results: CMC immediately cross-linked through hydrazone bonds (Figure 1) to
form optically clear, pliable hydrogels on both glass slides and rat eyes. In situ
cross-linked CMC hydrogel prolonged the duration of topical anesthesia by at least
9 days, significantly longer than unmodified CMC (p<0.05).
Conclusions: In situ cross-linked hydrogels of CMC can potentially be used to
dramatically prolong the duration of topically-delivered ophthalmic drugs.
Vida Rahmani1A, Frances Lasowski1B, Heather Sheardown1A. AChemical

Engineering, BSchool of Biomedical Engineering, 1McMaster University,
Hamilton, ON, Canada.
Purpose: A minimally invasive device is ideal for the delivery of therapeutics to
treat childhood ocular conditions, such as roscovitine to prevent retinoblastoma and
atropine to retard myopia progression. A polymeric, transscleral delivery vehicle is
proposed to reduce side effects by localizing drug delivery, creating a prolonged,
controlled delivery profile.
Methods: The proposed model materials for these applications are a 90/10 mixture
of poly(2-hydroxyethyl methacrylate)/ trimethyl siloxy silane (HEMA-TRIS) and a
50/50 mixture of N,N-dimethylacrylamide/trimethyl siloxy silane (DMAA-TRIS),
synthesized by UV. Some materials initially contained drug, which was released
prior to uptake studies. Both uptake studies and release studies, done at 37C, were
quantified using UV spectrophotometry.
Results: Drug uptake, Figure 1, was dependent on the loading concentration. Initial
drug incorporation into the disks did not result in greater uptake, as loading with
0.5 mg/ml in 1:2 Ethanol:PBS solutions yielded 0.0270.006 and 0.0280.007 mg
roscovitine per mg polymer for 0 mg and 21 mg of initial drug loading respectively.
HEMA-TRIS disks showed less uptake than DMAA-TRIS materials, such as
0.1654 mg and 0.2356 mg total uptake respectively after 120 hours in 0.08 mg/ml
roscovitine solution. Release profiles for drugs incorporated during synthesis,
Figure 2, show sustained release over two weeks with a moderate initial burst.
Conclusions: These results show that transscleral delivery via polymeric devices is
possible for the delivery of these therapeutics. Sufficient drug loading is likely
possible from the initial manufacture of the
lenses.
Commercial Relationships: Vida Rahmani, None; Frances Lasowski,

None; Heather Sheardown, None
Support: NSERC
Commercial Relationships: Houman D. Hemmati, In-situ crosslinked CMC

Hydrogels (P); Miguel Manzano-Garcia, None; Daniel S. Kohane, In situcrosslinked CMC Hydrogels (P); Robert Langer, In situ-crosslinked CMC
Hydrogels (P)
Support: Boston Keratoprosthesis Funds
Clever Contacts: Using Silicone Hydrogel Materials to Deliver Atropine and
Roscovitine for the Treatment of Childhood Ocular Conditions

Gentamycin Release from Collagen Wafers: A Method to Eliminate the Need
for Post Operative Eyedrops
Richard A. Eiferman1, Dale P. DeVore2, Bruce H. DeWoolfson3. 1Ophthalmology,
University of Louisville, Louisville, KY; 2DV Consulting, Chelmsford, MA;
3
Euclid Systems Corp, Herndon, VA.
Purpose: To determine the release kinetics of buffer supernatants collected from
collagen wafers containing gentamycin
Methods: Collagen wafers were incubated with 1.0 ml of 0.1M PBS, pH 7.0 at
37C. One milliliter solutions were collected at days 1, 3, 7, 9, 17 and 21 and
refreshed with 1.0 ml of PBS. Three individual samples per time period were
evaluated for gentamycin concentration utilizing a gentamycin EIA Competitive
Enzyme Immunoassay.
Results: At 24 hours, there was an initial burst of antibiotic release averaging 240
ug/cc. At day 3, the average was 83.5 ug/cc; at day 7, 30 ug/cc; at day 9, 12.5
ug/cc; and at day 17, 4.5 ug/cc. The wafer completely dissolved at day 21.
Conclusions: The collagen wafer released gentamycin in bacteriocidal levels for up
to 17 days. There was a very high release rate of antibiotic in the first 24 hours with
sustained levels lasting over two weeks. This in vitro study indicates that
subconjunctival placement of an antibiotic infused collagen wafer could supply

sufficient antibiotic in the post operative period eliminating the need for self
administation of eyedrops.
Commercial Relationships: Richard A. Eiferman, euclid (R); Dale P. DeVore,
euclid (C, P); Bruce H. DeWoolfson, euclid (I, E)
Support: None
Cellulose Based Tablet For Sustained Release Of Ilomastat
Abeer Mohamed Ahmed1, Alastair Lockwood1,2, Sahar Awwad1, Garima Sharma1,
Peng T. Khaw2, Steve Brocchini1,2. 1UCL School of Pharmacy, University of
London, London, United Kingdom; 2NIHR Biomedical Research Centre for
Ophthalmology, Moorfields Eye Hospital & UCL, London, United Kingdom.
Purpose: Ilomastat is a matrix metalloproteinase inhibitor (MMPi) that has been
shown to inhibit fibrosis after glaucoma filtration surgery in a rabbit model of
ocular fibrosis. To reduce scarring and fibrosis following glaucoma surgery and to
avoid dose dumping when injected, a sustained dosage form is required that allows
a prolonged local concentration of ilomastat to be maintained within the
subconjunctival space. Cellulose based excipients such as carboxymethylcellulose
(CMC) and hydroxypropylmethylcellulose (HPMC), which have been used in
ocular medicine were used to fabricate a small tablet for subconjunctival
implantation.
Methods: Ilomastat (1.0 mg) was dispersed in water (1.0 mL) and sonicated for 30
min to produce a homogenous suspension. An aqueous solution of CMC (2.0%) or
HPMC (2.0%) was prepared by dissolving the desired amount of the polymer in
water overnight. The suspension of ilomastat was then added to the aqueous
polymer solution (2%, 200.0 L) and homogenized using Ultra-Turrax
homogenizer for 2.0 min. The mixture was freeze dried. The lyophilised powder
was pressed into a tablet using 3 mm punch and die. The release of ilomastat from
the tablet in phosphate buffered saline (PH 7.4) was determined at flow rate of 2.0
L at 35.0 C in a flow rig. The concentration of the released ilomastat was
measured using high performance liquid chromatography at 280 nm.
Results: Ilomastat was successfully fabricated into a tablet that contains a
dispersion of a crystalline form of ilomastat in a cellulose based polymer matrix.
The weight of the tablet was in the range of 4.5-6.5 mg. Fabrication of the ilomastat
tablet using cellulose derivatives (CMC and HPMC) resulted in a sustained release
of ilomastat over 10 days. The tablets were prepared using CMC (2.0%), HPMC
(2.0 %) and a mixture of CMC/HPMC (1:1). The release profile of ilomastat was
71.8, 52.9 and 66.3 % respectively after 242.8 h (10 days). The concentration of the
released ilomastat was in the range of 44.7-200.2 (CMC), 11.8-193.6 (HPMC) and
48.3-162.3 M (CMC/HPMC 1:1) over a period of 10 days, which was within the
therapeutic range (10-100 M).
Conclusions: Sustained release of ilomastat was achieved by its fabrication into a
cellulose based tablet. The release period is prolonged, which may be suitable for a
successful sub-conjunctiva implant for improvement of the outcome of glaucoma
filtration surgery.
Commercial Relationships: Abeer Mohamed Ahmed, None; Alastair
Lockwood, None; Sahar Awwad, None; Garima Sharma, None; Peng T. Khaw,
WO09/063222 (P); Steve Brocchini, WO09/063222 (I)
Support: Medical Research Council G801650, Fight for Sight, Freemasons Grand
Charity, NIHR Biomedical Research Centre for Ophthalmology,Helen Hamlyn
Trust.
Development Of Drug Loaded Nanofiber Sutures For Ophthalmologic
Application
Fabiana K. Kashiwabuchi1A, Murilo W. Rodrigues, Jr.1A, Shuming Zhang2A,
Himatkumar Patel1B, Jesus S. Vidaurri-Martinez1A, Qingguo Xu1B, Justin Hanes2B,
Jiangxia Wang1C, Hai-Quan Mao2A, Peter J. McDonnell, III1A. AOphthalmology,
B
Nanomedicine, CSchool of Public Health, 1Johns Hopkins Hospital, Baltimore,
MD; AMaterials Science & Engineering, BNanomedicine, 2Johns Hopkins
University, Baltimore, MD.
Purpose: To fabricate functional nanofibers containing antibiotic drugs and
evaluate the possible application of the fibers as sutures capable of drug delivery
after ophthalmologic surgery.
Methods: Levofloxacin and Poly (L-lactic acid) (PLLA) were used to create the
functional nanofibers, employing electrospinning process. Polymer and drug were
dissolved in chloroform and the electrospinning applied. For the drug release test,
four milligrams of fibers were weighed, placed in tubes filled with buffered saline
and kept in incubator shaker at 37C for 10 days. The entire drug release test was
carried out in triplicate. At selected intervals, supernatant was tested with High
Performance Liquid Chromatography to quantify the drug release. The fibers were
morphologically evaluated by Scanning electron microscope and the tensile
strength measured. Antibacterial efficacy was performed against Staphylococcus
epidermidis, by placing a piece of fiber on agar plate and incubating overnight at
37C.
Results: HPLC demonstrated drug release within the first hour followed by a
sustained release (p<.0001) for at least ten days. Inhibition zone testing showed
antibacterial efficacy against S. epidermidis. The suture average diameter was 60.2
13.6 m and the tensile strength 0.148 0.036 N.
Conclusions: Levofloxacin, a third-generation fluoroquinolone, was released from
the nanofibers and showed activity against S. epidermidis, one of the most common
bacteria residing on the ocular surface. The PLLA used in these experiments is a
bioabsorbable and biodegradable synthetic polymer and has been shown to have no
cellular toxicity. This study showed that drug loaded nanofibers sutures have
potential application as a new drug delivery system in ophthalmic surgery.
Commercial Relationships: Fabiana K. Kashiwabuchi, None; Murilo W.
Rodrigues, Jr., None; Shuming Zhang, None; Himatkumar Patel, None; Jesus
S. Vidaurri-Martinez, None; Qingguo Xu, None; Justin Hanes, None; Jiangxia
Wang, None; Hai-Quan Mao, None; Peter J. McDonnell, III, None
Support: None
Electrohydrodynamic Spray Drying Technique for Moxifloxacin
Microencapsulation Delivery Systems
Qiongyu Guo1, Ahmed Aly1, Oliver D. Schein2, Jennifer H. Elisseeff1. 1Biomedical
Engineering, Johns Hopkins University, Baltimore, MD; 2Ophthalmology, Johns
Hopkins Wilmer Eye Inst, Baltimore, MD.
Purpose: To develop an electrohydrodynamic spray drying technique to fabricate
moxifloxacin microparticle systems in order to achieve sustained antibiotic release
for ocular treatments.
Methods: Moxifloxacin-loaded PLGA microparticles were prepared using an
electrohydrodynamic spray drying (electrospraying) technique. The antibiotic,
Moxifloxacin HCl, was encapsulated in poly (lactic-co-glycolic acid) (PLGA) by
dissolving both reagents in different solvents and electrospraying the solution using
high voltages ranging from 11-13 kV. The microparticles were collected using
distilled water, stored at -80 C then lyophilized. The microparticles were then
encapsulated into bioadhesive hydrogels: chondroitin sulfate-polyethylene glycol
(CS-PEG) bioadhesive. The morphologies of the microparticles were examined
using scanning electron microscopy (SEM). The release of Moxifloxacin was tested
in vitro by submerging the vehicles in PBS buffer solution, taking samples at
different time intervals and refreshing the solution. Drug concentration was
determined using high performance liquid chromatography (HPLC).
Results: In order to achieve an optimal, controlled release of moxifloxacin, we
encapsulated the antibiotics in PLGA-based microparticles by carefully selecting
solvent systems for electrospraying processing. The release speed of moxifloxacin
using the solvent of methanol:dichloromethane (MeOH:DCM)=10:90 was found to
be close to the one using the solvent of MeOH:DCM=20:80, while the release
speed using the solvent of MeOH:DCM=30:70 was much slower than the other two
solvent ratios. All of these conditions showed an effective release over ten days
with the release concentration continuously higher than the minimum inhibitory
concentration (MIC) (Figure 1). In contrast, the Moxifloxacin loaded in hydrogels
was released rapidly within 24 hours.
Conclusions: This study fabricated surfactant-free antibiotic-loaded polymeric
microparticles using an electrospraying technique and achieved sustained release of
Moxifloxacin HCl over more than ten days. A delivery system which incorporates a
bioadhesive may potentially integrate antibiotic prophylaxis and wound
healing.
Commercial Relationships: Qiongyu Guo, None; Ahmed Aly, None; Oliver D.

Schein, None; Jennifer H. Elisseeff, None

Support: Congressionally Directed Medical Research Program under the U.S.
Army Medical Research and Materiel Command (Contract No. W81XWH-09-20173)
Internalization of L-Tyrosine Polyphosphate Nanoparticles by Human
Trabecular Meshwork Cells
Jeffrey Dunmire1, Rachida Bouhenni1, Yang Yun2, Deepak P. Edward3,4.
1
Ophthalmology, Summa Health System, Akron, OH; 2Biomedical Engineering,
University of Akron, Akron, OH; 3Ophthalmology, Johns Hopkins University,
Baltimore, MD; 4Ophthalmology, King Khaled Eye Specialist Hospital, Riyadh,
Saudi Arabia.
Purpose: L-tyrosine polyphosphate (LTP) based nanoparticles can effectively bind
to various chemical and biologic molecules, and have been considered as a means
for targeted drug delivery. We investigated the in vitro biocompatibility and
internalization of LTP nanoparticles in human trabecular meshwork (HTM) cells.
Methods: Primary HTM cells were seeded on 6-well culture plates 24 h prior to
the addition of nanoparticles. Culture medium was replaced with rhodamine
encapsulated LTP nanoparticles suspended in complete medium at 100, 10, 1, or
0.5 g/ml. Cells were incubated at 37C/5% CO2 in a humidified chamber for 48 h,
72 h, or 7 d. Negative controls included cells receiving unloaded LTP nanoparticles
and cells receiving no nanoparticles. Following incubation, cells were rinsed, fixed,
permeabilized, stained with both phalloidin-Alexa Fluor 488 and DAPI, and
visualized by fluorescence microscopy.
Results: Internalization of LTP nanoparticles by HTM cells was demonstrated, to
varying degrees, at all time points for all concentrations of nanoparticle tested.
Nanoparticle uptake efficiency increased to greater than 75% by day 7 using 10
g/ml. Intracellular nanoparticles localized to the perinuclear region with few
remaining free in the cytoplasm. Cell density and morphology were unchanged at
all time points with 0.5, 1, and 10 g/ml of LTP nanoparticle. However, 100 g/ml
of LTP nanoparticle had the apparent consequence of stress-induced morphological
changes as evidenced by a more than 50% decrease in cell density and extension of
numerous cytoplasmic processes.
Conclusions: This in vitro data suggests that HTM cells can internalize loaded
LTP nanoparticles without affecting morphology or cell survival, except at high
concentrations. LTP nanoparticles could potentially be used for delivery of
chemical modulators of intra-ocular pressure, targeting cell systems that affect
aqueous humor outflow.
Commercial Relationships: Jeffrey Dunmire, None; Rachida Bouhenni,
None; Yang Yun, None; Deepak P. Edward, None
Support: None
Effect of Sustained Release Travoprost Delivered from a Punctum Plug on In
Vivo Miotic Activity over 13-Weeks
Charles D. Blizzard, Peter Jarrett, Amar Sawhney, Abbe Miller, Art Driscoll,
Michael Bassett, Leslie Jost. Ocular Therapeutix, Inc, Bedford, MA.
Purpose: To evaluate the effect of sustained release travoprost administered via a
hydrogel punctum plug on in vivo miotic activity in the dog iris sphincter muscle
contraction model.
Methods: Travoprost encapsulated in polylactide microparticles (PM), was
suspended in a multi-arm PEG precursor solution and injected into small bore
tubing prior to cross-linking. The travoprost in PM/hydrogel matrix was dried in
the tubing and cut into plugs. The travoprost plugs were inserted at Day 0 into the
inferior canaliculus of the right eye of 12 beagles and the contralateral left eye
served as a control. A pupil pen light with gauge and a ruler was used to measure
pupil diameter over 13-weeks. Average pupil diameter and standard error was
calculated for each group per time point.
Results: Upon insertion, the plugs hydrate and expand in diameter and shrink in
length to provide retention. The microparticles then slowly degrade and release the
travoprost into the tear fluid. At time zero, as shown in Figure 1, prior to plug
insertion there is no apparent difference in pupil diameter between the treatment
and control eyes. As the drug is released and it penetrates into the eye at sufficient
levels, the dog iris sphincter muscle contracts thereby achieving in vivo miotic
activity as demonstrated by the difference in pupil diameter compared to the
contralateral control eye over a period of 13-weeks.
Conclusions: In canines, prostaglandin F2-alpha drugs (e.g. travoprost) when
applied as eye drops stimulate and contract iris sphincter muscles. This in vivo
miotic activity has been used as an experimental model to monitor potency and
pharmacodynamic response. The results of this study demonstrate that travoprost
delivered into the tear fluid from a hydrogel plug retained in the canaliculus can
provide an extended duration pharmacodynamic response in a well established
animal
model.
Commercial Relationships: Charles D. Blizzard, Ocular Therapeutix, Inc. (E);

Peter Jarrett, Ocular Therapeutix, Inc (E); Amar Sawhney, Ocular Therapeutix,
Inc. (E); Abbe Miller, Ocular Therapeutix, Inc. (E); Art Driscoll, Ocular
Therapeutix, Inc. (E); Michael Bassett, Ocular Therapeutix, Inc. (E); Leslie Jost,
Ocular Therapeutix, Inc. (E)
Support: None
Intracorneal Delivery Of Bevacizumab Using Microneedles To Treat Injuryinduced Neovascularization
Yoo C. Kim1, Henry F. Edelhauser2, Mark R. Prausnitz1. 1Chemical and
Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA;
2
Ophthalmology, Emory University Eye Center, Atlanta, GA.
Purpose: Corneal transplantation is the most commonly performed tissue
transplantation. Neovacuarlization in the cornea after corneal transplant introduces
a significant increase in risk of graft rejection. For this reason, aggressive treatment
of neovascularization is necessary after corneal transplant surgery in order to
prevent rejection of the implanted cornea. This study examines the use of solid
microneedles, coated with bevacizumab, to reduce corneal neovascularization in a
minimally invasive way.
Methods: Solid metal microneedles 400 m in length were fabricated by
microfabrication. Bevacizumab was coated onto the microneedles using a rapidly
dissolving formulation. A 7-gauge silk suture was placed approximately 1 mm
from the limbus to induce corneal neovascularization in the eyes of New Zealand
White rabbits. The rabbits were then divided randomly into four groups: untreated
(Group 1), microneedle delivery (Group 2), topical eye drop (Group 3), and
subconjunctival injection (Group 4). All treatments were done at four days after
placement of the suture. For Group 2, a single bolus of approximately 1.1 g of
bevacizumab was given using four coated microneedles. For Group 3, a topical eye
drop of bevacizumab (25 mg/mL) was given three times per day for 14 consecutive
days. For Group 4, 100 L of bevacizumab (25 mg/mL) was given as a
subconjunctival injection. Digital photographs were obtained and the area of
neovascularization was measured for 18 days.
Results: Untreated eyes exhibited neovascularization that increased in area for up
to 10 days followed by decrease in vascularization until it plateaued. Eyes treated
with 1.1 g of bevacizumab using microneedles (Group 2) reduced
neovascularization compared to the untreated eye (Group 1) by 67.7% (day 10) to
40.8% (day 18). Eyes treated with 2500 g of bevacizumab using subconjunctival
injection (Group 3) reduced neovascularization compared to the untreated eye
(Group 1) by 61.8% (day 10) to 29.4% (day 18). Eyes treated with 1250 g of
bevacizumab by eye drops three times per day for 14 days (Group 4) reduced
neovascularization compared to the untreated eye (Group 1) by 44.3% (day 10) to
5.56% (day 18).
Conclusions: This study shows that microneedles can deliver a protein therapeutic
locally into the intrastromal space of the cornea in a minimally invasive way and
demonstrates that this approach can be effective to suppress neovascularization
after suture-induced injury using a much lower dose compared to other
conventionally used methods.
Commercial Relationships: Yoo C. Kim, None; Henry F. Edelhauser,
None; Mark R. Prausnitz, None
Support: NEI grants R24 EY017404, GAANN Fellowship to Y.C.K

Short-term Effects Of Dexamethasone Intravitreal Implant For Retinal
Macular Edema. A Consecutive Small Case Series
Giuseppe Lo Giudice1, Chiara M. Eandi2, Francesca Foltran3, Marco Tavolato4,
Veronica Maritan4, Silvia Babighian4, Alessandro Galan4. 1Ophthalmology, San
Antonio Hospital, Padova, Italy; 2Eye Clinic, University Torino, Torino, Italy;
3
Department of Environmental Medicine and Public Health, University of Padova,
Padova, Italy; 4Ophthalmology, San Antonio Hospital Padova, Padova, Italy.
Purpose: to evaluate the effectiveness and safety of OZURDEX
(dexamethasone intravitreal implant) 0.7mg in patients with macular edema (ME)
due to retinal vein occlusion.
Methods: this 6-month non randomized case series study collected 15
patients with ME due to branch retinal vein occlusion (7 eyes) and central retinal
vein occlusion (8 eyes) (BRVO and CRVO respectively). Ophthalmic examination,
best-corrected visual acuity (BCVA), intraocular pressure (IOP), fluorescein
angiography and optical coherence tomography (OCT), at baseline and each
follow-up visit were carried out. Outcome measures included BCVA, central retinal
thickness (CRT) by OCT and safety. Mean change of visual acuity (VA), OCT, and
IOP was assessed during the follow-up (6 months), using repeated measures
analysis of variance, after testing for normality. A linear regression model was also
used to evaluate the relationship between OCT and VA, as measured after 6 months
of follow-up.
Results: at baseline all eyes (15) with occlusive ME received DEX Implant
700g. Among these patients all had retinal vein occlusion > 3 months (mean 12.8
7.5). VA, IOP, and OCT at baseline and during the follow up period are
summarized in Figure 1. Mean change of VA between baseline and each time point
follow-up was not statistically significant (p = 0.2464) with only OCT
measurements that showed statistically significant differences with a repeatedmeasures ANOVA test (p<0.001) at 6 months follow-up. Statistical analysis
showed some evidence of benefit on CRT at earlier time points. The peak effects
were seen at two months, and there was substantial drop-off between two and three
months. Increases in IOP were similar and generally transient following each DEX
Implant (p = 0.0929). Simple linear regression analysis showed no statistically
significant negative relationship between OCT and VA measured after 6 months
(slope -0.00067, 95% CI: -0.0014 - 0.0000488 ).
Conclusions: in patients with ME due to BRVO and CRVO, DEX Implant 0.7 mg
produced substantial improvements in CRT, without, however, improving VA
significantly.
.
Commercial Relationships: Giuseppe Lo Giudice, None; Chiara M. Eandi,
None; Francesca Foltran, None; Marco Tavolato, None; Veronica Maritan,
None; Silvia Babighian, None; Alessandro Galan, None
Support: None
Spontaneous Separation of Pellet into the Vitreous: A Late Complication of
Retisert Implant
Levent Akduman1, Lyndell L. Lim2, Ebru N. Cetin1, Jamie Levy3. 1Ophthalmology,
Saint Louis University, Saint Louis, MO; 2Ophthalmology, Centre for Eye
Research, Melbourne Univ, East Melbourne, Australia; 3Ophthalmology, Royal
Victorian Eye and Ear Hospital, Melbourne, Australia.
Purpose: Separation of the Retisert pellet from its strut during exchange or
removal of the implant has previously been reported. We report two cases of
spontaneous pellet separation as a late term complication, and successful removal
with vitrectomy.
Methods: Two patients with chronic intermediate uveitis implanted with Retisert,
who later had spontaneous pellet dissociation, are described.
Results: Both patients had Retisert implanted 5.5 years previously. Their
presenting symptom was of a sudden, central huge floater. Clinical examination
revealed a dissociated Retisert pellet floating in the vitreous, and in one case
resultant retinal commotio and a retinal tear. Pellets were removed via a pars plana
vitrectomy with a large sclerotomy. The retinal tear in the second case was treated
with laser photocoagulation. Both cases recovered successful vision without further
complications.
Conclusions: Spontaneous pellet separation may occur with Retisert implants as
a late complication, where retinal tears may also occur as a result of the loose
pellet. Although both of these spontaneous dislocations and other cases of pellet
dislocation during exchange or removal of the implant have involved early versions
of the Retisert implant, it is yet to be determined whether these dislocations have
been the result of an initial manufacturing fault, or are related to their extended
intraocular exposure.
Commercial Relationships: Levent Akduman, Allergan (C, R); Lyndell L.
Lim, None; Ebru N. Cetin, None; Jamie Levy, None
Support: None
201 Targets for Ocular Neuroprotection: Lost in Translation Minisymposium
Monday, May 7, 2012, 8:30 AM - 10:15 AM
Floridian A Symposium
Program #/Board # Range: 1275-1280
Contributing Section(s): Anatomy Pathology,Biochemistry/Molecular
Biology,Glaucoma,Retinal Cell Biology,Retina+
Program Number: 1275
Loss of Synaptic Connectivity in Glaucoma: A Bump on the Road to
Translation
Adriana Di Polo. Pathology/Cell, University of Montreal, Montreal, QC, Canada.
There is now substantial evidence that retinal ganglion cells (RGCs) undergo
dendritic alterations, including potential loss of synapses, in glaucoma. In this
presentation, novel mechanisms and strategies that prevent RGC dendritic
retraction and loss of synaptic connectivity after axonal injury will be discussed.
The preservation of RGC dendritic morphology and functional synapses is a critical
element of the successful clinical translation of interventions to prevent vision loss
in glaucoma.
Commercial Relationships: Adriana Di Polo, Quark Pharmaceuticals, Inc. (F,
R)
Support: Canadian Institutes of Health Research
Choroidal Neovascularization: Role Of Neuroprotectin D1
Nicolas G. Bazan. Ophthal & Neuroscience, LSU Health Sciences Center, New
Orleans, LA.
Commercial Relationships: Nicolas G. Bazan, None
Support: None
Clinical Neuroprotection in Glaucoma: Unlikely to be Achieved by Use of a
Single Chemical?
Neville N. Osborne. Fundacin de Investigacin Oftalmolgica, Oviedo, Spain.
Our working hypothesis is that glaucoma is initiated by an alteration in the quality
of blood flow dynamics in the optic nerve head region causing both a compromise
in the normal retinal ganglion cell axon energy requirement as well as an influence
upon microglia, astrocytes and the lamina cribosa in the optic nerve head region
region. Ganglion cells are then particularly susceptible to additional insults such as
light and alterations in the constituents present in the extracellular retinal
compartments. This hypothesis therefore suggests that after the disease is initiated
ganglion cell apoptosis is initiated by both receptor and mitochondrial mediated
events and this will vary for different neurones. It also suggests that any
neuroproptection strategy will require the use of substances with multiple
beneficial modes of action which is unlikely to be achieved by use of a single
chemical such as memantine.
Commercial Relationships: Neville N. Osborne, None
Support: Fundacion BBVA, Spain
Control of VEGF Cytoprotection by Alternative Splicing
David O. Bates. Microvascular Research Laboratories, School of Physiology and
Pharmacology, University of Bristol, Bristol, United Kingdom.
Vascular endothelial growth factor is generated as 2 contrasting isoform families in
terms of their actions on vascular permeability, angigoenesis and vasodilatation,
but both families are cytoprotective on epithelial and endothelial cells. The proangiogenic VEGF165 isoform has been shown also to be neuroprotective in
hippocampal, dorsal root ganglia and retinal neurones. The contrasting VEGF165b
isoform also acts as an endogenous neuroprotective agent for hippocampal, retinal
and DRG neurons. Endogenous expression of human and rat VEGF165b is detected
in the hippocampal and retinal neurons in human tissue and forms a significant
proportion of total VEGF in rat brain. rhVEGF165b blocks glutamidergic
excitotoxicity in hippocampal neurons in vitro, dependent on VEGFR2 activation,
and P42p44MAPK. rhVEGF165b also inhibits both chemotherapy and
hyperglycaemia induced cytotoxicity of dorsal root ganglion cells. Exogenous
rhVEGF165b increases the survival of retinal ganglion cells in rat retinal ischemiareperfusion injury in vivo. Thus rhVEGF165b is an endogenous neuroprotective
agent that effectively protects both peripheral and central neurons both in vivo and
in vitro. rhVEGF165b may be therapeutically useful for pathologies involving
neuronal damage, including diabetic retinopathy and peripheral neuropathy, but
inhibition of all VEGF isoforms may be damaging to retinal neurons.

Commercial Relationships: David O. Bates, None
Support: Fight for Sight, Wellcome Trust
Autocrine Cytoprotection by VEGF on Muller Cells and Photoreceptors
Magali Saint-Geniez. Schepens Eye Research Institute, Harvard Med School,
Boston, MA.
Commercial Relationships: Magali Saint-Geniez, None
Support: None
The Impact of Cholesterol Deficiency on Retinal Degeneration: From Basic to
Clinical Research
Steven J. Fliesler. Departments of Ophthalmology & Biochemistry, University at
Buffalo/SUNY, Buffalo, NY.
The hallmarks and potential mechanisms underlying retinal degeneration associated
with cholesterol deficiency, particularly the Smith-Lemli-Opitz syndrome, will be
presented, along with a discussion that highlights how animal-based research in this
area has led to a potential therapeutic intervention for such diseases.
Commercial Relationships: Steven J. Fliesler, None
Support: NIH Grant EY007361
263 Gene Therapy and Delivery I
Monday, May 7, 2012, 1:45 PM - 3:30 PM
Presentation Time: 1:45 PM - 3:30 PM
Efficacy of Nanoparticle-based Gene Delivery for Rescuing Nr2e3 Associated
Retinal Degeneration
Nelly M. Cruz1, Yang Yuan2, Rinku Baid3, Uday Kompella3, Neena B. Haider1.
1
Schepens Eye Research Institute, Massachusetts Eye and Ear, Harvard Medical
School, Boston, MA; 2Genetics, Cell Biology & Anatomy, Univ of Nebraska
Medical Ctr, Omaha, NE; 3Department of Pharmaceutical Sciences, University of
Purpose: The nuclear hormone receptor Nr2e3 is a retinal-specific transcription
factor with critical functions in photoreceptor cell development and differentiation.
Mutations in NR2E3 have been associated with several eye disorders characterized
by progressive retinal degeneration, such as Enhanced S-cone syndrome (ESCS)
and Retinitis Pigmentosa. Currently, there are no effective treatment options for
this group of diseases. In order to develop a therapeutic strategy for Nr2e3associated retinal degeneration, we are using biodegradable Nile red poly(lactideco-glycolide) (PLGA) nanoparticles to deliver the Nr2e3 gene into retinal
degeneration 7 (rd7) mouse, which lacks Nr2e3 expression.
Methods: Nr2e3 loaded Nile red PLGA nanoparticles were introduced into
postnatal day 0 (P0), P10 or P21 rd7 eyes by intravitreal or subretinal injection,
followed by electroporation. The efficacy of delivery was assessed using Nile red
as a nanoparticle tracking dye, and green fluorescent protein expression as a marker
of transfected cells. To determine the effectiveness of the Nr2e3-containing
nanoparticles in rescuing the rd7 phenotype, animals were examined 2 months after
treatment for alterations in their clinical, histological, and morphological
phenotypes using indirect ophthalmoscopy, hematoxylin/eosin staining, and
electroretinography (ERG), respectively.
Results: While spotting of the fundus was clearly observable when rd7 mice were
injected with the blank vector, administration of the Nr2e3 loaded nanoparticles
reduced the severity of spotting and provided a partial recovery of the phenotype.
Under both photopic and scotopic conditions, improvements in ERG response were
observed when comparing the Nr2e3 containing nanoparticles to the blank vector.
Conclusions: We show that nanoparticle delivery of Nr2e3 in the rd7 mice
efficiently ameliorated clinical, morphological, and functional defects associated
with rd7 retinal degeneration. Following the administration of the Nr2e3 loaded
nanoparticles, we observed a partial rescue of the rd7 phenotypes, both by fundus
examinations as well as ERG analysis of photoreceptor function. The present study
provides promising results toward the development of therapies for human retinal
degenerations caused by loss of function of the NR2E3 protein.
Commercial Relationships: Nelly M. Cruz, None; Yang Yuan, None; Rinku
Baid, None; Uday Kompella, None; Neena B. Haider, None
Support: NIH Grant EY017653.
Efficient Transduction of Tyrosine-to-Phenylalanine Mutated AAV2 vectors

carrying Human ND4 gene and Biodistribution following Intravitreal Delivery
in a Rodent Model- a Gene Therapy for Leber Hereditary Optic Neuropathy
Rajeshwari D. Koilkonda1, William W. Hauswirth2, Vince Chiodo2, Sanford L.
Boye2, John Guy1. 1Ophthalmology, Bascom Palmer Eye Institute, Miami, FL;
2
Ophthalmology, University of Florida, College of Medicine, Gainesville, FL.
Purpose: In this study, we evaluated the ocular transduction efficiency and
biodistribution of self complimentary (sc) AAV2 (Y444+500+730)-vector genomes
carrying the human ND4 gene after intravitreal administration.
Methods: Eight week old Sprague Dawley rats (n=8; male=4, female=4) were
injected with scAAV containing wild type (wt)-human (h) P1ND4v2 (without
FLAG tag) packaged in triple Y-F (Y444+500+730) mutated capsids into the
vitreous cavity of right eyes (OD) (2X109vg in 4l). Contralateral eyes (OS)
remained un-injected. The animals were sacrificed 1m post injections (PI) and the
following organs were harvested for DNA extraction: blood, eyes, optic nerves,
right and left brain, heart, liver, skeletal muscle and gonads. Peripheral blood was
also analyzed at day 1 and day 8 PI. In order to better characterize the results for
the novel AAV2-triple Y-F capsid mutant, 2 rats (m=1, f=1) received scAAV2GFP vector (2X109vg in 4l) and served as AAV2 controls. To gauge the
expression of human ND4 in retinal ganglion cells (RGCs), another group of mice
(n=5) were injected with sc-AAV2-hP1ND4V2 (Triple Y-F) in both eyes, control
group remained un-injected. Confocal microscopy was performed on retinal whole
mounts at 10d PI by immunoflorescence.
Results: Expression-Confocal microscopy using hND4 antibody on retinal whole
mounts revealed punctuate and perinuclear expression of hND4 at 10d PI, which
co-localised with Thy1.2, indicating expression in RGCs. hND4 expression was
absent in un-injected controls. Biodistribution- The vector genome copy number
was represented as average of overall tissue score (AOTS) for each tissue. AOTS
for ocular tissues was, optic nerves, un-injected (OS): 0.83, injected (OD): 1.62,
eyes, un-injected (OS): 0.6, injected (OD): 3.28. None of the other organs such as
brain (left and right), liver, skeletal muscle, and gonads had the AAV genomes
(AOTS: 0), indicating AAV2 displayed narrow tropism. Peripheral blood showed
vector genome copies at day 1 (AOTS: 2.16) which however, dropped down to
minimum levels at day 8 (AOTS: 0.25). In addition, male and female animals had
similar AOT scores indicating AAV mediated transduction and biodistribution is
not gender specific. The vector genome copy number was almost similar using
either scAAV2-triple Y-F (Y444+500+730)-wt-hP1ND4v2 or scAAV2-GFP
vectors at 1m (AOTS: 1.45 and 1.5).
Conclusions: Biodistribution of sc-triple Y-F mutated AAV2 carrying the human
ND4 gene displayed a narrower tropism of ocular tissue, indicating safety in using
these vectors for future gene therapy on LHON patients with optic neuropathy.
Commercial Relationships: Rajeshwari D. Koilkonda, None; William W.
Hauswirth, None; Vince Chiodo, None; Sanford L. Boye, None; John Guy,
None
Support: R24EY018600
Enhanced Retinal Transduction Via The Vitreous With Aav8 Capsid Mutants
Christine N. Kay1, Sanford L. Boye1, Renee C. Ryals1, Jingfen Sun1, Andy W.
Neeley1, William W. Hauswirth2, Shannon E. Boye1. 1Ophthalmology, University of
Florida, Gainesville, FL; 2Dept of Ophthalmology, Univ of Florida Coll of
Medicine, Gainesville, FL.
Purpose: Several ongoing clinical trials for RPE65-associated Leber congenital
amaurosis (LCA2) have demonstrated the ability to subretinally target therapeutic
transgene to the retinal pigment epithelium (RPE) thereby restoring vision to these
patients. However, because most retinal degenerations are caused by mutations in
photoreceptor-specific rather than RPE-specific genes, development of
photoreceptor-targeted gene therapies would be of significant benefit. Of equal
importance is the need to develop a less invasive injection procedure, particularly
for diseased retinas that are more prone to injury during the subretinal injection
procedure. The purpose of this study was to evaluate the ability of adeno-associated
virus (AAV) capsid mutants to transduce photoreceptors following intravitreal
delivery to the mouse retina. These AAV capsid mutants originated from a serotype
(AAV8) with native tropism for photoreceptors.
Methods: HEK293 and 661W cells were infected with self-complimentary (sc)
AAV8-smCBA-mCherry capsid mutant vectors containing single or multiple
tyrosine mutations on their capsid surface at and MOI of 10,000. Fluorescenceactivated cell sorting (FACS) analysis was used to measure relative mCherry
fluorescence 3 days post-infection. Subsequently, scAAV8 capsid mutants
containing GFP cDNA were intravitreally injected into P60 C57Bl/6J wildtype
(WT) mice at a concentration ~9 x 10e9 vg/ul. Mice were sacrificed at 1 month
post-injection and eyes were enucleated, cryoprotected and sectioned. Frozen
sections were immunostained with an antibody raised against GFP and imaged with
confocal microscopy.
Results: AAV-mediated mCherry expression in HEK293 and 661W cells varied
depending on how many capsid mutations were present, with most exhibiting
higher efficiency relative to standard AAV8. Injection of AAV8 capsid mutants

resulted in robust expression in the outer retina of WT mice following delivery to
the vitreous.
Conclusions: The authors demonstrate that AAV capsid mutant vectors based on a
serotype with native tropism for photoreceptors, AAV8, has the ability to transduce
outer retina following intravitreal delivery. These results suggest that AAV8 capsid
mutant vectors could be used to deliver therapeutic transgene to photoreceptors,
thereby conferring therapy to various models of retinal degeneration associated
with photoreceptor defects.
Commercial Relationships: Christine N. Kay, None; Sanford L. Boye,
None; Renee C. Ryals, None; Jingfen Sun, None; Andy W. Neeley, None;
William W. Hauswirth, AGTC (Applid Genetic Technologies, Corp.)
(I); Shannon E. Boye, None
Support: Foundation Fighting Blindness, Career Development Award. CD-CL0711-0520-UFL
Assessment Of Adeno-associated Virus Serotypes In Transducing Human
Retina Using An Ex-vivo Tissue Culture System
Samantha R. De Silva1, Daniel M. Lipinski1, Peter Charbel Issa1, Mandeep S.
Singh1, Nathan J. Walker1, Mark W. Hankins1, Robert E. MacLaren1,2. 1Nuffield
Lab of Ophthalmology, University of Oxford, Oxford, United Kingdom;
2
Moorfields Eye Hospital NHS Foundation Trust, London, United Kingdom.
Purpose: Gene therapy for the treatment of retinal degenerations using adenoassociated virus (AAV) is a promising therapeutic option. In order to target further
conditions, new vectors must be developed and tested. Alongside pre-clinical
testing on mouse or primate models, the ability to test on human retinal tissue
would greatly advance the assessment of tropism and efficacy of new AAV vectors.
Methods: Retinal detachment surgery in some patients involves removing a small
area of retina (retinectomy). Ethical approval was obtained to extract and culture
this tissue, which would otherwise be disposed of (REC reference no.10/H0505).
Research was conducted in compliance with the Declaration of Helsinki.
Samples were obtained from 2 patients with their consent and quickly placed into
an organotypic culture system which was used to maintain the retinal tissue exvivo. AAV serotypes 2/2, 2/5 and 2/8 the latter containing the Y733F capsid
mutation were tested. All contained a construct encoding green fluorescent protein
(GFP) under a chicken beta actin (CBA) promoter. 10l of virus (of titre 1x1012
viral particles/ml) was added to each retinal sample and the samples were imaged
daily.
Results: We were able to maintain viable human retinal explants for 11 to 14 days
using the culture system. Photoreceptor transduction was demonstrated with all
vectors tested, with Mller cells also being transduced by AAV 2/2 and the Y733F
capsid mutant AAV2/8. The latter vector also successfully transduced bipolar and
horizontal cells.
Conclusions: Culturing human retinectomy tissue ex-vivo is an effective model for
testing AAV vectors on human tissue. We demonstrate effective human retinal
transduction using AAV 2/2 and 2/5, and also with the capsid mutant vector AAV
2/8 Y733F.
Commercial Relationships: Samantha R. De Silva, None; Daniel M. Lipinski,
None; Peter Charbel Issa, None; Mandeep S. Singh, None; Nathan J. Walker,
None; Mark W. Hankins, None; Robert E. MacLaren, None
Support: Wellcome Trust (094448/Z/10/Z), NIHR biomedical research centres,
Royal College of Surgeons of Edinburgh
Adeno-associated Virus Mediated Delivery of Complement and Kallikreinkinin System Inhibitors Reduce Vascular and Neuronal Complications in a
Model of Diabetic Retinopathy
Mehreen Adhi, Siobhan M. Cashman, Rajendra Kumar-Singh. Ophthalmology,
Tufts University School of Medicine, Boston, MA.
Purpose: Retinal vascular permeability and neuronal cell death have been
postulated as plausible mechanisms of visual dysfunction associated with
proliferative diabetic retinopathy (PDR) and diabetic macular edema (DME). We
aimed to investigate the effect of complement and kallikrein-kinin system (KKS)
inhibitors in diabetic retinopathy (DR), using a streptozotocin (STZ) induced
diabetic mouse model.
Methods: Using adeno-associated virus serotype 8 as an expression system, we
tested efficacy of three molecules: (i) human C1-esterase inhibitor (hC1INH), an
inhibitor of the classical complement pathway and KKS; (ii) soluble human CD59
(sCD59), an inhibitor of the terminal product of complement activation, the
membrane attack complex (MAC); and (iii) a novel molecule consisting of a
modified human tissue-factor pathway inhibitor (mhTFPI), an inhibitor of KKS.
Each of these viruses was injected into the vitreous of C57BL/6J mice. After 2
weeks of transgene expression, mice were rendered diabetic using STZ. After 4
days of STZ treatment, mice were injected intraperitoneally with 2.5% sodium
fluorescein and vitreous samples were collected to quantify leakage. Retinal
sections were stained for ganglion cell apoptosis (GCA) and MAC deposition.
Results: Fluorescein Angiography revealed a reduction in capillary dropout and a
3.4-fold, 1.6-fold and 1.2-fold reduction in leakage in hC1INH, sCD59 and
mhTFPI injected eyes respectively, when compared to control eyes. GCA was
significantly reduced in hC1INH, sCD59 and mhTFPI injected eyes compared to
controls. In addition, reduction in MAC deposition in the ganglion cell layer was
observed in hC1INH and sCD59 injected eyes compared to controls.
Conclusions: We have demonstrated reduction in both vascular and neuronal
complications in a model of DR using AAV-mediated delivery of natural inhibitors
of the complement system and KKS, and a novel inhibitor of KKS. To our
knowledge, this is the first study showing efficacy of MAC inhibition as a potential
treatment for DR. These approaches warrant further exploration as potential
therapies for visual dysfunction associated with PDR and DME, leading causes of
visual impairment in DR patients.
Commercial Relationships: Mehreen Adhi, None; Siobhan M. Cashman,
None; Rajendra Kumar-Singh, None
Support: None
Gene Transfer Into Corneal, Trabecular Meshwork And Retinal Cells Using
Pseudotyped Lentiviral Vectors
Daniel M. Lipinski1, Peter Charbel Issa1, Mandeep S. Singh1, Antonio Trabalza2,
Stuart M. Elison2, Nicholas D. Mazarakis2, Robert E. MacLaren1,3. 1Nuffield
Laboratory of Ophthalmology, University of Oxford, Oxford, United Kingdom;
2
Department of Gene Therapy, Faculty of Medicine, Imperial College London,
London, United Kingdom; 3Moorfields Eye Hospital, London, United Kingdom.
Purpose: The use of adeno-associated virus for therapeutic gene delivery into
dividing cells and for the delivery of large genes is limited. Lentivirus vectors
provide a feasible alternative as they typically integrate into the host genome and
have a larger coding capacity. Furthermore, lentivirus vectors may be pseudotyped
through substitution of heterologous surface glycoproteins in order to alter cellular
tropism. Herein, the ocular tropism of a novel lentivirus pseudotype, derived from
Venezuelan equine encephalitis virus (VEEV), was explored to determine its utility
for gene delivery in the eye.
Methods: HIV-1 lentiviral vectors expressing enhanced green fluorescent protein
(eGFP) were pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) or
VEEV-G (strain 3908) and concentrated 2000-fold. 1l of vector (max titre
2.63x10^9 TU293T/ml) was administered via subretinal, intravitreal or
intracameral injections in 5-week-old C57BL/6 mice (n=5 per group). In vivo
fluorescence imaging of the fundus or anterior chamber was performed by confocal
scanning laser ophthalmoscopy (cSLO) on days 1, 2, 7, 14 and 21 post-injection to
assess transgene expression. Eyes were removed post mortem for
immunohistochemistry (IHC) to determine cellular tropism.
Results: cSLO imaging one day post subretinal injection of VSV-G and VEEV-G
revealed retinal pigment epithelium (RPE) transduction, which was confirmed by
IHC. RPE65 expression was reduced in regions of RPE transduction compared to
neighbouring areas. cSLO imaging following intracameral VSV-G injection
revealed transduction of cells in the central and far peripheral cornea. Histological
localization of eGFP showed transduction of endothelial cells in the central cornea
and of trabecular meshwork cells in the iridocorneal angle. Intracameral VEEV-G
injection resulted in greater transduction of stromal keratocytes. Intravitreal VSV-G
and VEEV-G delivery resulted in RPE transduction only at the site of injection.
Conclusions: Efficient gene delivery to the RPE, cornea and trabecular meshwork
implicates lentiviral vectors as potentially useful tools for the treatment of ocular
disorders such as glaucoma and corneal dystrophies. As integrating vectors they
may be particularly useful for the transduction of corneal endothelium, and for the
expression of neuroprotective factors in dividing cells.
Commercial Relationships: Daniel M. Lipinski, None; Peter Charbel Issa,
None; Mandeep S. Singh, None; Antonio Trabalza, None; Stuart M. Elison,
None; Nicholas D. Mazarakis, None; Robert E. MacLaren, None
Support: Fight for Sight 1785
Selective Gene Transfer To The Retina Using Intravitreal Ultrasound
Irradiation
Shozo Sonoda1, Katsuro Tachibana2, Toshifumi Yamashita1, Makoto Shirasawa1,
Hiroto Terasaki1, Eisuke Uchino1, Ryo Suzuki3, Kazuo Maruyama3, Taiji
Sakamoto1. 1Department of Ophthalmology, Kagoshima University, Kagoshima,
Japan; 2Department of Anatomy, Fukuoka University School of Medicine,
Fukuoka, Japan; 3Department of Biopharmaceutics, Teikyo University Faculty of
Pharmaceutical Sciences, Sagamihara, Japan.
Purpose: To evaluate the efficacy of intravitreal ultrasound (US) irradiation for
green fluorescent protein (GFP) plasmid transfer into the rabbit retina using a
miniature US transducer.
Methods: Intravitreal US irradiation was performed by a slight modification of the

transconjunctival sutureless vitrectomy system utilizing a small probe. After
vitrectomy, the US probe was inserted through a scleral incision and placed 1-2
mm from the retina. A mixture of GFP plasmid (50 l) and bubble liposomes (BLs;
50 l) was injected into the vitreous cavity, and US was generated (frequency=3
MHz; duty=6%; intensity=0.15 W/cm2; time=60 s) to the retina using a Sonopore
4000 (NEPA GENE). The control group were not exposed to US. After 72 h, the
eyes were enucleated, and the gene-transfer efficiency was quantified by counting
the number of GFP-positive cells. Tissue damage was evaluated by slit-lamp
biomicroscopy and histological examinations.
Results: The retinas that received plasmid, BL, and US showed a significant
increase in the number (average SEM) of GFP-positive cells (324.9; n=7;
P<0.01). GFP-positive cells were limited to the area exposed to US, and were
found mainly in the outer nuclear layer. No GFP-positive cells were observed in the
control eyes (n=7). No apparent histological damage was detected by hematoxylin
and eosin staining in any treatment group.
Conclusions: Intravitreal retinal US irradiation can transfer the GFP plasmid into
the retina without causing any apparent damage. This procedure could be used to
transfer drugs directly to the retina and therefore has potential therapeutic value.
Commercial Relationships: Shozo Sonoda, None; Katsuro Tachibana,
None; Toshifumi Yamashita, None; Makoto Shirasawa, None; Hiroto Terasaki,
None; Eisuke Uchino, None; Ryo Suzuki, None; Kazuo Maruyama, None; Taiji
Sakamoto, None
Support: Japanese Grant-in-Aid for Young Scientists No.22791674
Development of human in vitro and murine in vivo tools for gene therapy
studies of Retinitis Punctata Albescens
Marie O. Pequignot, Aude Conscience, Lorenne Robert, Nicolas Cereso, Christian
P. Hamel, Vasiliki Kalatzis. Unite 1051, INSERM-Institute for Neurosciences of
Montpellier, Montpellier, France.
Purpose: We aim to perform pre-clinical gene therapy studies for Retinitis
Punctata Albescens (RPA). RPA is caused by mutations in the RLBP1 gene, which
codes for the visual cycle protein CRALBP. A CRALBP-deficiency results in a
malfunction of the retinal pigment epithelium (RPE) and leads to RPE and
photoreceptor (PR) death. The early apparition of characteristic clinical signs (e.g.
night blindness in childhood and small white dots observed by fundoscopy) allows
an early diagnosis thus providing a large therapeutic window. An Rlbp1-deficient
mouse model has been previously generated but it does not fully reproduce the
disease course. As a consequence, in parallel to this model, we are generating an in
vitro human retinal model of the RPA RPE for gene transfer studies.
Methods: We transferred the white Rlbp1-deficient mouse colony onto a
pigmented background. In parallel, three RPA patients volunteered for skin
biopsies from which we generated a stock of CRALBP1-deficient fibroblasts.
These cells were reprogrammed into induced pluripotent stem (iPS) cells.
Results: We are currently analyzing the phenotype of our pigmented Rlbp1-/- mice
to determine the functional tests that can be used as readouts for gene therapy
studies. In parallel, we obtained iPS-like clones for 2 of the 3 patient fibroblasts
and are currently characterizing their pluripotency status. The reprogrammation of
the 3rd patients fibroblasts is in process. We have set up the in vitro differentiation
of control iPS cells to RPE and are developing the tools necessary to characterize
its functions (e.g. phagocytosis, visual cycle analysis). The control and CRALBPdeficient RPE will constitute the human cellular model for gene transfer studies.
Conclusions: The human RPE model is potentially a unique tool as i) it allows a
better understanding of the disease by providing the possibility to perform tests that
cannot be done in patients, and ii) it represents a more biologically relevant model
than the mouse eye for gene therapy studies. Consequently, the validation of this
model will allow a considerable reduction in the future number of disease-specific
animals necessary for testing novel therapies, as well as providing a model for
testing novel therapies for diseases for which an animal model does not exist.
Commercial Relationships: Marie O. Pequignot, None; Aude Conscience,
None; Lorenne Robert, None; Nicolas Cereso, None; Christian P. Hamel,
None; Vasiliki Kalatzis, None
Support: Fondation de France, AFM, Inserm, Terre Plurielle, Retina France,
france Choroideremie, Choroideremia Research Fondation, Fondation pour la
Recherche Mdicale
Results of Safety and Tolerability Studies of UshStat, an EIAV-based
Lentiviral-vector Therapy for USH1B and the Elucidation of Retinal Cell
Types Responsible for USH1B Pathology
Marisa L. Zallocchi1, Linda Cheung1, You-Wei Peng1, Duane Delimont1, Katie
Binley2, Yatish Lad2, Kyriacos Mitrophanous2, Dominic Cosgrove1,3. 1Genetics,
Boys Town Nat'l Research Hosp, Omaha, NE; 2Medawar Centre, Oxford
Biomedica UK Ltd, Oxfordshire, United Kingdom; 3University of Nebraska
Medical Center, Omaha, NE.
Purpose: Usher syndrome type 1B (USH1B) is a common and severe form of

Usher syndrome, characterized by congenital deafness associated with retinitis
pigmentosa. UshStat is an EIAV-based lentiviral vector expressing Myosin VIIa
encoded by the USH1B gene. In UshStat this gene is expressed by the constitutive
CMV promoter so Myosin VIIa is produced mainly in the RPE and photoreceptor
(PR) cells. The safety and tolerability of UshStat was examined in a GLP study in
Rhesus macaques. Using the same EIAV vector platform myosin VIIa will be
expressed specifically in RPE or PR cells using tissue specific promoters to
determine which cell type is responsible for retinal pathology.
Methods: EIAV vectors have been developed that express human Myosin VIIa
using the CMV (UshStat) or RPE-specific (VMD2) or PR-specific (RK)
promoters. Safety and biodistribution assessments of UshStat were examined in
macaques following subretinal delivery of UshStat or buffer. Regular ophthalmic
examinations to monitor ocular inflammation and histopathological retinal
morphology were recorded up to 3 months. A further study in P-3 shaker-1 mice
will determine which retinal cell types are responsible for the USH1B disease
pathology using tissue restricted EIAV vectors and assessing light induced transducin translocation and PR degeneration.
Results: No UshStat-related adverse findings and no antibody responses against
any vector components were detected in the macaques. Biodistribution analysis
indicates that the vector is confined to the eye in the macaque. Long-term efficacy
studies in shaker-1 mice following UshStat subretinal delivery have shown
neuroprotection of PRs. The specificity of the VMD2 and RK promoters has been
validated in vivo using the GFP gene and the retinal specific EIAV-MYO7A
vectors have been subretinally delivered to shaker1 mice. The results will be
presented.
Conclusions: Subretinal delivery of UshStat vector in macaques was well
tolerated with no evidence for changes to retinal function. Previous studies show
that UshStat is efficacious in shaker-1 mice. Retinal specific EIAV-MYO7A
vectors will be used to determine which cell types are principally responsible for
ontogenesis of USH1B retinal pathology.
Commercial Relationships: Marisa L. Zallocchi, None; Linda Cheung,
None; You-Wei Peng, None; Duane Delimont, None; Katie Binley, Oxford
BioMedica UK Ltd (E); Yatish Lad, Oxford BioMedica UK Ltd (E); Kyriacos
Mitrophanous, Oxford BioMedica UK Ltd (E); Dominic Cosgrove, None
Support: NIH Grant R01 DC004844, Grant from the Foundation Fighting
Blindness
Rescue of Prominin-1 k.o. Mouse Phenotype by introducing wild-type
Prominin-1 using rAAV2/8 Vector
Dominic Eberle1, Stefan Weger2, Denis Corbeil3, Regine Heilbronn2, Marius Ader1.
1
CRTD, TU Dresden, Dresden, Germany; 2Charit, University Medicine FU Berlin,
Berlin, Germany; 3Biotec, TU Dresden, Dresden, Germany.
Purpose: Prominin-1 (PROM1, AC133, CD133, PROML1) is a 5-transmembranedomain glycoprotein located at plasma membrane protrusions and expressed in
various tissues. To date, 5 different mutations in human PROM1 are known
causing a variety of retinal degenerative disorders ranging from macular dystrophy
via Stargardt disease, cone-rod dystrophy and bulls eye maculopathy to retinitis
pigmentosa. Recent results indicate that PROM1 plays a central role in
photoreceptor outer segment disc morphogenesis. The PROM1 knock-out mouse
displays disorganized outer segment disc structure and retinal degeneration. In this
study we show a rescue of the knock-out phenotype by delivering wild-type
PROM1 to the host photoreceptor cells.
Methods: Young, post-natal day 15 PROM1 knock-out mice (AC133-/-) were subretinally injected with rAAV vectors containing GFP or PROM1 transcripts. GFP
was used for observation of target cells while PROM1 was used to rescue the
knock-out phenotype. We tested rAAV serotype 5 and 2/8 vectors according to
targeting specificity and transgene expression. PROM1 delivery was performed
using rAAV2/8 vectors containing an ubiquitously active chicken beta-actin
promoter driven transgene.
Results: We show that rAAV5 and rAAV2/8 have high photoreceptor targeting
specificity and display a strong, injection-site restricted transgene expression. In
few cases, single Mller-glia cells or second order neurons were targeted. PROM1
delivery by rAAV2/8 vectors to AC133-/- mice resulted in strong expression of
PROM1 protein at its native sub-cellular location, i.e. the tip of the connecting
cilium, 2 weeks after injection. Morphological differences between injected and
non-injected eyes first occur 3 weeks after injection. We show a relative
preservation of outer nuclear layer (ONL) thickness and photoreceptor morphology
in treated eyes, in comparison to untreated controls, that show a strong decline in
ONL thickness.
Conclusions: This study demonstrates a rescue of the PROM1 knock-out
phenotype by delivering wild-type PROM1 to host photoreceptor cells using
rAAV2/8 vector. Our results are in line with other studies showing promising
results for gene therapeutical strategies for the treatment of retinal degenerative
disorders and show the feasibility of gene therapeutical treatment of PROM1
mutations causing retinal degenerative diseases.

Commercial Relationships: Dominic Eberle, None; Stefan Weger, None; Denis
Corbeil, None; Regine Heilbronn, None; Marius Ader, None
Support: ProRetina, DFG
Evaluation Of Eye Physiopathology In The Harlequin Mouse Strain Aimed At
Developing A Gene Therapy Based On A Permanent Mitochondrial Function
Protection
Aicha Bouaita, Sbastien Augustin, Christophe Lechauve, Hlne CwermanThibault, Hong Liang, Franoise Brignole-Baudouin, Jos-Alain SAHEL, Marisol
Corral-Debrinski. Institut de la Vision, Paris, France.
Purpose: The Harlequin (Hq) mutant mouse, characterized by the depletion of the
mitochondrial Apoptosis Inducing Factor (AIF), represents a genetic model that
resembles human pathologies caused by respiratory chain complex I deficiency.
These mice exhibits photoreceptor and retinal ganglion cell (RGC) loss. We
demonstrated that RGC loss and complex I deficiency in optic nerves were durably
prevented by AAV2-AIF administration. Hence, our next objective was to shed
light on abnormalities in retinas and anterior eye segments, the extent of cell
dysfunction and the kinetics of their appearance for at last, develop a gene therapy
for preventing eye pathology in Hq mice.
Methods: We have performed in vivo imaging of eye structures, electroretinograms
(ERG) and immunochemistry studies. Besides, mitochondrial respiratory chain
complex activities in retinas and optic nerves were determined by spectrometric
assessments.
Results: Hq mice display abnormalities in the cornea, for instance the presence of
large superficial and hyper-reflective epithelial cells and outsized multinucleated
cells in the stroma. In some mice epithelium invaginations through the stroma were
obvious as well as neovascularisation. Photoreceptor loss was apparent in Hq mice
aged 6 months. In animals 12-month old, the retinal thickness was 2-fold reduced
as compared to controls. Functional assessment performed by ERG recordings in
mice 6-month old did show a significant decrease in scotopic and photopic
function; at 9 months the average amplitude of scotopic and photopic ERG B-wave
amplitudes were reduced by 95% relative to age-matched controls. All these
structural and functional changes were associated with a significant complex I
deficiency in Hq retinas relative to age- matched controls (a 70% reduction in its
enzymatic activity in mice older than 8 months).
Conclusions: Since we determined the kinetics of photoreceptor and corneal cell
loss, we designed an approach based on optimized gene expression (cotranslational import of the therapeutic protein by targeting the mRNA to the
mitochondrial surface) to prevent visual loss in Hq mice. We expect that subretinal
or intracameral administration of AAV2-AIF in 2-3 month old mice will preserve
cell integrity and prevent complex I defect.
Commercial Relationships: Aicha Bouaita, None; Sbastien Augustin,
None; Christophe Lechauve, None; Hlne Cwerman-Thibault, None; Hong
Liang, None; Franoise Brignole-Baudouin, None; Jos-Alain Sahel,
None; Marisol Corral-Debrinski, None
Support: INSERM,CNRS,ANR,SANOFI-FOVEA
Evaluation Of AAV-DJ Vector As A Therapeutic Delivery System For Ocular
Cells And Tissue
Matthew Hartzell, Maria Parker, Andrew Stempel, Trevor McFarland, Binoy
Appukuttan, John T. Stout. Opthalmology, Oregon Health and Science University,
Portland, OR.
Purpose: Gene therapy for the treatment of Leber Congenital Amerosis (LCA)
using an AAV vector is currently under a Phase I trial. The development of adenoassociated viral (AAV) vectors that can deliver to multiple ocular cell types with
high efficiency via subretinal or intravitreal delivery methods is essential for
effective therapy. The AAV-DJ (type2/type8/type9 chimera) was engineered from
shuffling 8 different wild type native viruses (Kay et al 2008). The goal of this
study is to investigate whether the chimeric AAV-DJ-GFP vector is capable of
transducing a variety of ocular cell types in vitro and in vivo.
Methods: The chimeric AAV- DJ vector serotype (AAV-DJ) encoding enhanced
green fluorescent protein (GFP) was produced (Vollum Viral Core). One microliter
of virus with a titer of 4x10e12 GC/ml was added to the following cell lines: HEK
293T, mouse Muller glia (C57MIO), human Muller glia (MIO-M7), primary
human and primary pig trabecular meshwork, human retinal pigment epithelial
(ARPE 19), rhesus retinal/choroidal endothelial (RF/6A), human primary iris
endothelial /fibroblast, retinoblastoma cells (Y79), human choroidal endothelial
and ocular melanoma (Mel 202). Cells were cultured and GFP expression was
measured by fluorescent microscopy at 18 and 42 hours.
1.5ul of AAV-DJ was injected intravitreally in each eye of a C57BL/6 mouse. Mice
were euthanized on post-injection day 5. Retinal flat mounts were prepared. GFP positive cells were viewed using fluorescent microscopy.
Results: With the exception of Y79 retinoblastoma cells, all cell types cultured
were transduced with high efficiency and GFP expression was observed at all time
points. Multiple GFP positive cells were observed within the neural retina after
intravitreal injection.
Conclusions: The 2-8-9 chimera AAV-DJ vector can transduce multiple ocular
derived cells in culture. The AAV-DJ vector has the potential to be useful for
ocular cell studies as well as gene therapy experiments.
Commercial Relationships: Matthew Hartzell, None; Maria Parker,
None; Andrew Stempel, None; Trevor McFarland, None; Binoy Appukuttan,
None; John T. Stout, None
Support: NIH;NEI;RO1 EY019042
Gene therapy for Dominant Optic Atrophy: a first pre-clinical trial on the
OPA1delTTAG mouse
Guy Lenaers, Marie Seveno, Lucie Elzire, Emmanuelle Sarzi, Vasiliki Kalatzis,
Christian P. Hamel. Institut des Neurosciences de Montpell, INSERM U1051,
Montpellier Cedex 5, France.
Purpose: Dominant Optic Atrophy (DOA) is an inherited mitochondrial disease
affecting the Retinal Ganglion Cells (RGCs), caused by mutations in one allele of
the OPA1 gene, encoding an intra mitochondrial dynamin. It is now well accepted
that in non-syndromic patients, haplo-insufficiency is the primary
pathophysiological mechanisms. Our pre-clinical project aims to obtain the proof of
principle that the micro-injection of an AAV2 vector expressing OPA1 in an Opa1
mouse model can prevent RGC degeneration and the evolution of the vision
deficiency.
Methods: We have constructed a new Opa1 mouse model with the
c.2708delTTAG mutation in exon27, that reproduces the most frequent mutation
found in patients with DOA (30% of all cases), and shown that it progressively
looses RGCs. We have constructed an AAV2-pOPA1 vector including the human
OPA1 minimal promoter and cDNA, and performed micro-injection in 2 and 9
months old Opa1 and wild-type animals. Vision parameters (Eye fundus, ERG,
VEP, Visual acuity) were followed every two months.
Results: Analysis and follow-up of 2 months old animals micro-injected with the
AAV2-pOPA1 vector and with a control AAV2-eGFP vector showed that: 1) the
surgery is not armful for the animal vision, 2) the expression of the eGFP is easily
detectable by fluorescent eye-fundus examination, 3) whereas the specific
expression of the human OPA1 protein can difficultly be differentiated from the
endogenous Opa1 expression, 4) visual parameters (VEP and visual acuity) were
significantly decreased in Opa1 animals, 5) treatment with the AAV2-pOPA1
vector has not yet induced a significant improvement of Opa1 mouse vision, after a
9 months follow-up. Immuno-histological analysis of the retina and optic nerve are
in process. 9 months old micro-injected animals are under investigation.
Conclusions: We have developed the concept of gene therapy for Dominant Optic
Atrophy and consequently set up the different tools to perform eye surgery and
analyse the possible consequences of AAV2 micro-injections on the visual
parameters. Although we can expect that the ectopic expression of human OPA1
can rescue the haplo-insufficiency found in Opa1 mouse model, our actual results
require further observations to confirm this hypothesis.
Commercial Relationships: Guy Lenaers, None; Marie Seveno, None; Lucie
Elzire, None; Emmanuelle Sarzi, None; Vasiliki Kalatzis, None; Christian P.
Hamel, None
Support: Association Franaise contre les Myopathies
LCA Gene Therapy In Somatic-Cell-Derived Induced Pluripotent Stem Cells
Erin R. Burnight, Emily E. Kaalberg, Bonita L. Moses, Jennifer A. Halder, Heather
T. Daggett, Robert F. Mullins, Edwin M. Stone, Budd A. Tucker. Ophthalmology
and Visual Sciences, University of Iowa, Iowa City, IA.
Purpose: Mutations in the CEP290 gene are major contributors to Leber
Congenital Amaurosis (LCA), the most severe form of inherited retinal
degenerative disease. CEP290-associated LCA is in herited in an autosomal
recessive manner and is thus a good candidate for gene-replacement therapy.
Reprogrammed somatic cell technologies provide researchers with the ability to
study human disease and therapeutic correction in vitro. The purpose of this study
is to generate iPSCs and subsequently photoreceptor precursor cells from a mouse
model of and patients with LCA. These cells will be used for the study of
therapeutic gene correction in iPSCs.
Methods: Fibroblast-derived iPSCs were generated from the retinal degenerative
mouse model CEP290rd16 and patients with molecularly confirmed CEP290associated LCA using a lentiviral reprogramming vector. To determine if cells were
fully reprogrammed, iPSCs were examined for the presence of pluripotency marker
transcripts and proteins. Mouse and human iPSCs were differentiated into
photoreceptor precursors using our previously developed step-wise differentiation
protocol. Lentiviral vectors expressing GFP under the control of retina-specific
promoters were developed to generate reagents for cell-specific therapeutic

transgene expression.
Results: CEP290-associated LCA mouse and human iPSCs were generated and
assessed for pluripotency marker expression. rt-PCR demonstrated that iPSCs
expressed the pluripotency markers Nanog, c-Myc, Klf4, Sox2, and Oct4.
Immunocytochemical analysis confirmed pluripotency marker expression.
Immunocytochemistry with antibodies targeted against OTX2 and Blue Opsin
showed successful generation of retinal progenitor cells (RPCs) and photoreceptor
precursors, respectively. Lentiviral vectors expressing GFP from the retinalspecific promoters OTX2, CRX, NRL, and RhoK were generated and tested in
primary human RPCs.
Conclusions: We show successful somatic cell reprogramming and differentiation
of mouse and human CEP290-associated LCA iPSCs into photoreceptor
precursors. Lentiviral vectors expressing human CEP290 from retina-specific
promoters have been generated and will be used in subsequent gene replacement
therapies and transplantation studies. This work will contribute to our overall goal
of using both gene and cell based therapies to restore vision in patients with LCA.
Commercial Relationships: Erin R. Burnight, None; Emily E. Kaalberg,
None; Bonita L. Moses, None; Jennifer A. Halder, None; Heather T. Daggett,
None; Robert F. Mullins, None; Edwin M. Stone, None; Budd A. Tucker, None
Support: Grousbeck Family Foundation
Circadian Rhythm in a Photo-Insensitive Mouse Model with Retinal
Expression of Exogenous Light-Gated Ion Channels
Trevor S. Lee1A, Deniz Dalkara1A, Leah Byrne1A, Stephan G. Jarjisian1B, Meike
Visel1A, Lance J. Kriegsfeld1B, John G. Flannery1A. AHelen Wills Neuroscience
Institute, BDepartment of Psychology and Helen Wills Neuroscience Institute,
1
University of California, Berkeley, CA.
Purpose: Gnat1-/-, Cnga3-/-, Opn4-/- triple-knockout (TKO) mice exhibit
disrupted signal transduction in their rod-, cone- and melanopsin-based
photoreception pathways. The activity-rest patterns of these mice have been shown
to be insensitive to environmental light-dark conditions and their circadian rhythm
(CR) does not entrain even to intense illumination. In this study, we investigate the
potential for therapeutic recovery of light-induced CR modulation via AAVmediated expression of light-gated ion channels (LGICs) in retinal ganglion cells
(RGCs) of TKO mice.
Methods: TKO mice were injected either (1) intravascularly as neonates, with an
AAV9 vector or (2) intravitreally at P15, with an AAV2 vector. The vectors
delivered cDNA encoding for GFP-conjugated I107V-mutated Chimera EF
(ChIEF) or an engineered photoswitch-coupling iGluR6 channel (LiGluR), driven
by hSyn promoter.
Wheel-running actograms were recorded from singly-housed adult mice, under
bright constant light and 12-hour biphasic light-dark conditions. In the final phase
of behavior data collection, the mice were kept under constant dim red (nonentraining) light conditions before being light-pulsed at their circadian time 16 and
immediately perfused. Immunohistochemistry was performed to map retinal
expression of the exogenous LGICs and to measure neuronal activity in the
suprachiasmatic nucleus (SCN) via c-fos labeling.
Results: The treated mice predominantly failed to respond to the presented light
conditions. Some treated mice showed actogram phase shifts, but not entrainment,
coincident with the transition between light-cycle programs. Fluorescence labeling
revealed extensive expression of the exogenous LGICs across RGCs. However,
SCN c-fos levels were not elevated relative to uninjected TKO mice.
Conclusions: Broad expression of hSyn-driven LGICs across RGCs is insufficient
to enable CR photoregulation in genetically light-insensitive mice under the
illumination conditions tested. LiGluR may transiently influence CR regulation
under dramatic changes in bright light conditions, but confirmation was
confounded by photoswitch turnover and re-administration. The lack of SCN
activation could also indicate promoter or vector tropism incompatibility in the
intrinsically photosensitive RGCs or other cell types critical for CR
photoregulation.
Commercial Relationships: Trevor S. Lee, None; Deniz Dalkara, None; Leah
Byrne, None; Stephan G. Jarjisian, None; Meike Visel, None; Lance J.
Kriegsfeld, None; John G. Flannery, None
Support: NIH Grant EY016994 and the Foundation for Fighting Blindness
A Potential Therapy for Macular Degeneration by Modulating Complement
Regulatory Protein Expression in the Retinal Pigment Epithelium
Roxana A. Radu, Zhichun Jiang, Gabriel H. Travis, Shanta Sarfare.
Ophthalmology, UCLA/Jules Stein Eye Inst, Los Angeles, CA.
Purpose: Accumulation of autofluorescent A2E-lipofuscin pigments within retinal
pigment epithelium (RPE) cells is seen in several forms of macular degeneration,
including age-related-macular degeneration (AMD) and Stargardt disease. In a
recent study, using the Stargardt disease-mouse model, the abca4-/- mouse, we
observed for the first time increased oxidative stress and complement activation in
vivo due to accumulation of A2E-lipofuscin fluorophores. Paradoxically, this was
accompanied by reduced expression of negative complement regulatory protein
genes (CRPs) in the RPE cells. In the current study, we over-expressed one or more
CRPs in the RPE of the abca4-/- mouse. We hypothesized that these regulatory
proteins will protect RPE cells from inappropriate attack by the complement
system, and thereby prevent photoreceptor loss.
Methods: We prepared mouse and human recombinant adeno-associated viruses
(AAV) expressing various complement- and inflammatory-related protein genes.
The AAV-CRP genes were delivered to the subretinal space of the Balb/C (WT)
and albino abca4-/- (KO) mice, via a trans-scleral/trans-choroidal approach under
direct visualization. Control injections were performed with AAV-null or AAVGFP viruses. Fundus photographs were taken before, immediate and at different
time points following the subretinal injection. The expression levels for CRPs
genes were measured by qRT-PCR and complement activation was evaluated by
immunocytochemistry. Visual retinoids and lipofuscin pigments were quantitated
by high-performance liquid chromatography.
Results: Both WT and KO AAV-CRRY-injected mice showed several-fold
increase in CRRY expression levels compared to control eyes. The expression of
the targeted protein was confined to the RPE based on immunofluorescence
analysis. Surprisingly, modulating the CRRY expression levels in the KO RPE
cells lead to two-fold increase of other CRP genes such DAF1, CD59a and CD59b.
More importantly, over-expression of CRRY significantly reduces the C3 breakdown fragment (C3b) accumulation in the RPE cells by immunohistochemistry.
Conclusions: Preliminary data suggest that by modulating the ocular immune
response via CRP-gene-based therapy, we can enhance the RPE defensive
mechanisms against aberrant complement attack and chronic inflammation.
Ongoing analysis of the AAV-CRP-injected mice are focusing on retina histology
and photoreceptor function.
Commercial Relationships: Roxana A. Radu, None; Zhichun Jiang,
None; Gabriel H. Travis, None; Shanta Sarfare, None
Support: EY000331
Correction of Cryptic Splicing in Usher Syndrome Using Antisense
Oligonucleotides
Jennifer J. Lentz1, Francine M. Jodelka2A, Anthony J. Hinrich2A, Kate E.
McCaffrey3, Hamilton E. Farris1, Nicolas G. Bazan1, Dominik M. Duelli2B, Frank
Rigo4, Michelle L. Hastings2A. 1Neuroscience Center, LSUHSC, New Orleans, LA;
A
Cell Biology and Anatomy, BCellular and Molecular Pharmacology, 2Rosalind
Franklin University of Medicine and Science, North Chicago, IL; 3Cell Biology and
Anatomy, Rosalind Franklin University, North Chicago, IL; 4Isis Pharmaceuticals,
Carlsbad, CA.
Purpose: Usher syndrome (Usher) is the leading cause of combined blindness and
deafness. All Usher patients develop retinitis pigmentosa, with the age of onset, and
the severity of deafness and presence of vestibular defects differing among
subtypes. Patients with Usher 1 suffer retinitis pigmentosa beginning in early
adolescence with congenital deafness and vestibular dysfunction. An obstacle to
developing treatment strategies for the disease has been the lack of animal models
that develop both auditory and visual defects. Recently, a mouse model for Usher
that does exhibit both phenotypes was developed based on the human mutation in
the USH1C gene responsible for Usher type 1C. The Ush1c216AA knock-in mice
exhibit retinal degeneration that begins after deafness and vestibular dysfunction.
Abnormal electroretinograms (ERGs) are evident as early as 1 month of age,
however, the loss of rod photoreceptors begins between 6 and 12 months of age.
The Ush1c.216G>A (c.216G>A) mutation introduces a cryptic 5 splice site that is
used preferentially over the normal site, producing a truncated mRNA and protein
product. This mouse model provides a valuable tool to investigate therapeutic
strategies for Usher and other diseases associated with mutations in splice sites.
Antisense oligonucleotides (ASOs) are powerful tool that can be used to correct
aberrant splicing and may be a useful therapeutic approach to treat Usher.
Methods: Antisense oligonucleotides (ASOs) were used to block the c.216G>A
cryptic 5 splice site in vitro and in vivo. ASOs that most effectively blocked
cryptic splicing of Ush1c.216A minigene transcripts were subsequently tested in
cell lines generated from Usher1C patients (216AA) and the c.216AA mice. ASOs
were also injected into c.216AA neonatal mice and correction of splicing in the
retina and cochleae were quantitated by RT-PCR and western blot. Hearing and
visual function were evaluated by auditory-evoked brainstem response (ABR) and
ERG analyses, respectively.
Results: ASOs effectively blocked cryptic splicing and increased the amount of
normal splicing in an Ush1c.216A minigene system, in cells from 216AA Usher 1C
patients and the c.216AA mice. A single systemic treatment with ASOs to neonate
mice corrected splicing and protein expression in the retina and cochlea. ASOtreated mice had no circling behavior characteristic of the 216AA mice. Mice are
currently being evaluated for restoration of hearing and vision by ERG and ABR
analysis.
Conclusions: Our results demonstrate that ASOs can effectively block cryptic

splicing of the c.216A transcript in vivo. These results suggest the therapeutic
potential of ASOs in Usher syndrome and other diseases caused by mutations that
disrupt splicing.
Commercial Relationships: Jennifer J. Lentz, None; Francine M. Jodelka,
None; Anthony J. Hinrich, None; Kate E. McCaffrey, None; Hamilton E.
Farris, None; Nicolas G. Bazan, None; Dominik M. Duelli, None; Frank Rigo,
Isis Pharmaceuticals (E); Michelle L. Hastings, None
Support: NEI EY005121; Research to Prevent Blindness, Inc.
AAV-mediated Gene Therapy Restores Retinal Function And Vision In The
PDE6-deficient Dog
Lolita Monnier1, Elsa Lhriteau1, Michel Weber2, Guylne Le Meur2, Jack-Yves
Deschamps3, Nathalie Provost1, Lyse Libeau1,2, Caroline Guihal1, Philippe
Moullier1,4, Fabienne Rolling1. 1Institut de Recherche Therapeutique 1, Laboratoire
de Therapie Genique INSERM UMR 649, Nantes, France; 2Service
d'Ophtalmologie, CHU-Htel Dieu, Nantes, France; 3ONIRIS, Nantes-Atlantic
College of Veterinary Medicine, Food Science and Engineering, Emergency and
Critical Care Unit, Nantes, France; 4Department of Molecular Genetics and
Microbiology, University of Florida, College of Medicine, Gainesville, FL.
Purpose: Defects in the subunit of rod cGMP phosphodiesterase 6 (PDE6) are
associated with autosomal recessive retinitis pigmentosa (RP), a childhood blinding
disease with early retinal degeneration and vision loss. To date, there is no
treatment for this pathology. Previous studies have tested gene replacement therapy
in the rd10 mouse model of PDE6-RP using adeno-associated virus (AAV)
serotype 5 or 8 vectors expressing murine pde6. They documented restoration of
rod function and preservation of retinal morphology. The aim of our preclinical
study was to test AAV-mediated gene replacement therapy in the rod-cone
dysplasia type 1 (rcd1) dog, a naturally occurring large animal model of PDE6
deficiency that strongly replicates the human pathology.
Methods: We generated AAV2/5 and AAV2/8 vectors expressing canine pde6
cDNA under the control of the photoreceptor-specific rhodopsin kinase promoter.
One hundred microliter of each vector was subretinally injected to one eye of four
rcd1 dogs. The contralateral eye remained uninjected. Retinal morphology was
assessed by fundus imaging, optical coherence tomography, histology and
immunohistochemistry. Retinal function was tested on all treated dogs using
bilateral electroretinography (ERG) at 1, 3, 6, 9 and 12 months after treatment.
Moreover, vision was evaluated in all animals using behavioral testing under dim
light conditions.
Results: In vivo and post-mortem morphological analysis showed a significant
preservation of the retinal structure in transduced areas of both
AAV2/5hRK.cpde6- and AAV2/8hRK.cpde6-treated retinas. In all treated eyes,
substantial rod-derived ERG signals were recorded as soon as 1 month postinjection (30% of normal eyes) and remained stable for at least 12 months (duration
of the study). They were undetectable in untreated contralateral eyes. Most
importantly, dim light vision was restored in all treated dogs.
Conclusions: These results demonstrate for the first time that gene therapy
effectively restores long-term retinal function and vision in a large animal model of
progressive photoreceptor defects, the rcd1 dog.
Commercial Relationships: Lolita Monnier, None; Elsa Lhriteau,
None; Michel Weber, None; Guylne Le Meur, None; Jack-Yves Deschamps,
None; Nathalie Provost, None; Lyse Libeau, None; Caroline Guihal,
None; Philippe Moullier, None; Fabienne Rolling, None
Support: Association Franaise contre les Myopathies (AFM), Ministre de
l'Enseignement Suprieur et de la Recherche
AAV-mediated Rescue Of Rod Photoreceptors In The Rd3 Mouse Model Of
Leber Congenital Amaurosis Type 12 (LCA12)
Laurie L. Molday1A, P. Yan1A, H Djajadi1A, L. Szczgiel2A, S. Boye3, V. Chiodo3, M.
Sarunic2B, K. Gregory-Evans1B, W. W. Hauswirth3, R S. Molday1A. ABiochemistry
& Molecular Biology, BDept. of Ophthalmology & Visual Sciences, 1University of
British Columbia, Vancouver, BC, Canada; ADepts of Molecular Biology &
Biochemistry, BDept of Engineering Science, 2Simon Fraser Univ, Vancouver, BC,
Canada; 3Dept. of Ophthalmology, Univ. of Florida, Gainesville, FL.
Purpose: Mutations in RD3, a 23 kDa protein required for the stable expression
and trafficking of guanylate cyclases (GC1 and GC2) in photoreceptor cells, are
responsible for retinal degeneration in LCA12 patients, the rd3 mouse, and the rcd2
collie. The purpose of this study was to determine if AAV-mediated delivery of the
mouse Rd3 gene can prevent photoreceptor degeneration in the rd3 mouse.
Methods: AAV8(Y733F) containing the mouse Rd3 cDNA under control of the
rhodopsin kinase promoter (hGRK1) was injected into the subretinal space of the
right eyes of P14 rd3 mice; the uninjected left eyes served as controls. Eyes were
evaluated at various times post-injection by electroretinography (ERGs), optical
coherence tomography (OCT), histology, immunocytochemistry and SDS gels.
Results: GC1 and GC2 expression was observed in rods and cones of the Rd3AAV treated eye 7 days post-injection. The level of expression increased with time,
and at 1 month post-injection GC1 and GC2 expression was observed in the outer
segment layer which persisted for at least 4 months. The outer nuclear layer (ONL)
thickness in the treated eye stabilized at about 60% that of wild-type mice, although
a gradual loss in cones was evident. In the untreated eye, both rods and cones
degenerated rapidly with essentially complete loss in the ONL by 4 months. The
scotopic ERGs were maintained in the treated eyes, but rapidly disappeared in the
untreated eyes. Photopic ERGs were absent in both the treated and untreated eye at
all times.
Conclusions: Rd3-AAV gene replacement therapy restored GC1 and GC2
expression and outer segment localization in rod cells of the rd3 mouse. GC
expression resulted in sustained scotopic ERGs and rod cell survival. Although
GC1 was detected in cones after treatment, the photopic ERG remained
undetectable and progressive cone cell degeneration was observed.
Commercial Relationships: Laurie L. Molday, None; P. Yan, None; H.
Djajadi, None; L. Szczgiel, None; S. Boye, None; V. Chiodo, None; M. Sarunic,
None; K. Gregory-Evans, None; W. W. Hauswirth, AGTC. inc (P); R. S.
Molday, None
Support: CIHR/IRSC, Macular Vision Research Foundation, Foundation Fighting
Blindness, Canada
Cone Targeted AAV-mediated Gene Therapy Restores Cone Function in the
Cngb3 Knockout Mouse, a Model of Human Achromatopsia 1
Wei Shi1,2, Song Mao2, Wentao Deng2, Jie Li2, Xuan Liu2, Sanford L. Boye2, Guojie Ye3, Willaim W. Hauswirth2, Ji-jing Pang2. 1Beijing Tongren Eye Center,
Beijing, China; 2Ophthalmology, University of Florida, Gainesville, FL; 3Applied
Genetic Technologies Corporation, Alachua County, FL.
Purpose: Mutations in the gene encoding the beta-subunit of the cone cyclic
nucleotide-gated channel (CNGB3) cause cone function loss in mammals including
humans. We tested two AAV5-hCngb3 vectors with different cone targeting
promoters to see if gene replacement therapy would result in restoration of cone
function in the Cngb3 knockout mice, a model of human Achromatopsia 1
(ACHM1).
Methods: Human CNGB3 cDNA in conjuction with cone-targeting promoter
mCARpro or IRBP/GNAT2 was packaged into AAV serotype 5 (AAV5mCARpro-hCngb3 or AAV5-IRBP/GNAT2-hCngb3 at1013 viral genomecontaining particles /ml). At postnatal day 14, 1 l of either vector was injected
subretinally into one eye of groups of 20 Cngb3 knockout mice, respectively. The
untreated, contralateral eyes served as controls. Dark- and light-adapted ERGs were
recorded periodically from 6 weeks to 6 months after treatment. 6 months after
injection, both treated and control eyes were harvested for histochemical studies.
Results: At 6 weeks post-treatment both treated and untreated eyes of Cngb3
knockout mice showed normal rod-derived ERGs. In untreated control eyes, conederived ERG signals were nearly unrecordable. In both AAV5-mCAR-hCngb3 and
AAV5-IRBP/GNAT2-hCngb3 treated eyes, restored light-adapted cone-derived
ERG waveforms were first recorded 6 weeks after treatment and remained stable
for at least 6 months. ERG amplitudes were about 2/3 of those of normal uninjected
C57BL/6J mice. Immunohistochemistry showed human CNGB3 staining in the
inner segments of many cones in treated eyes but not in cones from partner
untreated eyes. Anti-M-cone or S-cone opsin staining also showed that S-opsins
were preserved in treated eyes but not in untreated eyes of Cngb3 knockout mice.
Conclusions: Both AAV5-mCAR-hCngb3 and AAV5-IRBP/GNAT2-hCngb3
restore cone function and prevent S-cone degeneration for at least 6 months in
Cngb3 knockout mice, a model of ACHM1. However since studies in an
accompanying abstract show that in addition to cones, mCARpro in AAV5 also
expresses its transgene in the RPE while the IRBP/GNAT2 promoter is coneexclusive, the latter may be preferable for future studies in humans.
Commercial Relationships: Wei Shi, None; Song Mao, None; Wentao Deng,
None; Jie Li, None; Xuan Liu, None; Sanford L. Boye, Applied Genetic
Technologies Corporation, UF#13859 (P); Guo-jie Ye, AGTC (E); Willaim W.
Hauswirth, AGTC (P); Ji-jing Pang, None
Support: NIH grant EY021721 and grants from the FFB, MVRF, and RPB, Inc.
Trans-splicing of Rhodopsin mRNA: Modeling and Therapeutic Strategy for
Retinitis Pigmentosa
Adeline Berger1, Stphanie Lorain2, Melissa Desrosiers1, Peggy Fabre1, Ccile
Peccate2, Thomas Voit2, Luis Garcia2, Jos-Alain Sahel1, Alexis-Pierre
Bemelmans1. 1Institut de la Vision, INSERM/UPMC Univ Paris 06/CNRS/CHNO
des Quinze-Vingts, Paris, France; 2Institut de Myologie, INSERM/UPMC Univ
Paris 06, Paris, France.
Purpose: To implement new gene therapy strategies for autosomal dominant
Retinitis Pigmentosa, we applied Spliceosome-Mediated RNA Trans-splicing to

correct mutations of the rhodopsin (RHO) gene. This technology promotes a
splicing event in trans between the endogenous mutated rhodopsin pre-mRNA and
an exogenous pre trans-splicing mRNA (PTM) containing a partial RHO cDNA
devoid of mutation. Targeting the first intron of the RHO gene should thus allow
the repair of any mutations present in exon 2 and the following exons. A major
advantage of this approach is that the expression level of the "repaired" protein
depends only on the endogenous gene expression regulation.
Methods: We constructed several vectors expressing wild-type or P347L alleles of
the human RHO gene driven by an ubiquitous promoter. We introduced these
vectors in HEK293 cells to create cellular models of RHO mutation. We then
designed three different PTM targeting the first RHO intron. We evaluated these
PTM in our cellular model, using a silent mutation inserted in the exogenous
mRNA which allows us to precisely quantify trans-splicing efficiency by
conventional molecular biology techniques (PCR and restriction digest).
Results: After transient transfection of vector encoding whole rhodopsin gene, we
observed by flow cytometry and immunocytofluorescence an expression of wildtype or P347L RHO in approximately 80% of the cells. Among the three PTM
tested so far, one has no activity, one leads to a trans-splicing efficiency of 12% of
RHO mRNA, whereas the most efficient one reached a trans-splicing efficiency of
34%. The level of trans-splicing was equivalent for wild-type and P347L alleles,
confirming that trans-splicing occurred independently from the mutation. The
phenotype of cells expressing wild-type or P347L alleles with or without transsplicing is currently being characterized.
Conclusions: We have shown that Spliceosome-Mediated RNA Trans-splicing
allows to partially repair RHO mutation at the transcriptional level in a cellular
model. The PTM showing the best activity in vitro will be introduced into an AAV
vector to be tested in vivo in an animal model expressing a mutated RHO allele. If
efficient, this new tool will be directly applicable to patients.
Commercial Relationships: Adeline Berger, None; Stphanie Lorain,
None; Melissa Desrosiers, None; Peggy Fabre, None; Ccile Peccate,
None; Thomas Voit, None; Luis Garcia, None; Jos-Alain Sahel, None; AlexisPierre Bemelmans, None
Support: Association Franaise contre les Myopathies
First Steps Towards Development Of A New Non-viral Gene Therapy, Based
On The Overexpression Of Anti-angiogenic Peptides For The Treatment Of
Diabetic Retinopathy.
Anna Salas Torras1, Andrea R. Carvalho1, Miguel A. Zapata Victori2, Laura
Distefano2, Sim Schwartz Navarro3, Jos Garca-Arum2. 1Ophthalmology, Vall
d'Hebron Research Institute, Barcelona, Spain; 2Ophthalmology, Hospital Vall
d'Hebron, Barcelona, Spain; 3CIBBIM-Nanomedicine, Vall d'Hebron Research
Insitute, Barcelona, Spain.
Purpose: The aim of the study is to evaluate the use of minicircle technology for
the expression of two anti-angiogenic factors, pigment epithelium-derived factor
(PEDF) and somatostatin (SST), for non-viral gene therapy of diabetic retinopathy
Methods: Three plasmid vectors carrying either PEDF, SST, or green fluorescent
protein (GFP) cDNA were produced by insertion into the expression cassette of the
pMC.BESPX vector, between the attP and attB sites. The vectors were
transformed into the E.coli strain ZYCY10P3S2T and the growth media was
supplemented with L-arabinose to induce formation of minicircles. The products,
consisting of DNA vectors devoid of the bacterial backbone, were analyzed by
restriction digest and were tested for in vitro transfection efficiency using the
retinal pigment epithelium cell line ARPE-19 and lipofectamine. GFP expression
was evaluated by direct visualization by fluorescence microscopy, and SST and
PEDF expression was analyzed by Western blot of cell lysates and conditioned
culture media. GFP minicircles were also tested in vivo by subretinal injection with
lipofectamine in a rat model. GFP expression was evaluated by direct observation
of whole retina under a confocal microscope.
Results: Restriction analysis of minicircle induction products showed bands
corresponding to the minicircle vectors for each gene, demonstrating the
elimination of the plasmid backbone in all the cases compared with the precursor
vectors. Transfection studies in the ARPE-19 cell line with the minicircles were
positive in all three cases: transfection with GFP minicircles produced green cells,
with the same transfection efficiency as that of the conventional plasmid; the cell
lysates and the culture supernatants from the ARPE-19 cells transfected with PEDF
and SST minicircles, analyzed by Western blot, showed a higher expression of the
peptides in both cases compared with the control non-transfected cells. The in vivo
transfection study of GFP minicircles in the rat model, analyzed at 72h posttransfection, showed significant expression of green fluorescent protein in the
transfected retinas compared with the control eye, where there was no fluorescence.
Conclusions: Our data suggest that minicircle vectors are capable of expressing
high and persistent levels of therapeutic product in vitro and in vivo in the retina
and have a great potential to serve as episomal vectors for the treatment of diabetic
retinopathy.
Commercial Relationships: Anna Salas Torras, None; Andrea R. Carvalho,

None; Miguel A. Zapata Victori, None; Laura Distefano, None; Sim Schwartz
Navarro, None; Jos Garca-Arum, None
Support: ISCIII PI11/00706
Dual AAV vector system for Usher syndrome (USH1B) gene therapy
Frank M. Dyka, Shannon E. Boye, Sanford L. Boye, Vince A. Chiodo, William W.
Hauswirth. Ophthalmology, University of Florida, Gainesville, FL.
Purpose: Usher syndrome type 1B is a severe autosomal recessive deaf-blindness
disorder caused by mutations in MYOSINVIIa (MYO7A). MYO7A is expressed in
photoreceptors and retinal pigment epithelium (RPE). Due to the relatively large
size of the coding gene (6.5 kb), lentiviral vectors have been used to deliver
MYO7A transgene to a mouse model of USH1B. However, lentivirus does not
efficiently transduce photoreceptors. Adeno-associated viral (AAV) vectors are
capable of transducing both photoreceptors and RPE, however AAV has a
relatively small packaging capacity (~5kb), thus standard AAV is unsuitable for
gene replacement therapy for USH1B. We have previously shown that full length
MYO7A expression is achieved using fragmented/heterogeneous AAV vector
system. However, due to the inability to characterize discrete genetic payloads
contained within, this system is unlikely to gain FDA approval. The purpose of this
study is to develop a dual AAV vector system with defined genetic components
that is capable of delivering full length MYO7A to cells in vitro and in vivo.
Methods: Human MYO7A was cloned in AAV vector pairs, where one vector
contains a hybrid CMV/chicken actin promoter and the 5 portion of MYO7A
cDNA sequence and a second vector contains the 3 portion followed by a polyA
signal. Three vector pairs were constructed, where one shares an overlapping
sequence of 1350 bp of coding sequence. The other pairs utilize either natural
splice sites and intron of MYO7A, or splice donor and acceptor sites of alkaline
phosphatase. All vectors were packaged in AAV2 and titer matched for infection of
HEK293 cells (10,000 MOI each). Cells were collected 3 days post infection for
analysis on immunoblot.
Results: Co-infection with dual MYO7A AAV vectors expressed full length
MYO7A in vitro with equal or higher efficiency than that of the heterogeneous
vector. Of the dual vector systems tested, the overlapping system was the most
efficient and mediated expression of only full length MYO7A.
Conclusions: The results show that MYO7A can be expressed using dual AAV
vector systems. The system containing overlapping portions of MYO7A was both
highly efficient and specific. Because AAV has emerged as the most preferred
clinical vector, and because it efficiently transduces both photoreceptors and RPE,
our results suggest that a dual AAV vector system may be an option for the
treatment of USH1B.
Commercial Relationships: Frank M. Dyka, 61/560,437 (P); Shannon E.
Boye, 61/560,437 (P); Sanford L. Boye, 61/560,437 (P); Vince A. Chiodo, None;
William W. Hauswirth, 61/560,437 (P), AGTC, Inc. (I)
Support: FFB-myosin7a gene therapy for USH1B
AAV-Mediated Delivery of RdCVF and RdCVFL in a Mouse Model of Retinal
Degeneration
Leah C. Byrne1, Deniz Dalkara2, Emmanuelle Clerin3, Steven K. Fisher4, Jose A.
Sahel, Jr.3A, Thierry D. Leveillard5, John G. Flannery6. 1Helen Wills Neuroscience,
UC Berkeley, Berkeley, CA; 2Helen Wills Neuroscience, Univ of California,
Berkeley, Berkeley, CA; AUMR-S 968, 3Institut de la Vision, Paris, France;
4
Neuroscience Research Institute, Univ of California Santa Barbara, Santa Barbara,
CA; 5Genetique, Institut De La Vision, Paris, France; 6Helen Wills Neuroscience
Institute, University of California, Berkeley, Berkeley, CA.
Purpose: Rod-derived cone viability factor (RdCVF), a truncated thioredoxin-like
protein, is secreted and has been shown to have a protective effect on the survival
of cones in rodent models of rod-cone dystrophy, while RdCVFL, a full length
alternative splicing product encoded by the same gene, contains a thioredoxin fold
and may be involved in oxidative signaling. RdCVFL has been shown to interact
with the microtubule binding protein Tau, and in vitro to protect against
phosphorylation of Tau through oxidative stress. Here we evaluate the effects of
AAV-mediated delivery of RdCVF and RdCVFL in the rd10 mouse model of
retinal degeneration.
Methods: AAV vectors were used to deliver RdCVF or RdCVFL in neonatal rd10
mice via tail vein injections or via intravitreal injections at P14. A CAG promoter
drove ubiquitous expression of RdCVF or RdCVFL while a rho promoter restricted
expression of RdCVFL to photoreceptors. A viral 2A peptide was used to
simultaneously allow expression of a fluorescent reporter gene. Electroretinograms
were recorded to evaluate rescue of rod and cone-mediated responses. The density
of cones was quantified to determine the effect of expression on cone survival.
Western blotting was used to observe the effects of expression of RdCVFL on
levels of Tau phosphorylation.

Results: Injection of AAV-RdCVF and RdCVFL resulted in high levels of protein
expression. Delivery of RdCVF mediated functional rescue of cone photoreceptors
while delivery of RdCVFL resulted in a delay in the loss of the rod-driven scotopic
ERG, indicating its involvement in protecting rods.
Conclusions: Nxnl1 is a bifunctional gene encoding two alternative splicing
products. AAV-mediated delivery of these two products, RdCVF and RdCVFL,
mediates high levels of protein expression and rescue of photoreceptors in the
retina. RdCVF promotes survival of cones while the expression of RdCVFL delays
rod degeneration. The synergistic effect of co-expression of these two proteins is
being evaluated.
Commercial Relationships: Leah C. Byrne, None; Deniz Dalkara,
None; Emmanuelle Clerin, None; Steven K. Fisher, None; Jose A. Sahel, Jr.,
Patent holder on the use of RdCVF (P); Thierry D. Leveillard, Patent holder on
the use of RdCVF (P); John G. Flannery, None
Support: Bourse Chateaubriand, NIH Grant EY016994, and the Foundation for
Fighting Blindness
Overexpression of the NDUFA6 Subunit of Complex I Ameliorates
Neurodegeneration in Experimental Optic Neuritis
Jianwen Liu, Venu Talla, Sacide S. Ozdemir, Tsung-Han Chou, Vittorio Porciatti
Porciatti, John Guy. Bascom Palmer Eye Institute, University of Miami, Miami,
FL.
Purpose: We have previously shown that peroxynitrite mediates nitration of
nuclear-encoded complex I subunit NDUFA6 in the EAE animal model of MS and
that genetic knockdown of another supernumerary complex I subunit (NDUFA1)
mediates neurodegeneration that contributes to the permanent disability in optic
neuritis and MS. Here we overexpressed NDUFA6 to rescue visual loss and optic
neuropathy.
Methods: EAE was induced in female DBA/1J mice (n=20). Ten mice were
rescued by intravitreal injection of scAAV-NDUFA6 to which a FLAG epitope was
attached to the C terminus. Ten controls were injected with cherry to which a
mitochondrial targeting sequence (COX8) was appended to the N terminus
(scAAV-COX8-mcherry). Another group of 10 mice were injected with scAAVCOX8-mcherry but were not sensitized for EAE. Serial PERG and OCT evaluated
visual function and structure of the inner retina at 1, 3 and 6 months post injection
(MPI). All mice were sacrificed 6MPI for histopathology. Expression of NDUFA6FLAG in the retina and ONs were evaluated at 15 days post injection by RT-PCR,
immunofluorescence (IF) and western blotting (WB).
Results: Expression: Immunofluorescence revealed a typical punctate and
perinuclear expression of NDUFA6FLAG that co-localized with mitochondrial
porin and RGC thy1.2. RT-PCR and WB confirmed NDUFA6FLAG
overexpression in the retina and ONs. Rescue: PERG analysis at 3M and 6MPI
showed a 42% and 45% reduction in amplitude of EAE-mCherry compared to
control mCherry (p<0.005,). NDUFA6 injection rescued this amplitude by 100%
and 77.6% respectively (p<0.05). PERG latency was delayed by 15% and 21% in
EAE-mCherry compared to mCherry control (p<0.05), whereas the NDUFA6
injected mice rescued the delay by 100% and 76% respectively at 3M and 6MPI.
OCT images showed a significant thinning in EAE-mCherry retina compared to
unsensitized mCherry animals at 3M (16%) and 6MPI (15%) p<0.05, whereas
NDUFA6 rescued this thinning by 100% (p<0.05). The ultrastructural analysis of
the EAE ONs demonstrated different levels of degeneration in axon, myelin and
connective tissues. Degenerated axons showed a varied mitochondrial number,
shape and morphology. NDUFA6 ONs showed a relatively continuous myelin with
organized microtubules and normal looking mitochondria in the axons.
Conclusions: NDUFA6 gene therapy suppressed RGC degeneration and optic
neuropathy in experimental optic neuritis suggesting that it may also ameliorate
neurodegeneration in optic neuritis and MS patients.
Commercial Relationships: Jianwen Liu, None; Venu Talla, None; Sacide S.
Ozdemir, None; Tsung-Han Chou, None; Vittorio Porciatti Porciatti,
None; John Guy, None
Support: EY07982
A Novel Approach For Sustained Treatment Of Neovascularization By Flt23k
Integration Into Genome
Subrata K. Das, xiaohui zhang, Ling Luo, Hironori Uehara, Nirbhai Singh, Bonnie
Archer, Balamurali K. Ambati. Ophthalmology, Moran Eye Center, Salt Lake City,
UT.
Purpose: Vascular endothelial growth factor A (VEGF-A) and its receptors play
important roles in neovascularization. VEGFR1 (also known as Flt-1) is the most
potent receptor among others. Flt23K receptor (FLT domains 2 and 3 tagged with
KDEL) is able to bind and sequester VEGF-A and can be used to suppress
neovascularization. The delivery and long-term incorporation of the Flt23K is a
major hurdle. Our objective is incorporation of the Flt23K gene into the genome by
transpositional insertion, leading to long term reduction of neovascularization in

response to an injury without the need for repeated intraocular injections.
Methods: To achieve genomic integration of Flt23K gene, we utilized transposon
based helper-independent piggybac plasmid (piGENIE). Flt23K and DSRed
Express 2 cDNA were cloned in piGENIE. The plasmid lacking transposase (pBt
gene) gene was used as a control. To examine genomic integration and expression
of transgenes in vitro, we transfected the plasmids into HeLa cells and cultured up
to 10 passages. Fluorescence microscopy was done to check the transfection
efficiency with DS Red fluorescence. Also Flt23K expression was examined by
western blot with Anti-VEGF Receptor 1 antibody. Genomic integration was
analyzed using PCR based genome walking approach. For in-vivo study we used
laser induced retina model. Plasmids with 10% neuroporter were injected into the
vitreous space of C57BL/6J mice. Choroidal neovascularization (CNV) was
induced by laser photocoagulation one month after plasmid injection. CNV was
stained with Isolectin GS-IB4 one week after laser injury and CNV volume was
calculated with the confocal microscope software.
Results: Genome walking study in transfected HeLa cell indicated insertion of the
transgenes into genome. HeLa cells transfected with control plasmid did not
indicate any insertion of transgenes. Flt23K protein (25kDa) was detected only in
transfected HeLa cell lysate but not in control. CNV volume was sharply reduced
by 53% in piggybac injected eyes compared to control.
Conclusions: Our results indicate that nonviral gene therapeutic approach based on
Flt23K expression in invitro cell model was significantly pronounced and showed
distinctly reducing effective in choroidal neovascularization in C56BL/6J Mouse
model. Our approach can be extrapolated as novel way to treat pathological
neovascularization by long term expression of Flt23K.
Commercial Relationships: Subrata K. Das, None; xiaohui zhang, None; Ling
Luo, None; Hironori Uehara, None; Nirbhai Singh, None; Bonnie Archer,
None; Balamurali K. Ambati, None
Interleukin-10 Gene-transfected Regulatory Dendritic Cells Suppress Murine
Experimental Autoimmune Optic Neuritis
Ryusaku Matsuda1,2, Takeshi Kezuka1, Chiharu Nishiyama2, Yoshihiko Usui1,
Yoshimichi Matsunaga1, Yoko Okunuki1, Naoyuki Yamakawa1, Hiroshi Goto1.
1
Ophthalmology, Tokyo Medical Univ Hospital, Shinjuku-ku, Japan; 2Atopy
Research Center and Department of Immunology, Juntendo University School of
Medicine, Bunnkyou-ku, Japan.
Purpose: We have previously reported that calcitonin gene-related peptidetransfected mature dendritic cells (mDCs) suppressed murine experimental
autoimmune optic neuritis (EAON) and experimental autoimmune encephalitis
(EAE) via IL-10 production. (ARVO, 2009) Here, we investigated whether IL-10
gene-transfected mDCs suppressed EAON and EAE.
Methods: C57BL6/J mice were immunized with an emulsified mixture of myelin
oligodendrocyte glycoprotein (MOG35-55) to establish the EAON-EAE mouse
model. A plasmid expressing murine IL-10 was constructed, bone marrow from the
C57BL6/J mice (with/without GFP expression) was cultured to generate mDCs,
and mDCs were transfected with the mouse IL-10 gene using electroporation. The
IL-10 gene-transfected mDCs were injected intravenously immediately following
immunization (induction phase). Mice were sacrificed at day 28 after immunization
and the spleen, lymph nodes, and optic nerve were resected for
immunohistochemical (IHC) evaluation. Using flow cytometry, we analyzed
CD80/86 and, MHC class 2 expression in the spleen and lymph node cells.
Results: In the treatment group which IL-10 gene-transfected mDCs were
administered, the numbers of CD80/86-positive and MHC class 2 positive cells
were significantly lower in the spleen and lymph nodes as compared to those in the
control group (P<0.05). IHC results revealed that GFP-expressing mDCs existed
not only in the spleen and lymph nodes but also in the inflamed optic nerve. IL-10
gene-transfected mDCs thus appeared to regulate inflammation via primary and
secondary lymph organs and also at the local inflamed site.
Conclusions: IL-10 gene-transfected mDCs thus effectively suppressed the
development of EAON by downregulating the co-stimulatory signal molecules
CD80/86 and MHC class 2.
Commercial Relationships: Ryusaku Matsuda, None; Takeshi Kezuka,
None; Chiharu Nishiyama, None; Yoshihiko Usui, None; Yoshimichi
Matsunaga, None; Yoko Okunuki, None; Naoyuki Yamakawa, None; Hiroshi
Goto, None
Support: None
Recovery of ERG Components in the Cngb1-/- Model of RP Following Gene
Therapy
Vithiyanjali Sothilingam1, Naoyuki Tanimoto1, Marina Garcia Garrido1, Regine
Muehlfriedel1, Susanne Koch2, Martin Biel2, Stylianos Michalakis2, Mathias W.

Seeliger1. 1Division of Ocular Degeneration, Ctr for Ophthal Inst for Ophthalmic
Research, Tuebingen, Germany; 2Pharmacy-Pharmacology, Ludwig-MaximiliansUniversity Munich, Munich, Germany.
Purpose: The lack of the CNG channel subunit CNGB1 leads to a loss of lightmediated electrical activity of rod photoreceptors and eventually to retinitis
pigmentosa (RP). In the respective Cngb1-/- mouse model, visual function and
retinal morphology could be restored using rAAV-mediated gene replacement
therapy. Here we assess contributions of photoreceptor populations with different
functional status (i.e. from treated and untreated regions within the same eye) on
the sum responses of the Ganzfeld electroretinogram (ERG).
Methods: Cngb1-/- knock-out mice were treated via subretinal injection of rAAV
vector expressing murine CNGB1 under control of a rhodopsin promoter. Animals
were injected at two weeks postnatally (PW2), and examined functionally and
morphologically at PW10. ERGs were recorded under both scotopic and photopic
conditions using a Jaeger/Toennies Multiliner Vision and a Roland Consult
Ganzfeld system. In vivo retinal morphology was examined with optical coherence
tomography (OCT, HE Spectralis), and animals subsequently underwent
histological analysis.
Results: Cngb1-/- knock-out mice feature a small rod response even in the absence
of the CNGB1 subunit (Huettl et al., J Neurosci 2005). The respective ERG
component present in both treated and untreated eyes is in agreement with the
hypothesis that homomeric CNG channels, composed of the CNGA1 subunit only,
reach the outer segment membrane in a very small number. In treated eyes, we
found an additional signal component from a proportion of rods responding with
practically regular timing and sensitivity, which we attribute to the rescue area.
Since the treated region in this study amounts to approximately 1/3-1/4 of the
retina, signals were much smaller than normal rod ERGs. This difference was also
reflected in the mixed rod and cone ERG responses.
Although the rod ERG was strongly reduced, the morphological appearance of the
retina was not much altered. The major difference in the OCT was a preservation of
outer segment markers (OLM and IS/OS border) in treated eyes of Cngb1-/- mice.
Conclusions: Here we show that gene replacement therapy in Cngb1-/- mice results
in photoreceptor populations with different functional status. The respective ERG
components will be of high diagnostic importance and may serve as endpoints in
treatment trials.
Commercial Relationships: Vithiyanjali Sothilingam, None; Naoyuki
Tanimoto, None; Marina Garcia Garrido, None; Regine Muehlfriedel,
None; Susanne Koch, None; Martin Biel, None; Stylianos Michalakis,
None; Mathias W. Seeliger, None
Support: DFG Se837/5-2 (KFO 134), Se837/6-2, Se837/7-1 (KFO 134), EU
HEALTH-F2-2008-200234
LentiVector Platform, a Highly Effective Equine Infectious Anaemia Virusbased Lentiviral Gene Therapy Platform for Ocular Disease
Scott Ellis, James Miskin, Katie Binley, Jackie de Belin, Julie Loader, Michelle
Kelleher, Stuart Naylor, Kyriacos Mitrophanous. Oxford BioMedica UK Limited,
Oxford, United Kingdom.
Purpose: Oxford BioMedica has developed ocular gene therapies using its
proprietary LentiVector platform which is based on recombinant Equine
Infectious Anaemia Virus (EIAV). Currently we have four ocular therapies:
RetinoStat, StarGen, UshStat and EncorStat, for the treatment of age-related
macular degeneration, Stargardt macula dystrophy, Usher Syndrome 1B, and the
prevention of corneal transplant rejection respectively. RetinoStat , StarGen and
UshStat have received regulatory approval in the US and France (IND/CTA) and
are currently in clinical evaluation. The development of lentiviral vector-based
gene therapies to treat eye diseases is an attractive option due to the vectors innate
ability to express therapeutic genes for extended periods, either to correct inherited
disease, or to interfere with disease aetiology. The accessibility of the eye both for
administration and evaluating therapeutic benefit, combined with the anatomical
separation from the rest of the body preventing spread of the vector, are also
significant advantages.
Methods: The LentiVector platform has been extensively characterized in GLP
safety and biodistribution studies following subretinal administration, as part of the
RetinoStat, StarGen and UshStat programmes. These studies were conducted in
rabbits and NHP for up to 6 month duration.
Results: Subretinal administration of all three products caused only mild-tomoderate ocular inflammation (in the absence of prophylactic anti-inflammatory
medication) that was transient, completely resolving within 1 month. Subretinal
administration of vector caused no long-term detrimental changes within the eye.
Biodistribution studies of vector demonstrated that the lentiviral vector products
did not escape the ocular compartment, and as a result little or no antibody
responses were observed in these studies.
Conclusions: The EIAV-based LentiVector platform has been shown to be an
effective and safe system for the delivery of relatively large genes into target retinal
cells resulting in stable and long-term therapeutic expression.
Commercial Relationships: Scott Ellis, Oxford BioMedica Ltd (E); James

Miskin, Oxford BioMedica Ltd (E); Katie Binley, Oxford BioMedica Ltd (E);
Jackie de Belin, Oxford BioMedica Ltd (E); Julie Loader, Oxford BioMedica Ltd
(E); Michelle Kelleher, Oxford BioMedica Ltd (E); Stuart Naylor, Oxford
BioMedica Ltd (E); Kyriacos Mitrophanous, Oxford BioMedica Ltd (E)
Support: None
Design of a clinical study of gene therapy for Leber Hereditary Optic
Neuropathy
Helene Cwerman-Thibault, Sebastien Augustin, Christophe Lechauve, Jessica
Ayache, Manuel Simonutti, Jose-Alain Sahel, Marisol Corral-Debrinski. Institut de
la Vision - Inserm UMRS968, Paris, France.
Purpose: Since six years, we are involved in the development of a gene therapy to
prevent retinal ganglion cell (RGC) loss and optic atrophy. Our aim is to succeed in
the design of a clinical trial for Leber Hereditary Optic Neuropathy (LHON) due to
the ND4 (NADH dehydrogenase 4) substitution G11778A, affecting 70% of
patients. ND4 encodes a subunit of respiratory chain complex I; the mutation leads
to complex I deficiency. In this study we determined: (1) the kinetics of transgene
expression in rat RGCs after intravitreal administration of AAV2-ND4 and the
subcellular localization of the protein produced by the vector. (2) Since the inner
limiting membrane (ILM) could be a barrier for successful RGC transduction in
patients, we assessed 3 different routes of AAV administration in non-human
primate eyes.
Methods: Adult rats received intravitreal (IVT) injection of AAV2_ND4. RNA
was isolated from whole retinas to determine ND4 mRNA abundance at different
post-injection times. Mitochondrial crude extractions were examined by western
blot and Blue Native gels to assess if the human ND4 was efficiently translocated
into the organelle and may be detected in assembled complex I. For non-human
primates, 3 administration routes were compared using an AAV2_GFP vector: (1)
single IV injection; (2) injection after vitrectomy; (3) injection after vitrectomy and
ILM peeling. One month later, vector DNA level was evaluated in ocular and non
ocular tissues as well as GFP mRNA abundance; retinal structure was also studied
by histology and immunolabelling.
Results: Human ND4 mRNA was detected in rat retinas as early as 4 weeks after
vector administration; its level remains stable up to 8 months later. Preliminary data
indicated that ND4 protein localized to mitochondria. In non-human primates, we
detected both GFP mRNA and protein in RGCs. The safest route for AAV2
administration was the single IV injection since no abnormalities were evidenced in
any part of the eye. By contrast vitrectomy and ILM peeling induced inflammatory
cells in the anterior segment of the eye.
Conclusions: We are about gathering a thorough set of data both in rat and in nonhuman primates that will undoubtedly validate our AAV2_ND4 gene therapy.
Moreover, we prove both the safety and the efficiency of IV administration for
RGC transduction in non-human primates thus preparing the clinical trial for
LHON.
Commercial Relationships: Helene Cwerman-Thibault, None; Sebastien
Augustin, None; Christophe Lechauve, None; Jessica Ayache, None; Manuel
Simonutti, None; Jose-Alain Sahel, None; Marisol Corral-Debrinski, None
Support: AFM-Gnthon / ANR Emergence Bio / Fondation Voir et Entendre /
Inserm / CNRS / UPMC
Gene Transfer of Self Complimentary Adeno-associated Virus into Rabbit
Corneas After Photorefractive Keratectomy
Paulette M. Robinson1A, Sriniwas Sriram1A, William Hauswirth1B, Alfred S.
Lewin1C, Gregory S. Schultz1D. AOB-GYN, BMolecular Genetics and Microbiology,
C
Molecular Genetics & Microbio, DDept of OBGYN and Ophthalmology,
1
University of Florida, Gainesville, FL.
Purpose: Self complementary (double stranded) adeno-associated viruses
(scAAVs) have been shown to have a faster onset of gene expression because the
scAAV DNA is transcribed rapidly. In addition, scAAV generally have higher
transduction efficiency than conventional rAAV vectors. In this study, we
investigated scAAV-mediated gene transfer in rabbit cornea after photorefractive
keratectomy (PRK). Our goal was to characterize the time of first detectable
expression, determine the duration of transgene expression, and identify what cell
types were transfected.
Methods: Self complementary AAV expressing green fluorescent protein (scAAVGFP) was applied following PRK for two minutes. Corneas were removed at 0, 1,
2, 3, 4 , 7, 30 and 180 days after vector application and fixed in 2% formaldehyde
then cryosectioned. Slides were analyzed for direct fluorescence using confocal
microscopy. Images were analyzed using Optimas Imaging software and were
analyzed for statistical significance by student t-test using GraphPad prism.
Results: The GFP fluorescence was first detected 24 hours after application and the
peak fluorescence detected occurred at 7 days. Day 7 fluorescence was 22 times

greater than day 0. Levels of the fluorescence from day 180 were statistically the
same as day 0 levels of fluorescence. The transgene was expressed in all cell types
of the cornea; epithelium, keratocytes and endothelium.
Conclusions: These data indicate that topically applied scAAV vectors rapidly
express transgenes in all cell types in the cornea. Transduction of corneal cells with
scAAV vector expressing ribozymes or shRNA targeting profibrotic genes may
provide an optimal therapy to reduce scar formation in the cornea.
Commercial Relationships: Paulette M. Robinson, None; Sriniwas Sriram,
None; William Hauswirth, None; Alfred S. Lewin, None; Gregory S. Schultz,
None
Support: NIH Grant EY05587 and NIH Grant EY08571
AAV-mediated Cone Targeted Gene Therapy To Cpfl5 Mouse, A Model of
Human Achromatopsia 2 with Mutations in Cnga3
Ji-Jing Pang1, Song Mao1, Wei Shi1,2, Wentao Deng1, Shannon E. Boye1, Jie Li1,
Xuan Liu1, Sanford L. Boye1, Bo Chang3, William W. Hauswirth1. 1Ophthalmology,
University of Florida, Gainesville, FL; 2Beijing Tongren Eye Center, Beijing
Tongren Hospital, Capital Medical University, Beijing, China; 3Ophthalmology,
The Jackson Laboratory, Bar Harbor, ME.
Purpose: Mutations in the gene encoding the alpha-subunit of the cone cyclic
nucleotide-gated (CNGA3) channel cause cone function loss in mammals.
IRBP/GNAT2 promoter is a chimeric promoter in which sequence corresponding
to -1619 to -1411 of the human interphotoreceptor retinoid-binding protein (IRBP)
gene was directly fused to -151 to +126 sequence of human cone transducin alphasubunit (GNAT2) gene. mCARpro promoter is a 500 base pair mouse cone arrestin
promoter, described by Li et al. 2002. We tested whether these two newly
developed cone targeting promoters are cone specific and Cnga3 gene delivery can
restore cone function in cpfl5 (Cone Photoreceptor Function Loss 5) mice, a natural
model of human Achromatopsia 2 (ACHM2) with Cnga3 mutations.
Methods: At postnatal day 14 (P14), 1 l of AAV5-IRBP/GNAT2-GFP or AAV5mCARpro-mCherry vector (1013 genome containing viral particles/ml) was injected
subretinally into one eye of ten P14 C57 BL/6J mice, respectively; while 1 l of
AAV5-IRBP/GNAT2-Cnga3 vector (1013 genome containing viral particles/ml)
was injected subretinally into one eye of 20 cpfl5 mice. Uninjected contralateral
eyes were used as controls. Dark- and light-adapted ERGs, were recorded 2 months
after injection followed by harvest of injected and uninjected eyes for structural
studies.
Results: Frozen section stained with either M- or S-cone opsin showed that AAV5IRBP/GNAT2-GFP-mediated GFP expression was observed in almost all cones,
including both M- and S-cones, while AAV5-mCARpro-mCherry-mediated
mCherry expression was observed not only in cones but also in RPE cells of C57
BL/6J eyes. In AAV5-IRBP/GNAT2-Cnga3 treated cpfl5 eyes, restored lightadapted ERGs were observed at 2 months after injection; the b-wave amplitudes in
treated cpfl5 eyes were similar with those from the AAV5-IRBP/GNAT2-GFP
injected C57 BL/6J eyes, but were about 2/3 of those from uninjected C57BL/6J
eyes. In the contralateral uninjected cpfl5 eyes, the cone-driven ERGs were not
recordable. In the treated cpfl5 retinas, immunohistochemical CNGA3 staining was
apparent in the inner and outer segments of the cones with normal M- and S-cone
opsin distribution in outer segments of cones. However in untreated partner eyes,
no CNGA3 expression was observed and M- and S-cone degeneration was
apparent.
Conclusions: The AAV5-IRBP/GNAT2 vector targets cones, including both Mand S-cones, while AAV5-mCARpro vector drives expression in both cone and
RPE cells. AAV5-IRBP/GNAT2-Cnga3 mediated gene therapy corrects CNGA3
deficiency and restores cone function in a naturally occurring mouse model of
human ACHM2.
Commercial Relationships: Ji-Jing Pang, None; Song Mao, None; Wei Shi,
None; Wentao Deng, None; Shannon E. Boye, None; Jie Li, None; Xuan Liu,
None; Sanford L. Boye, UF#13859 (P); Bo Chang, None; William W.
Hauswirth, AGTC (P)
Support: NIH grant EY021721 and grants from the FFB, MVRF, RPB, and MSTC
(2010DFB33430).
AAV-mediated Expression Of Human Rdh12 in Mouse Retina
Debra A. Thompson1A,1B, Lin Jia1A, Jingyu Yao1A, Kecia L. Feathers1A, Selina A.
Azam2, Alexander J. Smith2, Robin R. Ali2. AOphthalmology and Visual Sciences,
B
Biological Chemistry, 1Univ of Michigan Med School, Ann Arbor, MI; 2Univ
College London Institute of Ophthalmology, London, United Kingdom.
Purpose: Inherited loss-of-function mutations in RDH12 result in severe retinal
degeneration often diagnosed in childhood as Leber congenital amaurosis (LCA).
The purpose of our studies was to initiate feasibility testing of AAV-vectormediated RDH12 gene-replacement therapy by evaluating outcomes in wild-type
and Rdh12-knockout mice.
Methods: A human RDH12 cDNA construct previously shown to exhibit RDH

activity in vitro was packaged as recombinant adeno-associated virus serotype 2/8
containing a rhodopsin-kinase promoter that drives expression in rods and cones.
The resulting construct, AAV2/8-hRK-hRDH12, was administered to adult Rdh12knockout and C57BL/6 mice by subretinal injection (1-2 l of 1 x 108-10 viral
genomes per l). Mice were euthanized at 3 wk post injection and RDH12
expression was evaluated by western and immunohistochemical (IHC) analysis.
Results: Using an antibody specific for mouse Rdh12, western analysis detected
the endogenous protein in retinas from wild-type but not Rdh12-knockout mice,
and IHC analysis showed labeling of the endogenous protein in photoreceptor inner
segments and outer nuclear layer in wild-type mice. In contrast, using an antibody
specific for human RDH12, the recombinant protein was detected in retinas from
both Rdh12-knockout and wild-type mice injected with AAV2/8-hRK-hRDH12. In
mice injected with ~109 viral genomes, the pattern of anti-human RDH12 reactivity
appeared similar to the endogenous protein in both the level and pattern of
expression. Similar results were obtained in mice injected at 5 and 11 wk of age.
IHC analysis of rhodopsin, S-opsin, and M/L-opsin showed that expression of
recombinant RDH12 did not disturb rod and cone cell integrity or diminish cell
numbers.
Conclusions: Subretinal injection of an AAV2/8 vector in which expression is
under the control of a rhodopsin-kinase promoter produces recombinant human
RDH12 in the mouse retina that appears correctly localized and does not disrupt
structural integrity. Future studies are needed to evaluate the ability of the
recombinant protein to reconstitute functional deficits resulting from RDH12 lossof-function. Our findings represent an important first step toward the development
of a novel therapy for a subset of LCA patients.
Commercial Relationships: Debra A. Thompson, None; Lin Jia, None; Jingyu
Yao, None; Kecia L. Feathers, None; Selina A. Azam, None; Alexander J.
Smith, None; Robin R. Ali, None
Support: Foundation Fighting Blindness, Research to Prevent Blindness, RP
Fighting Blindness UK, Medical Research Council, NHR Biomedical Research
Centre for Ophthalmology
Soluble BAFF-R Receptor (sBAFF-R) as a Potential treatment for Sjgren
Syndrome
Nevien Ismail-O'Keeffe1,2, Hongen Yin2, Amanda Perofsky2, John A. Chiorini2.
1
Biochemistry and Molecular Biology, Georgetown University, Washington, DC;
2
Molecular Physiology and Therapeutics Branch, National Institute of Dental and
Craniofacial Research, National Institute of Health, Bethesda, MD.
Purpose: Sjgren Syndrome (SS) is a systemic autoimmune disease that is
characterized by low salivary and lacrimal gland (LG) activity. One of the
immunological characteristics of SS is B cells hyperactivity and serum
autoantibodies. B cell-activating factor (BAFF) is a member of TNF super family,
which regulates B lymphocytes proliferation and maturation, and can promote B
cell survival both in vitro and in vivo. We studied the effect of neutralizing BAFF
by local gene transfer of a soluble form of its receptor in non obese diabetic (NOD)
mouse, which develop a Sjogrens syndrome like phenotype and are reported to
have elevated levels of BAFF expression in their salivary glands.
Methods: A recombinant Adeno-Associated virus (AAV2) was constructed
encoding a soluble BAFF receptor (sBAFF-R) and expression confirmed in vitro by
western and sandwich ELISA. The AAV2-BAFF-R or a control vector encoding
beta galactosidase was administered to the submandibular salivary glands (SG) via
retrograde cannulation of Whartons duct along with a reporter vector encoding
luciferase at a 1:10 ratio. Successful cannulation was confirmed by xenogen
imaging of the luciferase reporter vector. Salivary gland activity was measured by
monitoring drinking behavior of the mice over a 2 hours interval and by measuring
pilocarpine stimulated saliva production over a 20 min period at 18 weeks post
vector delivery.
Results: Statistical analysis of saliva collection and the lickometer data using
student t-test, showed no statistically significant difference in salivary gland
activity between the BAFF-R and the LacZ control groups suggesting BAFF-R
expression had little effect on gland function compared with control mice.
Immunological changes in the glands were assessed by counting the number of
filtrating foci in the glands showed little decrease in lymphocytic infiltration in the
BAFF-R group compared with the control group. Other immunologic changes
including cytokine and immunogolobulin levels will be assessed.
Conclusions: Although intracelluar inhibition of BAFF has been reported to affect
the Sjogrens syndrome like phenotype in NOD mice, expression of the sBAFF-R
had little effect on salivary gland activity and infiltration.
Commercial Relationships: Nevien Ismail-O'Keeffe, None; Hongen Yin,
None; Amanda Perofsky, None; John A. Chiorini, None
Support: None

A Human Cellular Model Of Choroideremia Generated Via Patient Ips Cells
Vasiliki Kalatzis1, Nicolas Cereso1, Marie O. Pequignot1, Lorenne Robert1, Aude
Conscience1, Fabienne Becker2, John de Vos2, Christian P. Hamel1. 1Hopital St
Eloi, BP 74103, INM Inserm U1051, Montpellier, France; 2Institute for Research in
Biotherapy, Inserm Montpellier, France.
Purpose: The first clinical trials of retinal gene therapy were carried out in 2008
with positive results. Consequently, the requirements for future preclinical studies
of other diseases will be less stringent, notably concerning the need for large
animal disease models. However, a growing number of diseases lack even an
appropriate small model, compromising their chances of one day reaching a clinical
trial. In such cases, a viable alternative would be to perform preclinical studies on
human cellular models of the pathogenic retina.
Methods: As a pilot project, we generated a human cellular model of the X-linked
disease choroideremia (CHM). CHM represents 2% of retinal dystrophies and is
characterised by night blindness in childhood leading to blindness by 50 y of age. It
is due to mutations in the CHM gene encoding Rab escort protein 1 (REP1). Mouse
and zebrafish REP1-deficient models are lethal. As it is impossible to obtain retinal
cells from a CHM patient, we generated these cells from skin fibroblasts via the
intermediate use of induced pluripotent stem (iPS) cells.
Results: We obtained the first bona fide iPS clone of a CHM patient carrying a
deletion of exon 8. We differentiated this iPS clone into a retinal pigment
epithelium (RPE) monolayer that expresses RPE-specific markers, is polarised, has
tight junctions and is capable of active transport. Ultra-structural studies
demonstrate the presence of microvilli on the surface and a characteristic RPE
subcellular organisation. We are currently studying the differences in phagocytosis,
melanosome trafficking and visual cycle differences between wild type and patient
RPE. This work will allow disease modelling in a human system as well as provide
the readouts for evaluating phenotype restoration following viral-mediated CHM
gene transfer.
Conclusions: The use of such innovative human disease systems are more
economical and less time-consuming alternatives to animal models, as well as more
biologically relevant for further understanding disease pathogenesis and for
preclinical therapeutic studies. Moreover, considering the current climate of retinal
gene therapy, preclinical trials on human cells will allow a more rapid transition to
phase I trials for diseases lacking an appropriate animal model.
Commercial Relationships: Vasiliki Kalatzis, None; Nicolas Cereso,
None; Marie O. Pequignot, None; Lorenne Robert, None; Aude Conscience,
None; Fabienne Becker, None; John de Vos, None; Christian P. Hamel, None
Support: Inserm, France Choroideremie, Choroideremia Research Fondation,
Fondation de France, AFM, FRM, Retina France, Terre Plurielle
AAV2/5 Mediated Gene Augmentation Rescues Photoreceptors in Canine
Models of RPGR-XLRP
William A. Beltran1, Artur V. Cideciyan2, Alfred S. Lewin3A, Simone Iwabe1,
Hemant Khanna4, Anand Swaroop5, William W. Hauswirth3B, Samuel G.
Jacobson2, Gustavo D. Aguirre1. 1Clinical Studies, Univ of Pennsylvania Sch Vet
Med, Philadelphia, PA; 2Dept of Ophthalmology, Scheie Eye Institute,
Philadelphia, PA; AMolecular Genetics & Microbio, BOphthalmology, 3University
of Florida, Gainesville, FL; 4Ophthalmology, University of Massachusetts Medical
School, Worcester, MA; 5N-NRL, Bldg 6, National Eye Institute, Bethesda, MD.
Purpose: Mutations in the RPGR gene are the most common cause of X-linked RP
in man. Two naturally-occurring canine models (XLPRA1 and XLPRA2) with
distinct deletions in RPGRORF15 recapitulate the spectrum of disease phenotypes
found in humans. Both models were used to test if AAV-mediated gene transfer of
human RPGRORF15 cDNA can rescue photoreceptors when delivered prior to
(XLPRA1) or after (XLPRA2) the onset of degeneration.
Methods: Two AAV2/5 vector constructs (titer: 1.51 x E11 vg/ml) carrying fulllength human RPGRORF15 cDNA under the control of either a hGRK1 or hIRBP
promoter were injected subretinally in XLPRA1 (150 ul at 28 weeks), and
XLPRA2 (70 ul at 5 weeks) dogs. Contra-lateral eyes were sham-injected with BSS
and served as controls. Photoreceptor structure and function was assessed by means
of non-invasive retinal imaging (cSLO/ SD-OCT) and ERG, and by
morphology/IHC at termination.
Results: In vivo retinal imaging showed preserved ONL thickness and IS/OS
structure in the treated retinal areas. Rod and cone function was greater in treated
than in control retinas. Morphology studies confirmed the rescue of photoreceptor
structure within the treated areas, as well as the reversal of rod and M/L cone opsin
mislocalization and the prevention of rod neurite sprouting. Potent expression of
the hRPGR transgene was found exclusively in photoreceptors located within the
treated areas.
Conclusions: These results show for the first time that gene transfer of the full
length human RPGRORF15 cDNA provides both structural and functional rescue
in an animal model of RPGR-XLRP.
Commercial Relationships: William A. Beltran, None; Artur V. Cideciyan,
None; Alfred S. Lewin, None; Simone Iwabe, None; Hemant Khanna,
None; Anand Swaroop, None; William W. Hauswirth, AGTC, Inc. (P); Samuel
G. Jacobson, None; Gustavo D. Aguirre, None
Support: EY-06855, -17549, -007961, -021721, Foundation Fighting Blindness,
Fight for Sight Nowak family grant, Macular Vision Research Foundation, Van
Sloun Fund , Research to Prevent Blindness, Inc.
Antisense Oligonucleotide-mediated Exon Skipping Improves Primary Cilia
Assembly In Fibroblasts Harbouring The Common Lca Cep290
C.2991+1655g>A Mutation
Jean-Michel Rozet1, Xavier Gerard2,3, Isabelle Perrault1, Eduardo Silva4, Karine
Bigot5, Sabine Defoort-Delhemmes6, Arnold Munnich1, Daniel Scherman3, Antoine
Kichler2, Josseline Kaplan1. 1Genetics U781, INSERM, Paris, France; 2Genethon,
Evry, France; 3CNRS UMR 8151-INSERM U1022, Paris, France;
4
Ophthalmology, University Hospital, Coimbra, Portugal; 5CERTO, Paris, France;
6
Ophthalmology, University Hospital, Lille, France.
Purpose: Leber congenital amaurosis (LCA) is a severe hereditary retinal
dystrophy responsible for congenital or early-onset blindness. The most common
disease-causing mutation (>10%) is located deep in intron 26 of the CEP290 gene
(c.2991+1655 A>G) where it creates a strong splice donor site that leads to the
insertion of a cryptic exon encoding a premature stop codon. The aim of this study
was to assess the feasibility of an antisense oligonucleotide (AON)-mediated exon
skipping strategy to correct this aberrant splicing.
Methods: Fibroblast cell lines of patients harbouring the c.2991+1655 A>G
mutation (n=4, 3/4 homozygous) and controls (n=3) were transfected using
antisense and sense 2O-methyl phosphorothioate-modified oligonucleotides
designed to target exon splicing enhancer (ESE) around the mutation. The skipping
was optimized for oligonucleotide sequences and concentrations, transfection
conditions and treatment time. The efficiency of skipping was assessed using qRTPCR, Western blot analysis using a polyclonal antibody recognizing the C-terminus
of the CEP290 protein and primary cilia counting.
Results: The level of expression of CEP290 messengers was unchanged when
control cell lines were transfected using the sense ONs or AONs (p>0.05).
Likewise, no change in expression was noted when patients cells were treated with
the sense ONs (p>0.05). Conversely, a highly significant increase in expression of
the wildtype CEP290 allele was obtained when cells were treated with AONs
(0.029<p<0.002) with expression levels reaching that of controls. Western blot
analysis evidenced increased levels of CEP290 in patients cell lines treated with
the AONs but not the sense ONs. Finally, following serum-starvation, primary cilia
expression was significantly reduced in patients fibroblasts compared to controls
lines (48.6% 6.5% vs 83.6%3.2 %; p=0.0097). Upon transfection with the
antisense ONs but not the sense versions, the proportion of ciliated cells increased
significantly in patients, reaching levels similar to controls (75.3%3.5% vs
78.3%3.4%; p=0.624).
Conclusions: Our results suggest that antisense ON-mediated exon skipping
resulted in a significant improvement of cilia assembly and/or maintenance.
CEP290 mutations are the most common cause of LCA. The results of this study
show therapeutic potential of exon skipping for the treatment of the mutation
c.2291+1655A>G which alone accounts for 10% of LCA cases.
Commercial Relationships: Jean-Michel Rozet, None; Xavier Gerard,
None; Isabelle Perrault, None; Eduardo Silva, None; Karine Bigot,
None; Sabine Defoort-Delhemmes, None; Arnold Munnich, None; Daniel
Scherman, None; Antoine Kichler, None; Josseline Kaplan, None
Support: RETINA FRANCE ASSOCIATION
Characterization Of A Novel Gene Therapy Reporter Vector (rAAV2/5-CBACNGA3-IRES-GFP) In A Mouse Model Of Achromatopsia
Srini Goverdhan1,2, Alun R. Barnard1, Mandeep Singh1, Peter Charbel Issa1,
Robert E. MacLaren1,3. 1Nuffield Laboratory of Ophthalmology, University of
Oxford, Oxford, United Kingdom; 2Vision Research Group / Southampton Eye
Unit, University of Southampton, Southampton, United Kingdom; 3Moorfields Eye
Hospital NHS Foundation Trust, London, United Kingdom.
Purpose: AAV mediated gene therapy holds promise as a potential clinical
treatment for a variety of inherited retinal degenerations. Quantifying the effects of
gene transfer on cone photoreceptor function in rodent models of human disease
can however be challenging as these cells are sparse. The CNGA3 (alpha 3 subunit
of the cone cyclic nucleotide-gated cation channel) knock-out mouse has a loss of
function and a slow degeneration, similar to a human cone dystrophy. The purpose
of this study was to assess a new AAV bicistronic expression cassette that both
replaces CNGA3 and expresses green fluorescent protein (GFP) in order to allow
the morphology of transduced cones to be studied in detail.
Methods: AAV2/5 vectors were generated with the gene encoding GFP coupled
bicistronically downstream to the gene encoding CNGA3 by an internal ribosome
entry site (IRES) and driven by a chicken beta-actin (CBA) promoter. Mice

received subretinal injections at 3 weeks with 2l of rAAV2/5-CBA-CNGA3IRES-GFP (5x1011 vector particles) in the study eye and 2l of rAAV2/5-CBAGFP (5x1011 vector particles) in the control eye. SLO autofluorecence retinal
imaging (Heidelberg OCT) was assessed 4 weeks post-injection for both CNGA3IRES-GFP treated and GFP control eyes.
Results: SLO autofluorecence showed comparable GFP expression for both
CNGA3-IRES-GFP treated and GFP control eyes, indicating that IRES-translation
from this construct was at least as good as a standard Kozac consensus. Histology
of retinal sections at 7 weeks allowed identification of cone photoreceptors by
CNGA3 immunostaining in the outer segment and GFP in the cytoplasm and inner
segment. The presence of cytoplasmic GFP allowed the morphology of transduced
cones and in particular, the cone pedicle synapses, to be studied in detail. In the
control eyes at this age only occasional surviving cone cells could be identified.
Conclusions: An IRES-GFP linked construct is useful for assessing the effects of
gene therapy on cone survival in mice in vivo by allowing SLO imaging to confirm
successful gene transfer. Histologically the presence of GFP assists in studying
proximal structural changes in the cone pedicle synapse after successful gene
transfer in individual cells, which would otherwise be difficult to trace from the
distal outer segments, where transgenic CNGA3 protein is localized.
Commercial Relationships: Srini Goverdhan, None; Alun R. Barnard,
None; Mandeep Singh, None; Peter Charbel Issa, None; Robert E. MacLaren,
None
Support: Health Foundation, Fight for Sight, Royal College of Surgeons of
Edinburgh, NIHR Biomedical Research Centre, HEFCE
The Development of Non-Viral Vectors for Choroideremia
Richard P. Harbottle, Elham Ostad-Saffari, Mariya Moosajee, Suet Ping Wong,
Dhani Tracey-White, Tanya Tolmachova, Miguel Seabra. Molecular Medicine,
Imperial College London, London, United Kingdom.
Purpose: Non-viral vectors are attractive alternatives to viral gene delivery systems
due to their low toxicity, relatively easy production, and great versatility. Recently,
we developed a novel plasmid vector employing a scaffold/matrix attachment
region (S/MAR) to provide functional episomal persistence within cells and
clinically relevant levels of transgene expression, in vivo, without vector toxicity.
We will provide evidence for the utility of S/MAR vectors in the eye.
Methods: We have employed our S/MAR vector technology for the development
of novel non-viral persistently expressing vectors for Choroideremia (CHM) gene
therapy. We have created a series of constructs with either the reporter genes EGFP
and Luciferase encoded along with REP1, with various promoters, with and
without an S/MAR element.
Results: We performed initial experiments to evaluate the expression of the
transgenes in vitro in a range of cell lines and demonstrated that the S/MAR
plasmids provide long-term transgene expression in vitro.
We subsequently optimised a procedure for the delivery and expression of these
non-viral constructs in vivo via subretinal injection of formulated DNA. We will
present data showing the longitudinal transgene expression of constructs measured
using a bioluminescence bioimager for over 12 months. Additionally, long term
expression of transgenes were also observed by rtPCR and Western blot analysis of
protein levels. We will also show that evidence for the persistent episomal
maintenance of these vectors in the eye.
We show that the S/MAR motif in these vectors is essential for providing long term
expression and maintenance in the eye.
We also demonstrate the lack of toxicity within the eye and show that fundus
examinations as well as detailed histological examinations of retinal sections do not
elicit an inflammatory response to our plasmids once subretinally injected in the
eye.
Conclusions: In conclusion, an ideal non-viral vector for gene therapy should be
non-toxic, have mitotic stability, allow non-integrative establishment and provide
persistent therapeutic expression levels. The S/MAR-containing vector that we
have developed and applied fulfils these requirements and provides proof of
principle for a new class of vector systems for ocular gene therapy.
Commercial Relationships: Richard P. Harbottle, None; Elham Ostad-Saffari,
None; Mariya Moosajee, None; Suet Ping Wong, None; Dhani Tracey-White,
None; Tanya Tolmachova, None; Miguel Seabra, None
Support: None
Sustained In Vitro Gene Expression Following Non-Viral Gene Transfer of
Large Transgene
Daniel C. Chung, Hadassah Janumala. FM Kirby Ctr Molecular Ophth, Scheie
Eye Institute, Philadelphia, PA.
Purpose: To demonstrate stable, long-term gene expression of large retinal
degeneration gene following non-viral phi C31 integrase mediated gene transfer in
cultured HEK293T cells.
Methods: Plasmid construct was developed carrying the full-length cDNA for the
Centrosomal Protein 290 (CEP290) gene, a large 8.2 kb gene that is responsible for
Leber Congenital Amaurosis (LCA) type 10, with an additional integrase
attachment site (attB) and marker gene (pCEP290.eGFP.attB). Human embryonic
kidney 293T (HEK293T) cells were plated on 2 chamber Tissue Tek slides and
100mm tissue culture dishes. Cultured cells were co-transfected with the CEP290
construct and an integrase containing plasmid (pInt) and controls without DNA
transfection and pCEP290.eGFP.attB only. All transfections were done using
1.5ug/ul of Lipofectamine 2000, following standard methods. Cell lysates were
obtained at 1, 2, 4 and 12-week time points, and Western blot analysis was
performed to evaluate CEP290 protein expression following phi C31 integrase
mediated gene transfer.
Results: Western blot analysis showed robust protein expression in both the lysate
of co-tranfected cells (pCEP290.eGFP.attb with pInt) and those of therapeutic
vector without integrase at week 1 and 2. At week 4, expression was limited to cells
that were co-transfected, whereas those transfected without integrase returned to
background levels. At 12 weeks after initial transfection, following multiple
passages of the transfected cells, protein expression for co-transfected cells did not
diminish. Non-transfected cells lysates showed background expression at all time
points.
Conclusions: Phi C3 Integrase is a uni-directional, site-specific integrating
recombinase that results in stable, efficient gene expression. It has a capacity to
accommodate large transgenes that some viral based vectors cannot package. PhiC
integrase mediated gene transfer of pCEP290.eGFP.attb to HEK 293T cells
demonstrates the ability for stable protein expression. Future studies for therapeutic
efficacy in animal models are being pursued.
Commercial Relationships: Daniel C. Chung, None; Hadassah Janumala,
None
Support: NIH K08 EY017024, Hope for Vision, Fidelity Charitable Trust, F.M.
Kirby Foundation
rAAV-mediated Gene Replacement Therapy Restores Vision and Delays
Degeneration in the CNGB1-/- Mouse Model of Retinitis Pigmentosa
Stylianos Michalakis1, Susanne Koch1, Regine L. Muehlfriedel2, Fred Koch1, Elvir
Becirovic1, Naoyuki Tanimoto2, Vithiyanjali Sothilingam2, Marina Garcia
Garrido2, Mathias W. Seeliger2, Martin Biel1. 1Pharmacy-Pharmacology, LudwigMaximilians-University Munich, Munich, Germany; 2Div of Ocular
Neurodegeneration, Ctr Ophthal Inst Ophthalmic Rsrch, Tuebingen, Germany.
Purpose: CNGB1 encodes the B1a subunit of the rod cyclic nucleotide-gated
(CNG) channel. Mutations in CNGB1 cause autosomal recessive retinitis
pigmentosa. Here, we used exon 26 CNGB1 knockout (CNGB1 -/-) mice to
evaluate rAAV-mediated CNGB1a gene replacement as a potential treatment for
this disease.
Methods: Due to the size of the CNGB1a cDNA (approx. 4 kb) optimization of the
regulatory elements within the expression cassette was necessary to comply with
the packaging limitations of rAAV vectors. We therefore replaced the BGH polyA
site and the WPRE element with a 221bp SV 40 polyA element. A short mouse
rhodopsin promoter element was used to drive rod-specific expression. Therapeutic
rAAVs (serotype 8) were injected into the subretinal space of 2-week-old CNGB1-/mice. The treatment success was monitored using immunohistochemistry,
electrophysiology and a water-maze test designed to specifically assess rodmediated vision.
Results: Using the optimized rAAV vectors we could successfully express fulllength CNGB1a in the knockout retina. Signs of a functional restoration were first
observed in the ERG at 6 weeks after injection. The CNGB1a protein was
exclusively found in rod photoreceptors and mainly localized to outer segments.
The treatment restored the expression of the previously down-regulated
endogenous CNGA1 protein, which co-localized with CNGB1a in rod outer
segments. In contrast to untreated mice, treated CNGB1-/- mice became able to
generate rod photoreceptor responses. Moreover, treated CNGB1-/- mice showed a
performance similar to wild type control mice in a rod vision behavioral test. In
contrast, untreated CNGB1-/- mice were not able to navigate to the escape platform
and showed no learning progress. In addition, the treatment led to a substantial
morphological preservation of rods. In particular, the treated area showed
significant preservation of rod outer segment structure and outer nuclear layer
morphology.
Conclusions: Using a capacity-optimized rAAV vector for rod-specific expression
of large (up to 4.2 kb) transgenes, we were able to successfully restore rod function
in CNGB1-/- mice. This work provides a proof-of-concept for the treatment of
retinitis pigmentosa due to a rod channelopathy by rAAV-mediated gene
replacement.
Commercial Relationships: Stylianos Michalakis, None; Susanne Koch,
None; Regine L. Muehlfriedel, None; Fred Koch, None; Elvir Becirovic,
None; Naoyuki Tanimoto, None; Vithiyanjali Sothilingam, None; Marina
Garcia Garrido, None; Mathias W. Seeliger, None; Martin Biel, None
Support: Deutsche Forschungsgemeinschaft

Use of a Short hPDE6b Promoter For Rod Gene Transfer in a Model of Severe
Retinal Dystrophy, the Rd10 Mouse
Yvan Arsenijevic1, Corinne Kostic2, Alberto Auricchio3, JongEun Ihm4. 1Unit of
Gene Therapy & Stem Cell Biology, Jules-Gonin Eye Hospital, Univ Lausanne,
Lausanne, Switzerland; 2Gene Therapy & Stem Cell Biol, Jules-Gonin Eye Hosp,
Univ Lausanne, Lausanne, Switzerland; 3Telethon Institute of Genetics and
Medicine and Dep. of Pediatrics, Federico II University, Napoli, Italy;
4
UGTSCB, Hospital de l'Ophthal, Jule-Gonins, Lausanne, Switzerland.
Purpose: Gene therapy of severe retinal dystrophies directly affecting
photoreceptor is still a challenge in terms of clinical application. One of the main
hurdles is to generate high transgene expression specifically in rods or cones. In the
present study, we are investigating the possibility to drive hPDE6b expression in
the Rd10 mouse retina using a specific sequence of the human PDE6b promoter.
Methods: Two 5 flanking fragments of the human PDE6b gene: (-93 to +53 (146
bp) and -297 to +53 (350 bp, see Di Polo and Farber, 1995) were cloned in
different plasmids in order to check their expression in vitro and in vivo. These
elements drove the activity of either luciferase (pGL3 plasmids) or EGFP (AAV2/8
backbone). Then, an AAV2/8 vector carrying the PDE6b cDNA was tested with
subretinal injections at P9 in the Rd10 eyes. Eye fundus, OCT, ERG recordings and
histological investigations were performed to assess the efficacy of the gene
transfer.
Results: The short PDE6b promoter containing 146bp (-93 to +53) showed the
highest activity in the Y-79 cells, as described previously (Di Polo and Farber,
1995). Subretinal administrations of AAV2/8-PDE6bpromoter-EGFP allowed a
rapid expression specifically in rods and not in cones. The expression is faster than
a vector containing the CMV promoter. The AAV2/8-PDE6bpromoter-PDE6b and
the control vector were injected at P9 in the Rd10 mouse retina and investigated 5
weeks post-injection. Out of 14 eyes, 6 presented an increased rod sensitivity of
about 300 fold, and increased a- and b-wave responses in ERG recordings. Flicker
stimulations revealed that cones are also functional. OCT images and histological
analyses revealed an increased ONL size in the injected area. The retina treated
with the therapeutic vector presented 4-6 rows of photoreceptors with
outersegments containing PDE6b. In the control eyes, only 2-4 rows of
photoreceptors with almost no OS were observed .
Conclusions: The 146 bp promoter sequence (-93 to + 53) is the shortest regulatory
element described to date which allows to obtain efficient rod-specific expression
in the context of somatic gene transfer. This first result is of great interest for AAV
vector design in general allowing more space for the accommodation of transgenes
of interest and good expression in rods. Moreover we showed the proof of principle
of the efficacy of AAV2/8-PDE6bp-PDE6b vector in the Rd10 mouse model of
severe photoreceptor degeneration without using neither AAV mutated capsids, nor
self-complementary vectors.
Commercial Relationships: Yvan Arsenijevic, None; Corinne Kostic,
None; Alberto Auricchio, None; JongEun Ihm, None
Support: FP7AAVEYE HEALTH-2007-B-223445
Pre-clinical Gene Therapy Studies In Choroideremia Mouse Models Using
AAV Vectors
Miguel C. Seabra1, Oleg Tolmachov1, Robert E. MacLaren2, Tanya Tolmachova1.
1
Molecular Medicine, Imperial College London, London, United Kingdom;
2
Nuffield Lab of Ophthalmology, University of Oxford, Oxford, United Kingdom.
Purpose: Choroideremia (CHM) is a progressive degeneration of the
photoreceptors, retinal pigment epithelium (RPE) and choriocapillaris caused by
defects in the CHM/REP1 gene. CHM/REP1 gene encodes Rab Escort Protein-1
(REP1), which participates in the lipid modification (prenylation) of Rab GTPases.
We showed previously that correction of prenylation defect in CHM cells could be
achieved with lentiviral vectors carrying CHM/REP1 cDNA transgene. The aim of
the present work was to perform preclinical gene therapy study using AAV vectors.
Methods: We generated AAV2/2 and AAV2/5 vectors expressing human
CHM/REP1 cDNA or EGFP under control of EFS (elongation factor-1 short) or
CAG (CMV-enhanced chicken beta-actin) promoter. WPRE was present on all
vectors. We transduced D17 cell line and primary fibroblasts derived from CHM
patients. REP1 expression was verified by Western blot and functionality was
confirmed by in vitro prenylation assay. AAV vectors expressing REP1 and GFP
were injected subretinally into eyes of 4-week old wild type and choroideremia
mice (Chm null/WT). The contralateral eye was not injected. Expression of EGFP
transgene in the RPE and neuroretina was observed by immunofluorescence. RPE
from injected and non-injected eyes was collected, and REP1 expression was
verified by Western blot.
Results: We observed expression of REP1 and GFP transgenes in D17 line and
CHM fibroblasts. Expression level was proportionally reduced with each cell
division. In D17 cells, expression from 2 promoters and 2 serotypes was similar,
while in CHM fibroblasts CAG vectors provided much higher level of transgene
expression, which was estimated as a 10-fold increase in comparison to the REP1
level in fibroblasts from controls. Extracts from the cells transduced with AAV2/2CAG-REP1 had increased prenylation activity in comparison to AAV2/2-CAGGFP-transduced and untransduced cells. In the mouse eyes injected with AAV2/2CAG-GFP, we observed signal in the RPE and neuroretina. In the RPE isolated
from the mouse eyes injected with AAV2/2-CAG-REP1, expression of REP1 was
increased in comparison to non-injected eye.
Conclusions: The CAG promoter provided stronger expression of the CHM/REP1
transgene in comparison to the EFS promoter. CHM/REP1 transgene delivered by
AAV vectors was functional and thus the AAV2/2-CAG-REP1 vector is suitable
for use in choroideremia human trials.
Commercial Relationships: Miguel C. Seabra, Named inventor on UK patent
application 1103062.4 filed on behalf of the University of Oxford (P) (P); Oleg
Tolmachov, None; Robert E. MacLaren, Named inventor on UK patent
application 1103062.4 filed on behalf of the University of Oxford (P) (P); Tanya
Tolmachova, None
Support: FIght for Sight, Choroideremia Research Foundation, Foundation
Fighting Blindness
Comparison Of Long-term Transduction Efficiency Of Reporter Genes After
Subretinal Injection Of The Adenovirus Vectors Of Serotypes 5, 28, And 35
Kazuhiro Ueyama1, Keisuke Mori1, Melissa Hamilton2, Hidekazu Omata1, Peter L.
Gehlbach3, Lisa L. Wei2, Shin Yoneya4. 1Ophthalmology, Saitama Medical
University, Iruma, Japan; 2GenVec, Inc., Gaithersburg, MD; 3Ophthalmology,
Johns Hopkins Wilmer Eye Inst, Baltimore, MD; 4Ophthalmology, Saitama
Medical School, Iruma, Japan.
Purpose: Adenovirus vectors (Ad) are efficient gene delivery vehicles and are
widely used in pre-clinical and clinical gene therapies. We have previously
demonstrated short-term transduction efficiency and intraocular localization of
reporter genes delivered by Ad of several serotypes with fiber modification
(Ueyama et al. #5194, presented in ARVO 2010). The aim of this study was to
evaluate the transduction efficiency of Ad vectors until six months for serotypes 5,
28 and 35 (abbreviation, Ad5, Ad28 and Ad35, respectively).
Methods: To determine the time course of luciferase activity, C57BL6 mice
received a single subretinal injection of Ad vector containing the construct
expressing luciferase at the concentration of 1x10e8 particle units. Nave animals or
animals with intraocular injection of empty adenoviral vectors served as negative
controls. Eyes were harvested at the date of 1, 7, 14, 28, 90 and 180 after injection
and assayed for luciferase activity.The assessed number of eyes was 4 to 6 eyes at
each virus and each timepoint, and the total number was 85.
Results: Ad5 expressed strongest within 7 days after injection, but decreased
rapidly. Ad28 sustained strong expression until 28 days, but decreased after 90
days. Ad28 had significantly stronger expression than Ad5 at 28 days after
injection (P=0.0127). Ad35 expressed strongest within 7 days after injection and
sustained throughout the 180 days-observation. Ad35 expressed stronger than Ad5
at the time of 28 days and 180 days after injection (P=0.0127, P=0.033
respectively).
Conclusions: The strength and duration of Ad expression differed between
serotypes. The prolonged gene expression mediated by Ad28 and Ad35 subretinal
injection may provide an advantage in delivering several therapeutic proteins.
Commercial Relationships: Kazuhiro Ueyama, None; Keisuke Mori, None;
Melissa Hamilton, GenVec (E); Hidekazu Omata, None; Peter L. Gehlbach,
None; Lisa L. Wei, GenVec (E); Shin Yoneya, None
Support: None
Evaluation Of Rod Photoreceptor Function And Preservation Following
Retinal Gene Therapy In The PDE6A Mutant Dog
Freya M. Mowat1, Joshua T. Bartoe1, Ashlee Bruewer1, Astra Dinculescu2, Sanford
L. Boye2, William W. Hauswirth2, Simon M. Petersen-Jones1. 1Small Animal
Clinical Sciences, Michigan State University, East Lansing, MI; 2Ophthalmology,
Purpose: The PDE6A mutant dog has a null mutation in the phosphodiesterase 6A
gene (PDE6A) that results in absence of rod function and rapid rod degeneration.
The affected dogs lack both gamma and beta subunits of PDE6 as well as the alpha
subunit. Our attempts to rescue this severe phenotype with an adeno-associated
viral vector serotype 5 (AAV5) vector proved unsuccessful. Our hypothesis was
that a next-generation AAV serotype 8 vector-type containing an engineered capsid
mutation would be successful in rescuing this phenotype.
Methods: 30-day old PDE6A mutant dogs received a unilateral subretinal injection
of AAV8 mut733 smCBA-cPDE6A. Approximately 100l of 4.19 x1011 vector
genomes/ml were delivered. Dark- and light-adapted ERGs were recorded and rodmediated vision was assessed via dim-light maze testing at 2-week intervals
following treatment. Animals were sacrificed 3-4 months after injection and
immunohistochemistry for retinal antigens including PDE, rod, cone and inner

retinal markers was performed on retinal cryosections from treated and untreated
eyes.
Results: There was evidence of rod function in the treated eyes. ERGs showed
improved threshold with a scotopic threshold response and small rod-shaped ERG
responses. Improvement in rod-mediated vision was detected in treated eyes using
maze testing. In treated eyes, rod photoreceptors were preserved in the injected
area, and PDE expression correlated with both photoreceptor preservation and
improved localization of rhodopsin to the rod outer-segments.
Conclusions: We have shown for the first time that AAV gene therapy can provide
partial rescue of rapid retinal degeneration in the PDE6A mutant dog, an important
model for a form of human retinitis pigmentosa.
Commercial Relationships: Freya M. Mowat, None; Joshua T. Bartoe,
None; Ashlee Bruewer, None; Astra Dinculescu, None; Sanford L. Boye, None;
William W. Hauswirth, AGTC (P); Simon M. Petersen-Jones, None
Support: Myers-Dunlap Endowment for Canine Health, Hal and Jean Glassen
Memorial Foundation, NIH grant EY021721.
copGFP dissolved in SonoVue microbubble, was injected into the anterior

chamberof New Zealand white rabbits or Tibetan monkeys after equal volume of
aqueous humor was drawn. The limbus corneae was then exposed to ultrasound for
1 min (Frequency = 1 MHz, Intensity = 1 W/cm2, Duty Cycle = 20%) . IOP was
monitored daily for 2 weeks by the TonoLab rebound tonometer. Frozen sections
for rabbit limbus corneaes were prepared to confirm gene expression.
Results: CNP gene transfer significantly (P<0.05) lowered rabbit and monkey IOP
starting 48 h after treatment. In rabbits, the maximum reduction (1.850.51 mm
Hg) was shown on day 5. In monkeys, the maximum reduction (3.170.95 mm Hg)
was observed on day 3. The rabbit IOP change correlated with gene expression at
the angle of anterior chamber, as well as the vicinal cornea and sclera.
Conclusions: Ultrasound-microbubble mediated transfection of CNP gene lowered
IOP and may be a safe and effective means for glaucoma gene therapy.
Commercial Relationships: Wenhan Yu, None; Haotian Xiang, None; Guo
Liu, None; Iok-Hou Pang, None; Jun Chen, None; Xuyang Liu, None
Support: NNSF 30872830 & 30872839

Differential Proteomics and Functional Research Following Gene Therapy in a
Mouse Model of RPE65 Leber's Congenital Amaurosis
Wensheng Li, Sr.1, Radouil T. Tzekov2, Qinxiang Zheng1, Xufeng Dai1, Ji-Jing
Pang3, Jia Qu1. 1Ophthalmology, Wenzhou Medical College, Wenzhou, China;
2
Ophthalmology, UMASS Medical School, Worcester, MA; 3Ophthalmology,
Purpose: To investigate the proteomic differences in retinal tissue after gene
therapy in retinal degeneration 12 (rd12) mice.
Methods: 1 l scAAV5-smCBA-hRPE65 was injected subretinally in right eyes of
rd12 mice at P14; left eyes remained uninjected as a control. Age-matched
C57BL/6J was set up as a comparison group. Electroretinograms (ERG) in darkand light-adapted conditions were recorded at P21 and P42. Total retinal protein
content of each group was extracted at P14, P21 and P42. Protein expression was
conducted by 2-DE. Proteins were identified by MALDI-TOF.
Immunohistochemistry (IHC) was performed to locate the identified proteins,
which were further verified by western blot and RT-PCR. The retinal reactive
oxygen species (ROS) were detected by IHC, while TUNEL was used to detect
apoptosis. Mller cells culture (MIO-M1) was used to understand the role of
PRDX6, a protein of interest.
Results: Four weeks after injection (P42), the treated rd12 eyes demonstrated
significant amplitudes increase in dark-adapted and light-adapted ERGs, while no
rod ERG response was detected in untreated contralateral eyes. Proteomic profiling
found differential proteins between untreated -rd12 and treated -rd12 or wild-type
eyes. Overall, 39 differential proteins were identified, 3 of which were selected for
further tests. Alpha A crystallin (CRYAA), heat shock protein 70 (HSP70) and
peroxiredoxin 6 (PRDX6) were up-regulated in untreated-rd12 eyes and not in
treated-rd12 eyes or wild-type eyes. IHC confirmed the existence of these 3
proteins in retina, as well as the up-regulation in untreated-rd12 eyes, while
Western blot confirmed the 2-DE results. RT-PCR results indicated that the
untreated -rd12 eyes had increased HSP70 and PRDX6 mRNA levels, while
CRYAA mRNA levels were decreased. ROS increased only in the untreated group,
mainly in the outer nuclear layer where TUNEL indicated signs of apoptosis. The
treated rd12 eyes did not show ROS increase or presence of apoptosis. MIO-M1
cells overexpressing PRDX6 displayed higher survival rate.
Conclusions: Proteomic analysis showed significant differences among groups at
P42 with many differential proteins identified. Gene therapy could restore retinal
function and protein expression in rd12 eyes. The up-regulation of CRYAA,
HSP70 and PRDX6 in treated rd12 eyes could play a role in preventing cell death.
PRDX6 could be an important antioxidant protector in the mouse retina.
Commercial Relationships: Wensheng Li, Sr., None; Radouil T. Tzekov,
None; Qinxiang Zheng, None; Xufeng Dai, None; Ji-Jing Pang, None; Jia Qu,
None
Support: This research was supported by grants of Major Projects of the National
Science and Technology of China (No: 2009ZX09503), Specilized Research Fund
for the Doctoral Program of Higher Education (No: 20

Comparison of Suprachoroidal delivery via an Ab-Externo approach with the
iTrack Microcatheter versus Vitrectomy and subretinal delivery for 3
different AAV Serotypes for Gene Transfer to the Retina
Gina Martorana1A, Monica Levine1A, Marc Peden1A, Shannon Boye1A, Zachary
Lukowski1A, Jeffrey Min1A, Craig Meyers1B, Sanford Boye1A, Mark Sherwood1A.
A
Ophthamology, BMolecular Genetics/Microbiology, 1University of Florida,
Gainesville, FL.
Purpose: Due to the invasive nature of subretinal injection and its potential to
cause retinal damage, especially when performed under fragile, degenerate retina,
there is impetus to develop less invasive delivery methods by which viral vector
can gain access to the posterior retina. In this study, we compared the efficacy
ofadeno-associated viral (AAV) the delivery to the suprachoroidal space using the
posterior iTrack illuminated microcatheter (iScience, CA) versus standard threeport pars plana vitrectomy/subretinal injection. We made additional comparisons
between the ability of three different AAV serotypes to transfect tissues, and
evaluated whether transduction was affected by vector concentration.
Methods: Eight age-matched New Zealand White rabbits were used to evaluate the
two delivery methods. The iTrack illuminated microcatheter was used to deliver
vector or balanced saline solution (BSS) to eleven of the eyes; the remaining five
eyes received vitrectomy/subretinal injection. The eyes were randomly assigned to
receive AAV2, AAV5, or AAV2(triple) vector containing 3 tyrosine-phenylalanine
mutations on its capsid surface. BSS served as the negative control. Within each
group, two vector doses were administered (either 1x1012 and 4x1012, or 1x1012
and 9x1012 particles/mL). Rabbits were sacrificed 4 weeks post injection, and the
eyes enucleated, sectioned and stained with antibodies directed against GFP and
PNA lectin (a cone marker).
Results: AAV-mediated GFP expression was found in eyes that received both
vitrectomy/subretinal and iTrack injections. Expression varied among individual
treatment groups. Immunostaining of sectioned eyes revealed strong, intermediate,
minimal or no GFP expression from AAV2(triple), AAV2, AAV5 or BSS-injected
eyes, respectively. Transduction profiles were not affected significantly by vector
concentration.
Conclusions: The ab-externo, suprachoroidal iTrack approach successfully
mediates AAV transduction of the posterior retina, similar to that seen with pars
plana vitrectomy/subretinal injection. AAV2(triple) and AAV2 viral serotypes
showed much stronger retinal transfection than the AAV5.
Commercial Relationships: Gina Martorana, None; Monica Levine,
None; Marc Peden, None; Shannon Boye, None; Zachary Lukowski,
None; Jeffrey Min, None; Craig Meyers, None; Sanford Boye, None; Mark
Sherwood, None
Support: None

Ultrasound-Microbubble Mediated CNP Gene Transfer Lowered IOP in
Rabbits and Monkeys
Wenhan Yu1, Haotian Xiang1, Guo Liu1, Iok-Hou Pang2, Jun Chen1, Xuyang Liu1.
1
Ophthalmic Lab. & Dept. of Ophthalmol., West China Hospital, Sichuan
University, Chengdu, China; 2Glaucoma Research, Alcon Research Ltd, Fort
Worth, TX.
Purpose: To evaluate C-type natriuretic peptide (CNP) gene expression delivered
via ultrasound-microbubble mediated gene transfection and its effects on rabbit and
monkey intraocular pressure (IOP).
Methods: Mixture (80 l), containing plasmids (150 ng/l) encoding CNP or

Assessment of Visual Function by Electroretinography following rAAV2CHM/REP1 Gene Therapy in a Mouse Model of Choroideremia
Alun R. Barnard1, Tanya Tolmachova2, Miguel C. Seabra2, Robert E. MacLaren1,3.
1
The Nuffield Laboratory of Ophthalmology & Oxford Biomedical Research
Centre, University of Oxford, Oxford, United Kingdom; 2Molecular Medicine,
National Heart and Lung Institute, Imperial College London, London, United
Kingdom; 3Moorfields Eye Hospital & NIHR Biomedical Research Centre for
Ophthalmology, London, United Kingdom.
Purpose: Choroideremia is an X-linked disease that leads to degeneration of the
choriocapillaris, retinal pigment epithelium and photoreceptors. It is caused by
mutations in the CHM/REP1 gene, which encodes Rab escort protein-1 (REP1).
Electroretinogram (ERG) recording was used to investigate the safety and efficacy
of recombinant adeno-associated virus (rAAV) mediated gene delivery of
CHM/REP1 in mice.

Methods: To examine if REP1 overexpression causes an alteration in retinal
function, subretinal injection of 1 l of high titer (1E+12 gp/ml) rAAV2-REP1 was
performed on wildtype mice (C57BL/6, 1 month old, n=5). In all cases, the
contralateral eye received an equivalent subretinal injection of rAAV2-EGFP as an
internal control for non-transgene specific effects (REP1 cDNA exchanged for
EGFP gene, but otherwise identical AAV2 expression cassette).
To explore the potential of CHM/REP1 gene therapy to ameliorate retinal function,
Chmnull/WT mice were treated with subretinal injection of 2-3 l of rAAV2-REP1 at
high (1E+12 gp/ml, n=4) and low (1E+11 gp/ml, n=5) doses at 1 month of age.
Subretinal injection of an isotonic solution was performed on the contralateral eye
of each animal, as an internal control for surgical sham effects. Six months after
injection retinal function was assessed by ERG.
Results: In rAAV2-REP1 treated eyes, dark-adapted ERG responses could be
recorded across a 7 log unit range of flash intensity. Responses from rAAV2-EGFP
injected control eyes were significantly smaller (reduced amplitude of the a- and bwaves). In a related experiment, performed using 1/10th of the viral concentration
(1E+11 gp/ml for both rAAV2-REP1 and rAAV2-EGFP), response amplitudes
were almost identical in both eyes and very similar to those obtained after high titer
AAV2-REP1 injection.
In both sham and high dose rAAV2-REP1 treated eyes of Chmnull/WT mice,
intensity-dependent, dark-adapted ERG responses could be recorded. At high
stimulus intensities, there was a small but significant increase in the amplitude of
responses from high dose rAAV2-REP1 treated eyes compared to fellow sham
treated eyes. Conversely, quantification of ERGs in low dose rAAV2-REP1 treated
Chmnull/WT eyes showed a slight reduction in amplitude of a- and b-waves compared
to fellow sham treated eyes overall.
Conclusions: No toxic effects of rAAV2-REP1 on retinal function could be
identified, even at the highest dose at which toxic effects could be seen following
delivery of a similar construct expressing EGFP. A significantly greater
improvement in ERG was seen in the high dose compared to low dose rAAV2REP1, indicating that 1E+11 to 1E+12 gp/ml represents the pharmacological
threshold for dosing in this mouse model.
Commercial Relationships: Alun R. Barnard, None; Tanya Tolmachova,
None; Miguel C. Seabra, None; Robert E. MacLaren, None
Support: NIHR Biomedical Research Centres; Fight for Sight; The Health
Foundation; Wellcome Trust; Royal College of Surgeons of Edinburgh;
Choroideremia Research Foundation USA; Foundation Fighting Blindness
Establishing A Course For AAV-mediated Gene Therapy For Leber
Congenital Amaurosis-1 (LCA1)
Shannon E. Boye1, Sanford L. Boye1, Issam McDoom1, Wei Chieh Huang2, Sukanya
Karan3, Igor V. Peshenko4, Alexander M. Dizhoor4, Wolfgang Baehr3, Samuel G.
Jacobson2, William W. Hauswirth1. 1Ophthalmology, University of Florida,
Gainesville, FL; 2Scheie Eye Institute, Philadelphia, PA; 3Ophthal & Vis Sci Lab
#S6881, Univ of Utah Sch of Med, Salt Lake City, UT; 4Pennsylvania College of
Optometry, Salus University, Elkins Park, PA.
Purpose: Ongoing clinical trials for RPE65 Leber congenital amaurosis (LCA2)
have established that AAV can be used to safely deliver therapeutic transgene to
the retinal pigment epithelium thereby restoring useful vision to patients. However,
most retinal degenerations are caused by mutations in genes expressed in
photoreceptors, a cell type yet to be targeted in clinical trials. Mutations in retinal
guanylate cyclase-1 (GUCY2D) are the leading cause of another form of severe
pediatric retinal dystrophy, LCA1. GUCY2D encodes GC1, a protein expressed in
the outer segments of rod and cone photoreceptors which is involved in the
recovery phase of phototransduction. Cones and rods of LCA1 patients lack
function and degenerate over time. The purpose of this study was to test whether
GUCY2D gene replacement would restore retinal function and preserve
photoreceptors in the GC1/GC2 double knock-out (GCdko) mouse which is
currently the only model with phenotypic fidelity to LCA1 (both cones and rods
lack function and degenerate).
Methods: AAV8(Y733F) vector containing the human rhodopsin kinase (hGRK1)
promoter was used to deliver murine GC1 (mGC1) to the subretinal space of
GCdko mice at multiple postnatal time points (P18-108). Contralateral eyes served
as uninjected controls. Retinal structure and function were monitored for at least 1
year with optical coherence tomography (OCT) and electroretinography (ERG),
respectively. Visual behavior was assessed with Morris Water Maze. At 1 year,
mice were sacrificed and retinas evaluated with IHC. Relevant proteins and RNA
transcripts were isolated via immunoblot and RT-PCR, respectively. Treated and
untreated GCdko eyes and wild type controls were compared for relative GC1
activity and calcium sensitivity.
Results: Retinal function was restored and retinal structure (both rods and cones)
preserved in GCdko mice treated with AAV8(Y733F)-mGC1 over the long term (at
least 1 year). Behavioral and immunohistochemical analyses are underway.
Conclusions: This is the first demonstration that AAV-mediated GC1 expression is
therapeutic over the long term in a true phenotypic model of LCA1. Together with
the knowledge that LCA1 patients exhibit preservation of retinal structure for
decades, these results strongly support the development of an AAV-based clinical

vector for the treatment of this disease.
Commercial Relationships: Shannon E. Boye, # 61/327,521 (P); Sanford L.
Boye, # 61/327,521 (P); Issam McDoom, None; Wei Chieh Huang,
None; Sukanya Karan, None; Igor V. Peshenko, None; Alexander M. Dizhoor,
None; Wolfgang Baehr, None; Samuel G. Jacobson, None; William W.
Hauswirth, # 61/327,521 (P), AGTC, Inc. (I)
Support: NNRI Wynn Gund TRAP
Single Vector Prostaglandin Pathway Gene Therapy for Glaucoma
Eric M. Poeschla, Tommy Rinkoski, Dyana Saenz, Pimprapar Wongsrikeao.
Molecular Medicine, Mayo Clinic College of Medicine, Rochester, MN.
Purpose: Expression of prostaglandin F2 (PGF2) biosynthesis and response
pathway component transgenes from integrated transgenes in the anterior chamber
of the eye has been shown to reduce intraocular pressure (IOP) and hence
represents a candidate gene therapy for glaucoma. Founding work used multiple
lentiviral vectors to express individual protein components of the PGF2 pathway.
A single vector that consolidates components would facilitate clinical grade and
clinical scale lentiviral production and its application to glaucoma.
Methods: A one vector system (1VS) was generated that encodes codon-optimized
mRNAs for COX-2 (coCOX-2) and the FP receptor (coFPR) from a bidirectional
promoter. This vector was first compared to a previously published efficacious
two-vector system (2VS) in vitro for expression level, cellular localization, and
biological activity of the PG system proteins. The single vector, the established
two-vector system, or a GFP-expressing control vector were also injected into the
anterior chambers of domestic cats in order to transduce the trabecular meshwork,
which regulates aqueous humor outflow.
Results: 1VS-transduced cells expressed both transgenes and confocal microscopy
confirmed appropriate intracellular localization. Production of PGF2 was robust
using both the 1VS and 2VS. A transcriptional reporter assay for coFPR activity
revealed 1VS signaling 8.5-fold higher than 2VS. In vivo in the cat, an accessible
and affordable mammal with a large, primate-similar anterior chamber that has
been instrumental for validating glaucoma pharmacologics, both vector systems
were effective at lowering IOP over the 4-month study period, and were not
significantly different in hypotensive effect.
Conclusions: A single lentiviral vector gene therapy was effective in expressing
PG pathway components and mediated sustained IOP reduction in vivo.
Commercial Relationships: Eric M. Poeschla, None; Tommy Rinkoski,
None; Dyana Saenz, None; Pimprapar Wongsrikeao, None
Lentiviral Vector Gene Replacement Therapy in Stargardt Disease and Usher
Syndrome type Ib
J T. Stout1, David J. Wilson1, Richard Weleber1, Mark Pennesi1, Maureen Toomey1,
Peter Francis1, Jose A. Sahel, Jr.2, Isabelle Audo3, Saddek Mohand-Said3, Stuart
M. Naylor4. 1Ophthalmology, Casey Eye Institute-OHSU, Portland, OR; 2UMR-S
968, Institut de la Vision, Paris, France; 3CHNO des Quinze-Vingts, Centre de
Recherche Institut de la Vision, Paris, France; 4Oxford BioMedica UK Ltd, Oxford,
United Kingdom.
Purpose: Single gene disorders are responsible for several conditions that lead to
profound vision loss including Stargardt disease and Ushers syndrome. These
diseases may be amenable to gene replacement therapy and trials are underway to
evaluate safety and efficacy of this therapeutic approach. The purpose of this report
is to describe the surgical technique and endpoint determination for lentiviral
vectors based on an EIAV-based platform in treating inherited retinal
degenerations.
Methods: Phase I/II trials are designed principally to evaluate the safety and
efficacy of gene replacement therapy in patients with Stargardt Disease and Usher
Syndrome type 1b. Eligible patients must have disease-causing mutations on both
chromosomes, confirmed by segregation analysis, for the gene ABCA4 (Stargardt
disease) or MYO7A (Usher 1B). Study design is that of dose escalation to determine
safety in an initial cohort followed by efficacy assessment in subsequent cohorts.
Lentiviral vectors (EIAV-ABCA4 for Stargardt, EIAV-MYO7A Usher 1b) are
administered subretinally following vitrectomy and efficacy endpoints consist of
visual acuity, OCT, GATE visual field, multifocal and full field ERG and spacing
of the cone mosaic (as assessed with adaptive optics).
Results: The Stargardt disease trial is underway following FDA and AFSSAPS
approval, FDA approval has also been received for the Usher trial. Thus far,
subretinal fluid reabsorption has been rapid and complete and without
inflammatory consequence.
Conclusions: No toxicity has been noted in Stargardt disease patients undergoing
gene replacement therapy with EIAV-ABCA4 in the first cohort. The lentiviral
vector platform has the potential advantage of permanent expression of gene

products implicated in retinal disease and in particular those that are too large for
other available vectors.
Commercial Relationships: J. T. Stout, Oxford Biomediuca is sponsoring the
clinical trial (F); David J. Wilson, Oxford Biomedica is sponsoring the trial (F);
Richard Weleber, Oxford Biomedica is sponsoring the trial (F); Mark Pennesi,
Oxford Biomedica is sponsoring the trial (F); Maureen Toomey, Oxford
Biomedica is sponsoring the clinical trial (F); Peter Francis, Oxford Biomedica is
sponsoring the clinical trial (F); Jose A. Sahel, Jr., Oxford Biomedica is
sponsoring the clinical trial (F); Isabelle Audo, Oxford Biomedica is sponsoring
the clinical trial (F); Saddek Mohand-Said, Oxford Biomedica is sponsoring the
clinical trial (F); Stuart M. Naylor, Employee of Oxford Biomedica (E)
Support: Oxford Biomedica
265 Aqueous Humor / Intraocular Pressure
Monday, May 7, 2012, 1:45 PM - 3:30 PM
Relationships between Episcleral Venous Pressure and Ocular and Systemic
Variables in Healthy Subjects
Arthur J. Sit, Nitika Arora, Jay W. McLaren. Ophthalmology, Mayo Clinic,
Rochester, MN.
Purpose: Episcleral Venous Pressure (EVP) is an important determinant of
intraocular pressure (IOP) according to the Goldmann equation. While previous
studies have investigated the parameters that affect IOP, the parameters that affect
EVP alone are poorly understood. In this study we examined the relationships
between EVP and physiologic parameters including blood pressure, ocular, and
systemic variables in healthy volunteers.
Methods: EVP was measured in 74 eyes of 37 healthy volunteers (ages: 46 to 80
years) by using a computerized, slitlamp mounted, episcleral venomanometer based
on the pressure chamber technique. Venous pressure was assumed to be equal to
the pressure in the chamber when the vein first began to collapse as the chamber
was inflated at a constant rate. Age, central corneal thickness (CCT, by ultrasonic
pachymetry), IOP, refractive error, height, weight, and blood pressure in the seated
position were measured. Body mass index (BMI) was calculated. Correlations
between EVP and these variables were examined by using generalized estimating
equation models to account for the possible correlations between eyes from the
same subjects.
Results: Means and correlations between the physiologic variables and EVP are
listed in Table 1. EVP was not significantly correlated with age, CCT, IOP, BMI or
pulse pressure (p>0.3). EVP was correlated with refractive error (p=0.03), systolic
blood pressure, and diastolic blood pressure (p=0.002 and p<0.001 respectively).
Conclusions: In normal subjects, EVP is weakly correlated with refractive error,

systolic and diastolic blood pressures, but is not correlated with the other variables
that we measured. This relationship suggests that IOP may be affected by blood
pressure through EVP. Changes in blood pressure must be considered when
investigating the effects of systemic treatments on aqueous humor dynamics. The
lack of a correlation between EVP and IOP suggests that other aqueous humor
dynamics parameters are primarily responsible for the variations in IOP within
healthy subjects.
Commercial Relationships: Arthur J. Sit, None; Nitika Arora, None; Jay W.
McLaren, None
Support: American Health Assistance Foundation, Research to Prevent Blindness,
Mayo Foundation for Medical Education and Research
Enhancement of -Opioidergic-Receptor Activity Provides Retina
Neuroprotection Against Glaucomatous Injury
Yasir Abdul, Shahid Husain. Ophthalmology, Medical University of South
Carolina, Charleston, SC.
Purpose: This study was designed to determine the neuroprotective activity of opioid-receptors and associated cellular mechanisms against glaucomatous injury
in a chronic glaucoma rat model.
Methods: Brown Norway rats were used to elevate intraocular pressure (IOP) by
injecting 50 L of 2 M hypertonic saline into the circumferential limbal veins. IOP
was recorded as the average of 6-8 consecutive measurements prior to surgery
(baseline IOP) and weekly after treatment, using a calibrated Tonolab tonometer.
Animals were treated with -opioid-receptor agonist, SNC-121 (1
mg/kg; i.p.), daily for 7 days. Pattern electroretinograms (PERG) and retinal
ganglion cells (RGCs) in flat-mounts were counted at week-8 post injury. The
expression of TNF- and MMP-9 were determined by immunohistochemistry
(IHC) on day-7, post injury.
Results: Significant IOP elevation was seen as early as 7 days, and maintained for
up to 8 weeks, post injury. PERG amplitudes were significantly reduced in ocularhypertensive eyes (14.030.69 volts) when compared to normal eyes (18.380.69
volts) at week-8, post injury. PERG deficits in hypertensive eyes were
significantly improved by SNC-121 treatment (17.351.76 volts; P<0.05). There
was a 26% loss of RGCs in the hypertensive eye when compared to the normal eye
at week-8, post injury. The loss in RGCs was fully blocked when animals were
treated with SNC-121. To dissect out the early cellular events during glaucomatous
injury, retinal samples were analyzed for TNF-, MMP-9, and NF-B at day-7,
post injury. In ocular hypertensive eyes TNF-, MMP-9, and NF-B were severalfold up-regulated. Both TNF- and MMP-9 were inhibited in SNC-121 treated
ocular hypertensive eyes.
Conclusions: These data provide concrete evidence that enhancement of opioidergic-receptor activity by exogenous ligand provides retina neuroprotection
against glaucomatous injury. Mechanistic data provide clues that TNF- and MMP9 are produced in the early phase of injury and suppressed by activation of opioid-receptors. These findings support the idea that enhancement of -opioidergic
activity in the eye may present a viable neuroprotective strategy for the treatment of
glaucoma.
Commercial Relationships: Yasir Abdul, None; Shahid Husain, None
Support: NIH (EY-019081)
A Novel Hypothesis: The Dorsomedial Hypothalamus Regulates Circadian
Fluctuations of Intraocular Pressure
Brian C. Samuels1, Nathan M. Hammes1, Philip L. Johnson2, Anantha Shekhar2,
Stuart J. McKinnon3, R. R. Allingham3. 1Ophthalmology, Eugene & Marilyn Glick
Eye Inst, Ind Univ, Indianapolis, IN; 2Psychiatry, Indiana University School of
Medicine, Indianapolis, IN; 3Ophthalmology, Duke Eye Center, Durham, NC.
Purpose: Fluctuation in intraocular pressure (IOP) has recently been identified as
an independent risk factor for progression of glaucoma. We hypothesize that
circadian fluctuations in IOP are regulated in part by neurons in the dorsomedial
hypothalamus (DMH), a currently undescribed function of this nucleus. The
purpose of this study was to determine whether site-directed chemical stimulation
of the DMH evokes increases in IOP.
Methods: The femoral artery of isoflurane-anesthetized Sprague-Dawley rats (250300g; n=6) were cannulated for continuous monitoring of heart rate (HR) and blood
pressure (BP), while the cisterna magna space was cannulated to monitor
intracranial pressure (ICP). The femoral artery and ICP lines were connected to
high sensitivity pressure transducers attached to a PowerLab data acquisition
system (AD Instruments). An iCareLab tonometer was used to record IOP every 2
minutes throughout the study. Using a stereotaxic approach, a microinjection of the
GABA-A receptor antagonist bicuculline methoidide (BMI; 30pmol/75nL) was
targeted to the DMH to stimulate the neurons. The resulting increases in HR, BP,
ICP, and IOP were recorded.
Results: Chemical stimulation of the DMH evoked significant increases in HR,
mean arterial pressure (MAP), and ICP with peak values occurring 5-10 minutes
after injection. A significant increase in IOP peaked approximately 30 minutes
post-injection.
Conclusions: These data are the first to support the notion that stimulation of
neurons in the region of the DMH mediate increased IOP. The DMH receives
strong direct and indirect projections from the suprachiasmatic nucleus (Chou et al.,
2003) and has extensive efferent projections to autonomic sympathetic relays.
Therefore, the DMH neurons are ideally situated to modulate circadian fluctuations
in IOP. Broadening our understanding of this pathway may shed light on the
mechanisms that underlie circadian fluctuations in IOP and may provide novel
therapeutic targets for glaucoma therapy.
Commercial Relationships: Brian C. Samuels, None; Nathan M. Hammes,
None; Philip L. Johnson, None; Anantha Shekhar, None; Stuart J. McKinnon,
None; R. R. Allingham, None

Support: AHAF NGR G2011012 (B. Samuels, PI) and NIH/NCRR KL2
RR025760 (A. Shekhar, PI)
Blocking of TNF--Induced Signaling Events by -Opioid Agonist in the Optic
Nerve
Naseem Akhter, Shahid Husain. Ophthalmology, Medical University of South
Carolina, Charleston, SC.
Purpose: Current study examined if enhancement of -opioidergic activity opposes
the production of TNF- within the optic nerve against glaucomatous injury.
Moreover, studies also determined if TNF--induced secretion of MMP-2, MMP-3,
or NF-kB expression in optic nerve head (ONH) astrocytes is blocked by -opioidreceptor activation.
Methods: Brown Norway rats were used to elevate intraocular pressure (IOP) by
injecting 50L of 2M hypertonic saline into the circumferential limbal veins. IOP
was recorded as the average of 6-8 consecutive measurements prior to surgery
(baseline IOP) and weekly after treatment, using a calibrated Tonolab tonometer.
Immediately after saline injections, animals were treated daily with -opioidreceptor agonist, SNC-121 (1mg/kg; i.p.), for 7 days. Retinas were collected at 3, 7,
and 42 days post injury. Primary cultures of human ONH astrocytes were isolated
and purified by immunopanning. Cells were treated with SNC-121 (1M), 15
minutes prior to TNF- (25 ng/mL) treatment for 6 hours. The expression of TNF, MMP-2, MMP-3, and NF-kB was determined by Western blotting.
Results: In ocular hypertensive eyes, expression of TNF- was significantly
(P<0.05) up-regulated in the optic nerve when measured between 3-42 days post
injury. TNF- production was completely inhibited when animals were treated with
the selective -opioid-receptor agonist, SNC-121. To dissect out the TNF-induced signaling mechanisms, ONH astrocytes were treated with TNF- for 6
hours in the presence or absence of 1M SNC-121, followed by analysis of MMP2, MMP-3, and NF-B expression. TNF- treatment increased the secretion of
MMP-2 and MMP-3 by 870215 and 588168%, respectively, at 6 hours when
compared with controls. The MMP-2 and MMP-3 secretion was reduced to 1718
and 23848%, respectively, at 6 hours, when cells were pre-incubated with 1M
SNC-121. Additionally, TNF- increases NF-kB (p65) expression by 615%,
which was inhibited significantly (P<0.05) in the presence of SNC-121.
Conclusions: These data provide evidence that TNF- is produced from glial cells
in the early phase of glaucomatous injury, and remain significantly elevated during
disease progression. Mechanistic data in ONH astrocytes provide clues that opioid-receptor activation opposes the secretion of MMP-2 and MMP-3 by
inhibiting the upstream regulator transcription factor, NF-kB (p65).
Commercial Relationships: Naseem Akhter, None; Shahid Husain, None
Factors Affecting The Long-term Efficacy Of Latanoprost In Congenital
Glaucoma
Maurizio G. Uva1A, Antonio Longo1A, Michele Reibaldi1A, Valentina Cifalin1A,
Alfredo Pulvirenti1B, Carlo Rapisarda1A, Salvatore Faro1A, Teresio Avitabile1A,
Alfredo Reibaldi1A. AInstitute of Ophthalmology, BDepartment of Mathematics and
Computer Science, 1University of Catania, Catania, Italy.
Purpose: To investigate the factors affecting the long-term efficacy of latanoprost
in primary congenital glaucoma (PCG) and Childhood glaucoma (ChG).
Methods: Patients with PCG or ChG treated with latanoprost 0.005% eyedrops
were re-examined. At study visit and from clinical charts were evaluated:
intraocular pressure, length of glaucoma control with latanoprost treatment, need of
further medication or glaucoma surgery, systemic and topical side effects. A
congenital glaucoma severity score was calculated by clinical parameters.
Results: Forty-six eyes of 27 patients (35 with PCG; 11 with ChG) had been
treated with latanoprost. Eleven eyes had received previous surgery. Mean followup was 9147 months. Latanoprost therapy was successful over the long term in 18
eyes (39%), 6 eyes (13%) had required an additional therapy, 19 eyes (41%) had
received surgery. For 3 PCG eyes (7%), latanoprost had been discontinued. In the
eyes with successful glaucoma control by latanoprost, mean IOP reduction was
8.51.8 mmHg (35.6%). Factors allowing the prediction of the success of treatment
(glaucoma control with latanoprost) were established through a decision tree
induction (with Chi-Squared Automatic Interaction Detection -CHAIDsegmentation technique). These are: glaucoma diagnosis at age greater than 4
months (success rate: 37% in eyes with glaucoma diagnosis at age lower/equal than
4 months, vs 63% in eyes with glaucoma diagnosis at age greater than 4
months,chi-square p=.004), and in this latter group, no previous glaucoma surgery
(success rate: 48% in eyes without previous glaucoma surgery vs 15% in eyes with
previous glaucoma surgery, chi-square p=0.029). The length of glaucoma control
by latanoprost was related to the age at treatment (linear regression r=0.519,
p<0.001), and, in children treated at age<1 year, to severity score (linear regression
r= -0.522, p=0.015). The mean length of treatment was lower in PCG than in ChG
eyes (3840 and 7847 months, t-test P=0.023). No patient discontinued the
treatment because of side effects (in 4 eyes mild conjunctival hyperaemia; in 1 eye
increase of iris pigmentation and eyelash changes, both regressed 3 years after
discontinuation because of low IOP; in 1 patient irritation of the upper airways that
regressed with systematic nasolacrimal occlusion on instillation of drops).
Conclusions: Long-term treatment with latanoprost in primary congenital
glaucoma is effective in about a third of the treated eyes; factors related to longterm success of the treatment were age at the diagnosis greater than 4 months, and
no previous glaucoma surgery; the glaucoma control by latanoprost was longer in
eyes with less severe glaucomatous alterations and in ChG.
Commercial Relationships: Maurizio G. Uva, None; Antonio Longo,
None; Michele Reibaldi, None; Valentina Cifalin, None; Alfredo Pulvirenti,
None; Carlo Rapisarda, None; Salvatore Faro, None; Teresio Avitabile,
None; Alfredo Reibaldi, None
Support: None
Ocular Surface Alteration and Topical Antiglaucomatous Therapy: Symptoms
vs Clinical Signs
Teresa Rolle, Daniela Curto, Elena Isaia, Francesco Damiani, Pietro Lanzafame,
Alessandro G. Actis. Clin Physiopathol-Section of Opht, University of Torino,
Torino, Italy.
Purpose: To evaluate the ocular surface disease in glaucomatous and ocular
hypertensive patients treated with IOP-lowering drugs with preservative
(benzalconium chloride) and to analyze the correlations between symptoms and
clinical signs.
Methods: This was a prospective observational study. 106 consecutive patients
with open angle glaucoma or ocular hypertension were enrolled in the study. Each
patient completed the Ocular Surface Disease Index questionnaire; Schirmer test,
tear break-up time and lissamine green staining of the conjunctiva were performed.
For each patients we considered the eye that showed worse results for each test. A
multivariate regression analysis was used to investigate the relationship between
OSDI and clinical tests, OSDI and the number of antiglaucomatous drugs. 2 test
was used to evaluate the correlation between OSDI and the duration of treatment. P
values < 0.05 were considered significant.
Results: 102 patients (96,2%) reported symptoms in at least 1 eye; 24 (22.6%) mild
to moderate; 78 (73,6%) severe. OSDI score was related with the number of drugs
used (p<0.01). No correlations were found with the duration of treatment. Schirmer
test was altered in 89 patients (84%): 57 patients (53.8%) showed a mild- moderate
decrease in tear production and a severe deficiency was observed in 32 patients
(30,2%). Tear break-up time was reduced in 80 (75.5%) patients: mild to moderate
in 71 (67%) and severe in 9 (8.5%). Conjunctival lissamine green staining showed
positive results in 60 (56.6%) patients: mild to moderate in 46 (43.4%) and severe
in 14 (13.2%). Agreement on the results of the 3 clinical tests was obtained in 43
patients (40.6%) followed by tear break-up time and Schirmer test in 30(28.3%).
Poor correlation was observed between symptoms and clinical signs: a significant
correlation was found only for OSDI vs Schirmer test (p<0.05).
Conclusions: A large number of glaucomatous and ocular hypertensive patients
had an abnormal OSDI and the score increases with the greater the number of
glaucoma drugs prescribed. A large proportion of patients who had a severe OSDI
had a normal or mild to moderate alterations of clinical tests. It can be assumed that
other factors (psychological, environmental , severity and awareness of the
disease) can influence OSDI.
Commercial Relationships: Teresa Rolle, None; Daniela Curto, None; Elena
Isaia, None; Francesco Damiani, None; Pietro Lanzafame, None; Alessandro G.
Actis, None
Support: None
Characterization Of Patients Undergoing Tube Shunt Or Trabeculectomy
Surgery For Uveitis-related Intraocular Pressure Elevation
Jonathan J. Tsang, Umair Iqbal, Ralf R. Buhrmann, Chloe C. Gottlieb. University
of Ottawa Eye Institute, Ottawa, ON, Canada.
Purpose: To compare demographic data, including uveitis and glaucoma diagnosis,
of patients with uveitis-related intraocular pressure elevation undergoing tube shunt
or trabeculectomy surgery.
Methods: Patients were identified by an electronic search of one surgeons billing
records for glaucoma filtering procedure codes. Inclusion criteria were diagnosis of
uveitic glaucoma or elevated intraocular pressure associated with uveitis and either
tube shunt or trabeculectomy surgery. A chart review was conducted and
information tabulated for: age, gender, uveitis diagnosis, angle status.
Results: Of 37 patients diagnosed with uveitis, 23 (62%) were male and 14 (38%)
were female. The mean age was 60 years (range: 18 to 95 years). Open angle
glaucoma (OAG) was present in 25 cases (67.6%). There were 17 males with OAG
(68%), while only 8 females (32%) had OAG. Six patients had angle closure

glaucoma (ACG) (16.2%). The number of males with ACG was 5 (83.3%) and that
of females with ACG was 1 (16.6%). One patient had mixed mechanism glaucoma
(2%). The angle status of 2 uveitic glaucoma cases (5.4%) was unknown. Three
patients had ocular hypertension (OHT, 8.1%), of which 1 had angle closure and 2
had an unknown angle status. The number of females with OHT was 2 (66.6%) and
that of males was 1 (33.3%).
Conclusions: In the patients studied, the age range was broad and the majority of
patients were male. OAG diagnosis was more frequent than ACG diagnosis. Both
OAG and ACG were more prevalent in males while uveitis related OHT with no
glaucoma was diagnosed in more females than males. Only a minority of patients
developed OHT without glaucoma. One patient with OHT had angle closure and
the status of angle in the other 2 patients was unknown.
Commercial Relationships: Jonathan J. Tsang, None; Umair Iqbal, None; Ralf
R. Buhrmann, None; Chloe C. Gottlieb, None
Support: None
(MT1/MT2), 4-PPDOT (MT2) and Prazosin (MT3), the first did not modify the
effect of agomelatine while the selective MT2 antagonist 4-PPDOT reduced IOP to
85.6 1.6 % and Prazosin to 87.2 1.9 % (n=18). The effect of agomelatine was
compared to other commercial compounds such as Xalatan, Trusopt, Timolol and
Alphagan, in this animal model. Among them only Trusopt and Timolol were more
effective reducing IOP their values being 71.0 2.0 % and 73.6 1.8 %
respectively.
Conclusions: Agomelatine present interesting properties reducing IOP. This is
interesting since this compound belongs to melatonin analogues and not ot classical
hypotensive drugs. The development of new melatonin compounds should expand
the limited repertoire of drugs currently used for the treatment of glaucoma.
Commercial Relationships: Jesus J. Pintor, None; Antonio Bergua,
None; Alejandro Martinez-Aguila, None
Support: SAF2010- 16024, BSCHUCM (GR58/08), RETICS RD07/006/0004 and
(PR34/07-15785).

The Relationship of Body Mass Index and Blood Pressure to Intraocular
Pressure in Patients with Glaucoma
Sandra Ngo1A, Alon Harris1A, Brent A. Siesky1A, Ingrida Januleviciene2, John
Abrams1A, Barbara M. Wirostko3, Yara Catoira1A, Annahita Amireskandari1A,
George Eckert1B, Erin Stewart1A. AOphthalmology, BDivision of Biostatistics,
1
Indiana University School of Medicine, Indianapolis, IN; 2Eye Clinic, Kaunas
University of Medicine, Kaunas, Lithuania; 3Ophthalmology, University of Utah,
Park City, UT.
Purpose: To investigate the relationship between intraocular pressures (IOP), body
mass index (BMI) and systemic blood pressure in patients with open angle
glaucoma (OAG).
Methods: IOP and systemic blood pressure were examined in patients with OAG
at baseline and after 2 years follow up. 31 normal weight, 39 overweight and 27
obese patients were analyzed according to body mass index calculations at each
visit. Blood pressure was measured using automated ambulatory measurements
after five minutes rest and IOP was measured using Goldmann applination
tonometry. Associations between of changes in IOP and blood pressure were
analyzed using Pearson correlation coefficients.
Results: IOP decreased from baseline to two-year measurement in normal weight
(-1.5, 95%CI -2.7-(-0.4)), in overweight (-1.9, 95%CI -3.4-(-0.4)), and in obese (2.5, 95%CI -3.9-(-1.2)) OAG patients. Systolic blood pressure (SBP) decreased
from baseline to two-year measurement, although not reaching statistical
significance, in normal weight (-3.8, 95%CI -12.3-4.7), in overweight (-2.3 95%CI
-8.3-3.8), and in obese (-3.9, 95%CI -10.9-3.2). In normal weight patients, there
was a positive correlation between changes in IOP and SBP (r=0.36, p=0.0431).
However, no correlations between IOP and SBP changes were observed in an
overweight population (r=-0.08, p=0.65) or an obese population (r=0.09, p=0.64).
No significant correlations were found with diastolic blood pressure.
Conclusions: In normal-weight individuals with OAG, changes in SBP are
positively correlated to changes in IOP after two years. This relationship between
IOP and SBP was not observed for overweight or obese individuals.
Commercial Relationships: Sandra Ngo, None; Alon Harris, Pfizer (C); Brent
A. Siesky, None; Ingrida Januleviciene, None; John Abrams, None; Barbara
M. Wirostko, Altheos (C), Merck (C), Pfizer (C); Yara Catoira, None; Annahita
Amireskandari, None; George Eckert, None; Erin Stewart, None
Support: Unrestricted grant from Research to Prevent Blindness and Pfizer
Pharmaceuticals

Assessment Of Intra-ocular Pressure Lowering Effect Of Novel Gel
Formulations Of Latanoprost And Timolol In Nonhuman Primates Following
Topical Delivery
Robin J. Goody1, Steve Whittaker1, Steve Henry1, Rohn Brookes1, Michael
Struharik1, Franck Bermon2, Philippe Margaron2, Heinz Polzer3, Matthew S.
Lawrence1. 1RxGen, Inc, Hamden, CT; 2Iris Pharma, La Gaude, France;
3
MedProject Pharma, Oberhaching, Germany.
Purpose: This study was designed to evaluate the ability of novel gel formulations
containing latanoprost and timolol to lower intraocular pressure (IOP) in sedated
nonhuman primates.
Methods: Following prescreening to confirm IOP-lowering response to
latanoprost, 12 adult African green monkeys were recruited for a multi-week crossover dosing regimen comprising 4 separate weeks of dosing, on which 4
consecutive days of once-daily topical dosing (50 l) occurred. Dosing weeks were
separated by 10 day washout periods. Dosing sequence was assigned in a Latin
square design to 4 treatment groups, based on baseline IOP measures. On study day
1 animals received either gel vehicle, latanoprost 0.0025%/timolol 0.5% gel
(formulation 1), Xalacom (latanoprost 0.005%/timolol 0.5%) or latanoprost
0.005%/timolol 0.5% gel (formulation 2) after collecting baseline IOP measures.
Follow-up IOP measures were performed immediately prior to and 6 hours after
dosing on the first and fourth day of dosing on each study week. Statistical analyses
were conducted on absolute IOP values. Studies were conducted in compliance
with the ARVO Statement for the Use of Animals in Ophthalmic and Vision
Research.
Results: Administration of Xalacom resulted in significant IOP lowering at 6 hours
after dosing on day 1 of dosing (2.0 mmHg; p<0.0001) compared with response to
vehicle treatment in the same animals (0.2 mmHg). Administration of either gel
formulation also resulted in significant IOP lowering (formulation 1=2.9 mmHg,
p=0.0054; formulation 2=2.4 mmHg, p=0.0004) compared with vehicle at 6 hours
after dosing on day 1. No significant differences were observed between Xalacom
and either gel formulation. A similar magnitude of IOP lowering activity was
observed 24 hours after administration of the third of 4 daily doses for either gel
formulation and for Xalacom, suggesting a sustained IOP lowering effect. No
further significant IOP lowering was observed at 6 hours after administration of the
fourth daily dose.
Conclusions: The gel formulations tested demonstrated similar IOP lowering
activity to Xalacom in a nonhuman primate model. Results suggest the gel
formulations may facilitate delivery and provide effective and sustained IOP
lowering activity at a lower latanoprost strength, meriting further study.
Commercial Relationships: Robin J. Goody, None; Steve Whittaker,
None; Steve Henry, None; Rohn Brookes, None; Michael Struharik, None;
Franck Bermon, Iris Pharma (E); Philippe Margaron, Iris Pharma (E); Heinz
Polzer, MedProject Pharma (E); Matthew S. Lawrence, None
Support: None

Topical Application of Anti-depressant Compound Agomelatine, a melatonin
analogue, Reduces Intraocular Pressure
Jesus J. Pintor1, Antonio Bergua2, Alejandro Martinez-Aguila1. 1Bioquimica y
Biologia Molecular IV, E U de Optica UCM, Madrid, Spain; 2Department of
Ophthalmology, University of Erlangen-Nuremberg, Erlangen, Germany.
Purpose: To investigate the possible hypotensive effect of the anti-depressant
compound agomelatine, a melatonin analogue, in New Zealand rabbits
Methods: New Zealand white rabbits, weighting 3-4 Kg, were used for IOP
studies. Agomelatine (Santa Cruz) was formulated in isotonic saline containing 1%
DMSO (Sigma) and tested at different concentrations from 10-12 M to 10-4 M and
was applied to the cornea at a fixed volume of 10 L in the treated animals. The
control animals received the same volume of saline + 1% DMSO. IOP was
measured by means of a TonoVet contact tonometer supplied by Tiolat Oy.
Luzindole, Prazosin and 4PPDOT were used as antagonists of melatonin receptors.
Results: Agomelatine applied at a concentration of 100 uM reduced IOP to 79.2
1.4 % compared to control (100 %). This maximal effect was obtained 180 min
after the application of the compound (n=8). Concentration-response curve for this
melatonin analogue presented a pD2 value of 9.7 0.3, which was equivalent to an
EC50 of 0.19 nM. Among the tested melatonin receptor antagonists, luzindole

Studies On A Sensitive Hplc Method For The Determination Of
Raceanisodamine In Rabbit Aqueous Humor And The Pharmacokinetics Of
Its Eyedrops
Lan Ding. Department of Ophthalmology, Eye and ENT Hospital, Shanghai, China.
Purpose: To assess the ocular absorption of raceanisodamine, an analytical HPLC
method was developed for the determination of raceanisodamine in rabbit aqueous
humor and to study its pharmacokinetics in rabbit aqueous humor.
Methods: Proteinaceous material in rabbit aqueous humor is extracted by ethyl
acetate. An aliquot of the supernatant is analyzed by HPLC. The chromatography
was performed on a reversed-phase encapped column (Dikma, 250*4.6 mm, i.d. 5
m C18 ) at 30C with a mobile phase consisting of acetonitrile in 10mM
monopotassium phosphate solution with 0.47 mlL perchloric acid acetate (pH 3.0,

11.5: 88.5, v/v). The flow rate was 1. 0 mlmin-1. Raceanisodamine was monitored
with UV detector at 214 nm.
Results: Good linearity between concentration of raceanisodamine (c, ngmL-1 ) in
aqueous humor and peak area was obtained for y=0.226x+0.008,R=0.996(n=3)in
the range from 0.1-0.15 ngmL-1. The lower limit of quantification (LOD) of the
method was 20.0 ngmL-1, indicating that the method is adequate for sensitive
pharmacokinetic studies. The average recoveries were determined as 122.8%,
97.06% and 106.7% at a concentration of 0.2, 1, 3 ngmL-1. With in-day and interday coefficients of variation were lower than 5%. 24 healthy rabbits were given the
eye drops to study the pharmacokinetic bioavailability of the raceanisodamine eye
drops. The concentration-time course followed a 1-compartment open model and
the major parameters were listed: tmax: (10.003.01)h, Cmax: (12401.918.34)
ngmL-1, t 1/2 (44.772.19)h, AUC 0-8: (29065.1644.78) ngmL-1, MRT:
(30.70.63)h.
Conclusions: This method is selective, sensitive and reproducible for the
determination of raceanisodamine in rabbit aqueous humor and is suitable for the
pharmacokinetic study of raceanisodamine in ophthalmic system.
Commercial Relationships: Lan Ding, None
Support: The Science and Technology Commission of Shanghai Municipality
grant 09DZ1906700
Prednisolone Induces Time- And Dose-dependent Ocular Hypertension In
Rats
Bing Li, Erica Perez, David Cantu-Crouch, Yu Wang, Shutong Cao, Laura Klekar,
Jamie Raines, David Earnest, Richard Ornberg, Ganesh Prasanna. Glaucoma
Research, Alcon Research Ltd, Fort Worth, TX.
Purpose: To develop a simple steroid-induced ocular hypertension model in rats.
Methods: Prednisolone pellets (0.1 mg, 1.5 mg, 2 x10 mg and 25 mg) were
implanted subcutaneously in Male Lewis rats and intraocular pressure (IOP)
readings were taken in a masked manner using a TonoLab rebound tonometer.
Body weights were monitored weekly. Correlation of plasma and aqueous humor
(AH) steroid levels and IOP elevation were made following implantation of pellets
(n=8 rats in each group). Ultrastructural changes to the anterior chamber angle and
trabecular meshwork (TM) and expression of select protein markers were assessed
using a) light microscopy, b) electron microscopy and c) immunohistochemistry of
-smooth muscle actin (-SMA), collagen IV and collagen VI.
Results: Prednisolone levels in plasma and AH increased in a dose-dependent
matter. No prednisone pellet treated group (except 50 mg) showed >20% weight
loss. Compared with baseline IOP and that for placebo rats, rats implanted with
prednisolone pellets (2 x 10 mg and 25 mg), exhibited significant IOP elevation
(15-28%; p<0.001) starting on weeks 2-3 post-implantation and was maintained till
the end of pellet release (21 or 60 days). In prednisolone implanted rats, topical
ocular dosing with Xalatan caused a biphasic response in IOP, with a transient
25% increase at 1 hour and 21% decrease at 24 hour post-dose. When compared to
placebo rats, prednisolone-implanted rats demonstrated ultrastructural changes to
the TM consisting of more compact layering of TM lamellae and reduced intralamellar spaces due to thickened beams or matrix deposition. Schlemms canal
(SC) appeared to be less prominent/ reduced in prednisolone rats. -SMA
immunolabeling was greatly increased (++) in the TM tissues in prednisoloneimplanted rats (++ in 63-75% rats) compared to placebo rats (++ in 0-42% rats).
Conclusions: Prednisolone treatment elicits reproducible ocular hypertension in
male Lewis rats. Ultrastructural changes in -SMA are indicative of a steroid effect
on TM as previously shown in human anterior chamber tissues (glaucomatous or
steroid-treated) and maybe involved in resistance to AH outflow. IOP in
prednisolone-implanted rats was responsive to topical dosing of a prostaglandin
analog. Implanting pellets is a simple technique and less labor intensive than
topical ocular dosing for delivering steroids over a long period of time. A
prednisolone-induced model of ocular hypertension in rats could be useful for
further investigations of the patho-mechanisms involved in glaucoma.
Commercial Relationships: Bing Li, Alcon Research Ltd (E); Erica Perez,
Alcon Research Ltd (E); David Cantu-Crouch, Alcon Research Ltd (E); Yu
Wang, Alcon Research Ltd (E); Shutong Cao, Alcon Research Ltd (E); Laura
Klekar, Alcon Research Ltd (E); Jamie Raines, Alcon Research Ltd (E); David
Earnest, Alcon Research Ltd (E); Richard Ornberg, Alcon Research Ltd (E);
Ganesh Prasanna, Alcon Research Ltd (E)
Support: None
Activation Of Erk Signaling Pathway By Bradykinin B2 Receptors In Isolated
Human Primary Ciliary Muscle Cells
Rajkumar V. Patil, Linya Li, Naj Sharif. Pharmaceutical Research, Alcon Research
Ltd, Fort Worth, TX.
Purpose: Bradykinin (BK) stimulation of B2 kinin receptors has been shown to
initiate signaling in trabecular meshwork cells and increase conventional outflow
facility. The ciliary body, which regulates aqueous outflow via uveo-scleral
pathway, also expresses both B1 and B2 kinin receptors and is a potential target for
kinin action. Here we investigated whether BK and related kinin peptides can
initiate signaling pathways involving ERK1 and ERK2, upstream of matrix
metalloproteinase (MMP) secretion in isolated primary human ciliary muscle (hCM) cells. The results support the possibility that B2 kinin receptors in ciliary body
may contribute to the regulation of aqueous outflow.
Methods: Primary h-CM cells were cultured in 96-well plates and incubated
overnight, at 37C in CO2 atmosphere. Cells were then starved off serum overnight
before being challenged with BK agonists or antagonists at room temperature.
Treated cell lysates were then evaluated for phosphorylated ERK1/2 using HTRF
technology based on a sandwich immunoassay using an anti-phospho-ERK1/2
antibody labeled with d2 and an anti-ERK1/2 antibody labeled with Eu3+-Cryptate.
The fluorescence signals were recorded at 620 nm for the donor and 665 nm for the
acceptor.
Results: BK treatment of h-CM cells caused an increase in ERK1/2
phosphorylation. The stimulation of ERK1/2 phosphorylation was 1.86 0.26 fold
(n = 3) in the presence of 100 nM BK compared to basal ERK1/2 phosphorylation.
The optimal time of stimulation of ERK1/2 phosphorylation was 10 min using 50K
cells/well. In addition, BK analogs Met-Lys-BK, and RMP-7 (100 nM) also
increased ERK1/2 phosphorylation in h-CM cells by 1.57 0.04 and 1.55 0.09
fold, respectively. However, Des-Arg9-Bradykinin, a B1 receptor selective agonist
(0.1-1M), did not increase ERK1/2 phosphorylation in h-CM cells.
Phosphorylation of ERK1/2 was significantly blocked when h-CM cells were pretreated with 100 nM of a peptide B2 receptor antagonist, HOE-140, or a nonpeptide B2 receptor antagonist, WIN-64338, before being exposed to BK agonists.
HOE-140 and WIN-64338 alone had no effect on phosphorylation of ERK1/2 in hCM cells.
Conclusions: BK caused a concentration- and time-dependent increase in ERK1/2
phosphorylation, which was attenuated by two selective B2 receptor antagonists
(HOE140; WIN-64338) in h-CM cells. In addition, Des-Arg9 Bradykinin, a B1
receptor selective agonist did not show any effect on ERK1/2 phosphorylation in hCM cells. These observations suggests that B2 kinin receptors initiate signaling in
h-CM cells by activating ERK1/2 which in turn may regulate MMP release and
ultimately regulate the uveo-scleral pathway resulting in modulation of intraocular
pressure.
Commercial Relationships: Rajkumar V. Patil, Alcon (E); Linya Li, Alcon
(E); Naj Sharif, Alcon (E)
Support: None
Remote and Continuous Monitoring of Intraocular Pressure Using Novel
Photonic Principle
Zeev Zalevsky1,2, Israel Margalit1, Yevgeny Beiderman1, Alon Skaat3, Michael
Belkin3, Ralf-Peter Tornow4, Vicente Mico5, Javier Garcia5. 1Engineering, Bar-Ilan
Univ, Ramat-Gan, Israel; 2Graduate School in Advanced Optical Technologies,
Friedrich-Alexander Universitt, Erlangen-Nrnberg, Germany; 3Goldshleger Eye
Research Institute, Tel-Hashomer, Ramat-Gan, Israel; 4Augenklini, Erlangen,
Germany; 5Departamento de ptica, Universitat de Valncia, Burjassot, Spain.
Purpose: To present the initial experimental testing of a new measurement
principle for continuous, remote non-contact and monitoring of intra-ocular
pressure (IOP).
Methods: A photonic device involving a fast camera and a laser was tested in
rabbits eyes for continuous remote monitoring of the IOP. The device is based on
tracking the secondary speckle patterns trajectories produced by reflection of an
illuminating laser beam from the iris, cornea or sclera. IOP fluctuations change the
speckle distributions reflected from the rabbits tissues that are acting as a
transducer element of the sensing system. The distribution is continuously
measured and analysed. The device is inexpensive since it requires only a laser, a
camera and a computer.
The anterior chambers of the eyes were canulated by an anterior chamber
maintainer connected to a saline infusion bag. The IOP was varied by changing the
elevation of the bag in respect to the position of the eye. The changes in the speckle
pattern was continually monitored and analysed.
Results: Data from the photonic device were correlated with the IOP fluctuations
resulting from raising and lowering the infusion bag. The measurements show a
good correlation and sensitivity of the proposed device with IOP changes while
providing a high precision measurement (5% estimated error) for the best
experimental configuration. The results obtained via the photonic approach were
also compared with a reference IOP measurement obtained with Goldmann
tonometer.
Conclusions: The first experimental testing of a new photonic device has been
performed with rabbits showing a promising new direction for remote and
continuous monitoring of IOP. The system provides a high precision and noninvasive measurement method for IOP monitoring over prolonged periods.

Commercial Relationships: Zeev Zalevsky, None; Israel Margalit,
None; Yevgeny Beiderman, None; Alon Skaat, None; Michael Belkin,
None; Ralf-Peter Tornow, None; Vicente Mico, None; Javier Garcia, None
Support: None
Characterization Of Nuclear Import And Export Of GR Following Dex
Treatment In Human Trabecular Meshwork Cell-line
Adnan Dibas1A,2, Yong Park1A,2, Abbot F. Clark1B,2, Thomas Yorio1A,2.
A
Pharmacology & Neuroscience, BCell Biology and Anatomy, 1University of North
Texas Hlth Sci Ctr, Fort Worth, TX; 2North Texas Eye Research Institute, Fort
Worth, TX.
Purpose: To characterize the role of the cytoskeleton and importins in steroidinduced glucocorticoid alpha (GR) receptor nuclear import and export in human
trabecular meshwork cells.
Methods: Stably-transfected RFP-GR cell lines were developed. Nuclear
accumulation of RFP-GR in NTM5 cells treated with vehicle (ethanol),
dexamethasone (DEX) or RU486 was measured in cytosolic and nuclear fractions
by western blotting and laser confocal microscopy. Cytochalasin D, and colchicine
were tested for their abilities to affect GR-trafficking. Nuclear export of RFP-GR
was studied using confocal microscopy following DEX or RU486 removal.
Importin and exportin proteins redistribution following DEX treatment was
assessed by western blot.
Results: NTM5 cells transfected with RFP-GR showed a clear cytosolic
localization of receptor that underwent translocation to the nucleus after DEX
treatment. While neither cytochalasin D nor colchicine blocked DEX- or RU486induced RFP-GR nuclear translocation, both drugs blocked nuclear export of
RFP-GR following DEX and RU486 removal. Interestingly, both nuclear import
and export of DEX-induced RFP-GR was much faster than RU-486-induced
nuclear shuttling. Importin-13 protein appears to translocate from nucleus to
cytosol following DEX treatment.
Conclusions: RFP-GR receptor behaves similarly to the wild type GR with its
cytosolic localization and shuttling to nucleus after DEX or RU486 treatment.
DEX-induced nuclear import and export of RFP-GR occurred at a much faster
rate that RU-486-induced shuttling of RFP-GR. It appears that the cytoskeleton
network is needed for nuclear export but not import of RFP-GR as only the
disruption of the cytoskeleton blocked the nuclear export of RFP-GR following
DEX removal.
Commercial Relationships: Adnan Dibas, None; Yong Park, None; Abbot F.
Clark, None; Thomas Yorio, None
Support: NEI
The Effect Of Benzalkonium Chloride On The Intraocular Pressure Lowering
Efficacy Of A Local Rock-inhibitor (AMA0076)
Karolien P. Hollanders1A, Davine Sijnave1A, Tine Van Bergen1A, Sarah Van de
Velde1A, Evelien Vandewalle1A, Lieve K. Moons1B, Dirk Leysen2, Ingeborg
Stalmans1A. ALab of Ophthalmology, BBiology Dept, Zoological Inst, 1KU Leuven,
Leuven, Belgium; 2Amakem NV, Diepenbeek, Belgium.
Purpose: To elucidate the effect of Benzalkonium chloride (BAK) on the
intraocular pressure (IOP) lowering efficacy of a local ROCK-inhibitor AMA0076
(Amakem NV) in the rabbit eye.
Methods: Topical administration of AMA0076 (0.1%, 0.3% and 0.5%) was tested
in a normotensive New Zealand White rabbit model (5 rabbits/group). The IOP
lowering efficacy of the compound was determined with or without the addition of
0.01% BAK. The contralateral eye was used as a control and was treated with
vehicle (H2O PEG) in all groups. IOP was measured at baseline and at 1, 2, 3, 4, 5,
6, 7 and 8h post administration.
Results: Single topical administration of AMA0076 significantly lowered IOP in a
concentration dependent manner compared to the control eye (p<0.05).
Placebo/vehicle administration did not induce significant changes in IOP. The
maximal IOP lowering effect of AMA0076 0.1%, 0.3% and 0.5% containing
0.01% BAK was 38%, 45% and 53%, which was significantly stronger compared
to their BAK-free equivalents: 21%, 28% and 37% (p=0.005, p=<0.001; p=0.008
respectively).
Conclusions: The local ROCK inhibitor, AMA0076 was significantly more
effective in lowering IOP in the presence of 0.01% BAK.
Commercial Relationships: Karolien P. Hollanders, None; Davine Sijnave,
None; Tine Van Bergen, None; Sarah Van de Velde, None; Evelien Vandewalle,
None; Lieve K. Moons, None; Dirk Leysen, Amakem NV (E); Ingeborg
Stalmans, None
Support: This research is supported by a grant provided by Amakem NV.
Enhanced Ocular Bioavailability Of Carbonic Anhydrase Inhibitor

Formulation With Novel Nanopolymers
Tapan Shah, Jun Inoue. Formulation Development, Senju USA, Inc., Pasadena,
CA.
Purpose: The purpose of this paper is to present a novel ophthalmic composition
comprising a hyperbranched polymer with carbonic anhydrase inhibitor (CAI).
COSOPT and TRUSOPT are commercially available topical ophthalmic
solutions developed for treating Glaucoma by inhibiting aqueous humor secretion.
Due to poor and limited aqueous solubility of Dorzolamide at physiological pH, the
pH of these commercial ophthalmic solutions (cont. 2% Dorzolamide) has to be
kept at about 5.65. This low pH can lead to local eye irritation. In this study, 1.3%
(w/v) CAI topical solution composition with Timolol was formulated at pH 7.0
containing 2% commercially available Polyethyleneimine hyperbranched polymer
(HP) that has comparable topical availability of CAI. The main advantage is
significant patient compliance because of enhanced eye comfort at pH 7.0.
Methods: The solubility of CAI (Dorzolamide and Brinzolamide) at pH 7.0 with
HP at room temperature, and the stability at 40 degrees Celsius and 60 degrees
Celsius for 4 weeks were determined with UPLC (Waters Acquity system, a
gradient 1% Triethylamine in water: Acetonitrile method, performed at room
temperature, with the flow rate of 0.7 mL/min, at 254 nm wavelength and 10 L
injection volume, was used on BEH C18 1.7 m, 2.1x 50 mm column.). In vitro
cornea permeation profile of Dorzolamide for 3 hours was evaluated by all-glass
side-by-side diffusion cell study using extracted New Zealand rabbits cornea. A
standard solution containing COSOPT active ingredients at pH 7.0 was used as a
control sample.
Results: The solubility of CAI at pH 7.0 increased with HP dose dependently, and
by at a factor of 2.5 with addition of 2% (w/v) HP to the solution. The long term
stability results also indicated that the new formulation was stable without any
precipitation. Consequently, the total cornea permeation rate of Dorzolamide,
partition coefficient and permeability coefficient at pH 7.0 were 2.5 fold higher
compared to the control sample.
Conclusions: A new water soluble CAI-beta blocker ophthalmic solution at pH 7.0
containing nanopolymer with comparable efficacy was discovered. The improved
permeation in the presence of HP was mainly because of improved partitioning of
CAI into the intact cornea epithelium.
Commercial Relationships: Tapan Shah, Senju USA, Inc. (E); Jun Inoue,
Senju USA, Inc. (E)
Support: None
Effect of Commonly Used Topical Anesthetics on Intraocular Pressure
Measurements
Nathan Welch, Shane Havens, Donna Neely, Vikas Gulati. Ophthalmology,
UNMC, Omaha, NE.
Purpose: Topical anesthetics have been shown to affect episcleral venous pressure
in animal models. The purpose of this study was to examine the effect of
commonly used topical anesthetics on intraocular pressure (IOP) measurement.
Methods: Twenty volunteers with a normal anterior segment examination, not
currently using any topical medications, and with no history of prior ocular surgery
were included in the study. At baseline, IOP was measured without any topical
anesthetic using rebound tonometry (RT, ICare tonometer). Proparacaine
hydrochloride, 0.5% was administered in the right eye and benoxinate
hydrochloride, 0.4% (with 0.25% fluorescein sodium) in the left eye. IOP was
measured with RT at 1 minute and 5 minutes after administration of topical
anesthetics by a second masked observer. IOP was also measured by Goldmann
applanation tonometry (GAT) by a third masked observer at 5 minutes. Central
corneal thickness (CCT) was obtained with an ultrasonic pachymeter.
Results: In proparacaine treated eyes, both the 5 min RT IOP (15.0 4.2 mmHg)
and 5 min GAT IOP (15.2 4.6 mmHg) were significantly (ANOVA p value
<0.01) lower than the baseline RT IOP (16.6 4.2 mmHg). A similar trend towards
slightly lower IOP at 5 minutes seen in the benoxinate treated group did not reach
statistical significance (ANOVA p value = 0.11). Fifteen of 20 eyes in the
proparacaine group and 12 of 20 eyes in the benoxinate group had lower RT IOP at
5 min as compared to baseline. Bland Altman plots showed good correlation
between 5 min RT IOP and GAT IOP (mean difference = 0.2 2.1 mmHg). A
trend towards lower readings on GAT IOP (vs. RT IOP) was observed with thinner
CCT. GAT IOP readings tended to be higher than RT IOP with thicker corneas
(R2=0.13, p=0.13).
Conclusions: Topical anesthetic use was found to be associated with lower IOP
readings after 5 minutes. Lowering of episcleral venous pressure is one possible
explanation for the observed change. However, the exact mechanism mediating this
change needs to be further evaluated.
Commercial Relationships: Nathan Welch, None; Shane Havens, None; Donna
Neely, None; Vikas Gulati, None
Support: None

Intra Ocular Pressure Lowering Efficacy of AMA0076, a Locally Acting Rho
Kinase (ROCK) Inhibitor, in Dutch Belted Rabbits
Sarah Van de Velde1A, Tine Van Bergen1A, Evelien Vandewalle1A, Davine Sijnave1A,
Karolien Hollanders1A, Lieve K. Moons1B, Dirk Leysen2, Ingeborg Stalmans1A.
A
Lab of Ophthalmology, BBiology Dept, Zoological Inst, 1KULeuven, Leuven,
Belgium; 2Amakem NV, Diepenbeek, Belgium.
Purpose: To determine the intra ocular pressure (IOP) lowering efficacy of the
local ROCK inhibitor, AMA0076, in Dutch Belted rabbits.
Methods: Dutch Belted rabbits (5 rabbits/group) received topically a single dose of
AMA0076 in one eye. A concentration range of 0.031 - 0.625% AMA0076 was
tested. The contralateral eye served as control and was treated with saline. IOP was
measured at baseline and 30 min, 1, 2, 3, 4, 5, 6, 7 and 8h post topical
administration.
Results: Single topical administration of AMA0076 significantly lowered IOP in
all groups compared to the control eye (p<0.05). Mean baseline IOP during the
experiments was between 20.6 and 21.5 mmHg. Thirty minutes after topical
treatment, a concentration dependent reduction in IOP was demonstrated with a
IOP ranging from 3.9mmHg - 7mmHg compared to the control eye. Maximal IOP
reduction of each concentration following single dose administration of AMA0076
was observed 2 hours after instillation with a IOP ranging from 4.6mmHg and
7.5mmHg. The IOP lowering effect with the highest concentrations of AMA0076
(0.625% and 0.125%) was sustained during the experiment.
Conclusions: AMA0076 was effective in rapidly lowering IOP in a dose depended
and sustained manner after a single topical dose in Dutch Belted rabbits. This new
class of local ROCK inhibitors has potential therapeutic value for the IOP lowering
treatment of glaucoma.
Commercial Relationships: Sarah Van de Velde, None; Tine Van Bergen,
None; Evelien Vandewalle, None; Davine Sijnave, None; Karolien Hollanders,
None; Lieve K. Moons, None; Dirk Leysen, Amakem NV (E); Ingeborg
Stalmans, None
Support: This research is supported by a grant provided by Amakem NV.
Intraocular Pressure And Systemic Hypercapnia And Hyperoxia
Alanna Adleman1A, Monica Jong1A, Joseph A. Fisher1,2, Tien Wong1A, Richard W.
Cheng1A, Sunni R. Patel1A, Ayda M. Shahidi1A, Christopher Hudson3,1A.
A
Ophthalmology and Vision Sciences, 1University of Toronto, Toronto, ON,
Canada; 2Thornhill Research Inc., Toronto, ON, Canada; 3School of Optometry,
University of Waterloo, Waterloo, ON, Canada.
Purpose: To investigate the magnitude of change, if any, of intraocular pressure
(IOP) to systemic gas provocations in healthy participants.
Methods: The sample consisted of five healthy participants (mean age 24.6 2.7
years) with no ocular or systemic diseases and free from any blood flow-altering
medications. Following stabilization of end-tidal partial pressures of carbon dioxide
(PETCO2) and oxygen (PETO2), three IOP measures were obtained in a randomly
selected eye during each of a series of four inhaled gas conditions; baseline A
(PETCO2 = 38mmHg; PETO2 = 100mmHg), hypercapnia 42 (PETCO2 = 42mmHg;
PETO2 = 100mmHg), hypercapnia 46 (PETCO2 = 46mmHg; PETO2 = 100mmHg),
baseline B (PETCO2 = 38mmHg; PETO2=100mmHg). Measurements were then
obtained in the other eye during the following four inhaled gas conditions: baseline
C (PETCO2 = 38mmHg; PETO2 = 100mmHg), hyperoxia 250 (PETCO2 = 38mmHg;
PETO2 = 250mmHg), hyperoxia 500 (PETCO2 = 38mmHg; PETO2 = 500mmHg),
baseline D (PETCO2 = 38mmHg; PETO2 = 100mmHg). The gas blends were
achieved using the RespirAct (Thornhill Research Inc., Toronto, Canada)
attached to a sequential rebreathing circuit (HiOx-80, VIASYS Healthcare Inc.).
Results: The means and standard deviation of IOP at each gas provocation were:
baseline A = 12.4 1.9; hypercapnia 42 = 13.2 1.4; hypercapnia 46 = 13.0 1.4;
baseline B = 12.5 1.8; baseline C = 12.8 3.0; hyperoxia 250 = 12.1 2.2;
hyperoxia 500 = 12.0 1.7; baseline D = 11.9 3.1. Two repeated-measures
ANOVAs were performed which showed no significant differences in IOP between
hypercapnia and baseline (p=0.38), nor between hyperoxia and baseline (p=0.34).
Conclusions: Preliminary results of this study show that IOP does not appear to
vary significantly with systemic hypercapnia or hyperoxia.
Commercial Relationships: Alanna Adleman, None; Monica Jong, None;
Joseph A. Fisher, Thornhill Research Inc. (I, E); Tien Wong, None; Richard W.
Cheng, None; Sunni R. Patel, None; Ayda M. Shahidi, None; Christopher
Hudson, Thornhill Research Inc. (I)
Support: Ontario Research Fund
Shear Stress Effects on Schlemms Canal Cells
Nicole E. Ashpole1, W Daniel Stamer2. 1Biomedical Engineering, University of
Arizona, Tucson, AZ; 2Ophthalmology, Duke University, Durham, NC.
Purpose: Nitric Oxide (NO) is a free radical that is produced by the enzyme,
endothelial NO synthase (eNOS). eNOS activity and abundance is regulated by
shear stress in vascular endothelia, and the resulting increase in NO production has
a variety of physiological consequences including smooth muscle relaxation and
vasodilation. In humans, shear stress levels in Schlemms Canal (SC) are calculated
to be comparable to shear levels attained in large arteries, particularly at elevated
intraocular pressure (IOP). We investigated the relationship between NO
production and shear stress in cultured human SC cells.
Methods: Human Schlemms Canal endothelial cells were seeded into Ibidi flow
chambers at confluence, allowed to mature for one week and then assayed for
effects of continuous shear (0.1, 1, 5, 10 and 15 dynes/cm^2) on cell alignment, NO
production and eNOS expression. Cell alignment was assessed using phase-contrast
microscopy, NO production was measured directly using an NO electrode
(Innovative Instruments) paired with DAF-FM Fluorescence (Invitrogen), and
eNOS expression was evaluated using SDS-PAGE and Western blot analysis.
HUVECs were used as a positive control.
Results: Like HUVECs, SC cells aligned in parallel with the direction of flow, a
behavior that was time and shear-dependent. For example, SC alignment at 10.0
dynes/cm^2 began within three days, with the majority of cells aligned within a
week. By comparison, HUVECS fully aligned within 24 hours. The synthesis of
NO (0.05-3.2 nmol/10^5 cells) in HUVECS and SC cells was directly proportional
to the shear-stress magnitude. Likewise, the expression of eNOS was found to
increase in a shear-stress -dependent manner in both HUVECS and SC cells.
Conclusions: Human SC cells respond to shear stress similar to other vascular
endothelia; aligning with flow, up-regulating eNOS and increasing NO production.
Due to known effects of NO on vascular permeability, these results suggest that
shear stress and NO production in Schlemms Canal may participate in the
regulation of aqueous outflow across the inner wall.
Commercial Relationships: Nicole E. Ashpole, None; W Daniel Stamer, None
Plate assay for glaucoma drugs
Jason Y. Chang1,2, Brian S. McKay1, W Daniel Stamer1,3. 1Ophthalmology and
Vision Science, University of Arizona, Tucson, AZ; 2Bioengineering, Imperial
College London, London, United Kingdom; 3Ophthalmology, Duke University,
Durham, NC.
Purpose: The goal of this study was to develop a medium throughput assay to
screen small molecules, peptides and/or antibodies for their capacity to interfere
with vascular endothelial (VE) cadherin-cadherin binding at the level of Schlemm's
canal inner wall for development of novel conventional outflow drugs.
Methods: Chimeric human VE-cadherin-Fc proteins (Kd = ~8 mM in vivo) were
used in the development of the assay; consisting of free VE-cadherin-Fc chimeric
proteins (10nM-500nM) that were labeled with Alexa Fluor 488 hydrazide and
VE-cadherin chimeras that were bound to immobilized protein A coated 96-well
plates (1g/well). As a control for specificity we also tested immobilized Ncadherin FC chimeras, which do not bind VE-cadherin. Anti-VE-cadherin IgGs that
recognize either intracellular (#2158; Cell Signaling) or extracellular (9H7) VEcadherin epitopes (used at 30-100 ng/ml final in 2mM calcium binding buffer) or
calcium-free binding buffer were tested for ability to inhibit cadherin-cadherin
binding. A fluorescence microtiter-plate reader (493ex/517em; FlexStation 3;
Molecular Devices) was used to monitor binding interactions.
Results: Control experiments validated the calcium dependence of VE-cadherincadherin binding (5nM - 25nM protein concentration) (n=4, p<0.05). Results also
showed a linear relationship between VE-cadherin-cadherin binding activity and
increased protein concentration (0.01M - 5M, r2=0.993). As expected, labeled
VE-cadherin did not bind to the immobilized N-cadherin chimera. While both VEcadherin antibodies (intracellular and extracellular) were able to compete against
the VE-cadherin chimera for protein-protein binding at low concentrations of
chimeric protein (50nM), only the extracellular antibody inhibited VE-Cadherin
chimera binding at physiological proteins concentrations.
Conclusions: Our VE-cadherin assay possesses great potential for glaucoma drug
discovery and development. Results show that the assay can detect interference of
VE-cadherin-cadherin binding; although due to low (mM) affinity of VE-cadherincadherin binding, high concentrations of protein are required for specific VEcadherin binding. Future studies to improve the sensitivity of the assay and better
distinguish between specific and non-specific interactions are ongoing.
Commercial Relationships: Jason Y. Chang, None; Brian S. McKay, None; W
Daniel Stamer, None
Effects of Low- and High-dose Application of Vasopressin on Aqueous Humor
Dynamics

Barbara Bogner, Christian Runge, Clemens Strohmaier, Andrea Trost, Falk
Schroedl, Herbert A. Reitsamer. Ophthalmology/Optometry, Paracelsus Medical
University, Salzburg, Austria.
Purpose: Previous experiments have shown that vasopressin reduces IOP dosedependently. The purpose of the present study is to investigate the effect of
different doses of arginine-vasopressin (AVP) on aqueous flow (AF).
Ciliary blood flow (CilBF) and aqueous production are related to each other. To
distinguish between blood flow and secretory mediated effects of AVP on AF, two
doses of exogenously applied AVP were selected: The first dose did not change
CilBF (defined as low-dose); the second dose caused a significant reduction of
CilBF (defined as high-dose).
Methods: In anesthetized New Zealand white rabbits mean arterial pressure
(MAP), intraocular pressure (IOP) and orbital venous pressure (OVP) were
measured continuously by direct cannulation of the central ear artery, the vitreous
and the orbital venous sinus, respectively. CilBF was measured transsclerally by
laser-Doppler flowmetry. Aqueous flow (AF) was determined simultaneously by
fluorophotometry. After baseline measurements the effects of exogenously applied
AVP (0.08 ng/kg/min and 1.33 ng/kg/min) were recorded.
Results: At the selected low-dose of AVP CilBF was not changed, the high-dose of
AVP caused a significant reduction of CilBF (-35.684.00%). Low-dose and highdose AVP reduced the AF ~20%. Detailed results are summarized in table 1.
Conclusions: Both low- and high-dose AVP cause ~20% reduction of AF. It has
been shown, that CilBF reduction of more than ~25% of its normal level results in a
proportional decrease of aqueous production. The model of the relationship
between CilBF and AF suggests that low-dose AVP modulates aqueous production
due to the inhibition of secretory mechanisms in the ciliary epithelium, as CilBF is
not changed. High-dose AVP additionally causes a reduction in CilBF and
vasoconstriction. One would expect that there is a proportional decrease in AF.
However, AF is not further reduced. Therefore, additional factors (e. g. stimulatory
mechanisms or starling forces) might play a role. However, to clarify this was
beyond the scope of this study.
Commercial Relationships: Barbara Bogner, None; Christian Runge,

None; Clemens Strohmaier, None; Andrea Trost, None; Falk Schroedl,
None; Herbert A. Reitsamer, None
Support: Austrian Academy of Sciences, PMU-FFF, Fuchs Foundation, Adele
Rabensteiner Foundation, Lotte Schwarz Endowment
The Effect of Brimonidine on Aqueous Humor Dynamics During the Day and
Night in Ocular Hypertensive Patients
Shan Fan, Vikas Gulati, Donna Neely, Matt Maslonka, Carol B. Toris.
Ophthalmology, Univ of Nebraska Medical Ctr, Omaha, NE.
Purpose: This study evaluates the effects of brimonidine on daytime and nighttime
aqueous humor dynamics in patients with ocular hypertension (OHT).
Methods: Thirty patients with OHT (58.69.2 years of age) were enrolled in this
randomized, double-masked, crossover study. Participants self-administered 0.2%
brimonidine or placebo three times a day for 6 weeks. At the end of each 6 week
period, aqueous humor dynamics were studied at one daytime and one nighttime
visit. Measurements included aqueous flow by fluorophotometry, outflow facility
by tonography, episcleral venous pressure (Pev) by venomanometry and seated and
supine intraocular pressure (IOP) by pneumatonometry. Measurements of IOP were
made at 9 AM, 11 AM, 1 PM, 3 PM, 9 PM, 11 PM, 1 AM, and 3 AM. Uveoscleral
outflow (Fu) was calculated mathematically using the modified Goldmann
equation. The results were compared by two-tailed paired t-tests. P values< 0.05
were considered statistically significant.
Results: When treated with vehicle, nighttime supine Pev (11.21.6 mmHg) was
significantly higher than daytime seated Pev (10.21.5, p=0.002), and aqueous flow
and uveoscleral outflow were lower (by 0.79 l/min, p<0.01 and 1.06 l/min,
p=0.01, respectively) at night than during the day. Compared with vehicle
treatment, brimonidine significantly lowered seated IOP at 9 AM, 11 AM, 9 PM
and11 PM and supine IOP at 9 AM, 11 AM (by 1.5 to 2.2 mmHg, p<0.01) but had
no effect on IOP at 1 AM and 3 AM. Brimonidine significantly increased
uveoscleral outflow during the daytime (by 0.84 l/min, p<0.01) only and had no
effect on daytime and nighttime aqueous flow, outflow facility or Pev.
Conclusions: Brimonidine treatment for 6 weeks reduced habitual IOP during the
day but not at night. The daytime IOP reduction is associated with an increase in
daytime uveoscleral outflow. The lack of IOP effect at night can be explained by
failure to increase uveoscleral outflow, nighttime physiological changes in aqueous
humor dynamics in patients with OHT may diminish the pharmacological effect of
brimonidine making this drug ineffective at night.
Commercial Relationships: Shan Fan, None; Vikas Gulati, None; Donna
Neely, None; Matt Maslonka, None; Carol B. Toris, None
Support: an AGS MAPS grant (VG); Research to Prevent Blindness
Clinical Trial: University of Nebraska, NCT01144494
PEDF Modulation of Schlemm's Canal Barrier Function
Shannon M. Niere1, Emely Hoffman1, Kristin M. Perkumas1, R R. Allingham2,
Craig E. Crosson3, W Daniel Stamer4. 1Ophthalmology, University of Arizona,
Tucson, AZ; 2Ophthalmology, Duke University Eye Center, Durham, NC;
3
Ophthalmology, Medical Univ of South Carolina, Charleston, SC;
4
Ophthalmology, Duke University, Durham, NC.
Purpose: Pigment epithelial-derived factor (PEDF) has anti-angiogenic activity,
participating in the regulation of retinal endothelial barrier function; however its
role in other ocular endothelia are unknown. Since PEDF is a constituent of
aqueous humor, the goal of the present study was to measure levels in human
aqueous humor and examine PEDF effects on the permeability of Schlemm's canal
(SC) endothelia, part of the blood-aqueous barrier.
Methods: After informed consent, aqueous humor was obtained from patients with
no known ocular disease undergoing routine cataract surgery. Samples were
assayed for PEDF using ELISA. In a second assay, primary cultures of SC and
aqueous plexus (AP) endothelia were isolated from human donor and porcine eyes,
respectively. Cells were plated at confluence on Transwell filters in DMEM
containing 10% FBS and after one week, cells were put into serum-free media +/PEDF (0.3-1 M) and assayed for transendothelial electrical resistance (TEER)
using an ohmmeter.
Results: PEDF levels in aqueous humor were detected in 9/10 patient samples,
ranging from 0.4 to >100 ng/ml. At the upper end of the physiological range, PEDF
(1 M for 24 hours) increased net TEER of human SC endothelial monolayers from
14 1 to 17 1 ohm*cm2, while porcine AP cells increased from 212 to 302
ohm*cm2 (p<0.05). In both cell types, significant changes in TEER were not
observed at earlier time points, or using lower concentrations of PEDF.
Conclusions: PEDF is present in normal aqueous humor at a range of
concentrations, suggesting a role for PEDF in the regulation of blood-aqueous
barrier at the level of Schlemm's canal and consistent with its function in bloodretinal barrier homeostasis. Interestingly, effects of PEDF were not immediate, but
required 24 hours; suggesting involvement in long-term control of blood-aqueous
barrier.
Commercial Relationships: Shannon M. Niere, None; Emely Hoffman,
None; Kristin M. Perkumas, None; R. R. Allingham, None; Craig E. Crosson,
None; W Daniel Stamer, None
Support: ey017007
DIDS Causes Src-Dependent Inhibition of Na,K-ATPase in the Nonpigmented
Ciliary Epithelium and Slows Aqueous Humor Secretion by the Intact Porcine
Eye
Mohammad Shahidullah, Nicholas A. Delamere. Physiology, College of Medicine,
Univ of Arizona, Tucson, AZ.
Purpose: Aqueous humor (AH) secretion is the result of net ion transport that
involves the coordinated operation of Na,K-ATPase in the nonpigmented ciliary
epithelium (NPE) with several other basolateral transporters and channels in the
ciliary epithelium bilayer. Here, we report on the ability of DIDS to reduce AH
secretion and present studies to examine the mechanism.
Methods: AH secretion rate in the intact eye was measured by fluorescein dilution
technique. NPE cells were cultured to confluence on permeable supports, treated
with drugs by adding to both sides of the membrane, and then harvested to measure
Na,K-ATPase activity or to detect protein phosphorylation. Na,K-ATPase activity
was measured either by rubidium uptake or by direct colorimetric estimation of
ATP hydrolysis. Protein phosphorylation was detected by Western blot analysis.
Results: In an intact pig eye preparation the rate of AH secretion was significantly
reduced by DIDS added to the blood-side perfusate. In primary cultured NPE,

DIDS inhibited ouabain-sensitive 86Rb uptake. Incubation of the cells with DIDS
activated Src family kinase (SFK) and ERK1/2 as judged by Western blot analysis.
DIDS and a second stilbene derivative, SITS, also inhibited Na,K-ATPase activity
as measured by ATP hydrolysis. PP2, an SFK inhibitor, prevented the DIDS effect
on 86Rb uptake, SFK and ERK1/2. In BCECF-loaded NPE, DIDS was found to
reduce cytoplasmic pH. Parallel studies showed PP2 eliminates activation of SFK
and ERK1/2 that occurs upon pH reduction.
Conclusions: The ability of DIDS to suppress AH secretion is consistent with
inhibition of Na,K-ATPase in the NPE following a drop in cytoplasmic pH, SFK
and ERK1/2 activation.
Commercial Relationships: Mohammad Shahidullah, None; Nicholas A.
Delamere, None
Effects of Head-Down and Head-Up Tilt on Episcleral Venous Pressure in a
Rabbit Model
Jeffrey W. Kiel, William J. Lavery. Ophthalmology, Univ of Texas Hlth Sci Ctr SA,
San Antonio, TX.
Purpose: In humans, changing position from upright to supine elicits an
approximately 10 mmHg increase in cranial venous pressure (CVP) caused by the
hydrostatic column effect, but episcleral venous pressure (EVP) and IOP rise by
only a few mmHg. This dissociation between CVP and EVP suggests a regulatory
mechanism controlling EVP. The aim of the present study was to determine if the
rabbit model is suitable to study the effects of postural changes on EVP despite its
short hydrostatic column.
Methods: In anesthetized rabbits (n=50), we measured ear artery pressure (BP),
IOP, and orbital venous pressure (OVP) by direct cannulation; carotid blood flow
(BFcar) by transit time ultrasound, heart rate (HR) by a digital cardiotachometer,
and EVP with a servo-null micropressure system. The protocol goal was to obtain
measurement of supine EVP for approx. 15 minutes, followed by approx. 15
minutes of EVP measurement in either head-down tilt (n=43) or head-up tilt (n=7).
The data (mean +/- standard error) were analyzed by paired t-tests.
Results: Head-down tilt caused significant increases in IOP, OVP, and EVP. Headup tilt caused IOP, OVP, and EVP to significantly decrease. From supine to headdown tilt, BP and BFcar were unchanged, IOP increased by 2.0 +/- 0.4 mmHg
(p<0.01), EVP increased by 2.4 +/- 0.4 mmHg (p<0.01), OVP increased by 2.5 +/0.2 mmHg (p<0.01) and HR decreased by 9 +/- 3 bpm (p<0.01). For head-up tilt,
BP decreased by 3.6 +/- 1.0 mmHg (p<0.05), IOP decreased by 3.0 +/- 1.0 mmHg
(p<0.05), EVP decreased by 1.6 +/- 0.7 mmHg (p = 0.06), OVP decreased by 1.5
+/- 0.2 mmHg (p<0.01), BFcar decreased by 8.4 +/- 2.5 mL/min (p<0.05) and HR
was unchanged.
Conclusions: Although the hydrostatic column in the rabbit is shorter than humans,
the rabbit model permits sufficiently sensitive measurements of the pressures and
systemic parameters likely involved in the EVP responses to posture change. The
present results indicate directionally similar EVP and IOP responses to tilt as occur
in humans and, as in humans, the responses are smaller than would be expected
from the change in the hydrostatic column height. We conclude the rabbit model is
appropriate for studying the mechanisms responsible for the relative immunity of
EVP and IOP to posture change.
Commercial Relationships: Jeffrey W. Kiel, None; William J. Lavery, None
Support: NGR of the American Health Assistance Foundation and the van Heuven
endowment
Effects of Atropine and Timolol on Rabbit Episcleral Venous Pressure during
Head-Down Tilt
William J. Lavery, Jeffrey W. Kiel. Ophthalmology, UT Health Science Center San Antonio, San Antonio, TX.
Purpose: In humans, cranial venous pressure increases when going from an upright
to supine position due to the hydrostatic column effect, but usually there is only a
small change in episcleral venous pressure (EVP) and IOP. This suggests a
regulatory mechanism may be isolating EVP and IOP from the effect of gravity
during posture change. Because the episcleral circulation is innervated by both
parasympathetic and sympathetic nerves, the goal of the present study was to test
the hypothesis that EVP during head-down tilt is under muscarinic cholinergic or
beta adrenergic neural control.
Methods: In anesthetized rabbits (n=19), we measured mean arterial pressure
(MAP), IOP, and orbital venous pressure (OVP) by direct cannulation; carotid
blood flow (BFcar) by transit time ultrasound, heart rate (HR) by a digital
cardiotachometer, and EVP with a servo-null micropressure system. Measurements
were made initially with the animals in the supine position, and then with the
animals tilted in a head down position after which the animals were given either
topical atropine (0.5 mg/kg, iv bolus, n=8) or timolol (5 mg/ml, 50 microliters
topical, n=11) while remaining in the head down position. The data (mean +/-
standard error) were analyzed by repeated measures ANOVA.

Results: In both groups, head-down tilt caused significant (p<0.05) increases in
OVP (0.9 0.1 to 3.1 0.3 mmHg), EVP (10.4 0.4 to 12.4 0.5 mmHg) and IOP
(13.8 0.8 to 16.2 0.8 mmHg) while MAP (72.3 0.9 to 71.6 1.0 mmHg),
BFcar (30.5 2.2 to 29.6 1.8 ml/min) and HR (293 6 to 290 6 bpm) were not
significantly different. Timolol cause significant decreases in BFcar (11% 3%),
HR (11% 2%) and IOP (17% 5%) but did not alter MAP or EVP. Atropine did
not alter any of the measured parameters.
Conclusions: Since neither atropine nor timolol affected EVP during head-down
tilt, we conclude that EVP during posture change is not regulated by cholinergic or
beta adrenergic neural control.
Commercial Relationships: William J. Lavery, None; Jeffrey W. Kiel, None
Support: Supported by the NGR of the American Health Assistance Foundation
and the van Heuven endowment
The Relationship between Central Corneal Thickness, Tonographic Outflow
Facility, and Intraocular Pressure
Pouya Alaghband1, Evgenia Kanonidou1, Laura Beltran-Agullo1, Darryl R.
Overby2, K Sheng Lim1. 1Ophthalmology, St Thomas' Hospital, London, United
Kingdom; 2Bioengineering, Imperial College London, London, United Kingdom.
Purpose: Central corneal thickness (CCT) is known to be associated with onset
and progression of glaucoma and can possibly potentiate the influence of
tonographic outflow facility (TOF) that maintain intraocular pressure (IOP). This
study investigates the correlation between CCT, TOF and IOP.
Methods: This is a retrospective analysis of patients who were recruited in our
aqueous studies from 2006-2009 at St Thomas Hospital, London, UK. All studies
were approved by local ethics committee within hospital.
Eighty nine newly diagnosed patients with OHT/POAG attending our department
have been offered to have baseline outflow facility measurement as part of their
glaucoma investigations since 2006. We identified patients who had reliable
baseline outflow facility. There were 34 healthy volunteers as part of these trials as
well.
Measurements: 1) Outflow facility measured by electronic Schiotz tonometer 2)
IOP measured by Goldmann applanation tonometry 3) CCT measured by handheld
ultrasonic pachymeter
Results: One hundred and twenty three patients were found to have a reliable
tonographic outflow facility. Sixty three were male and 60 female. There were 58
white individuals. Baseline characteristics of these patients as a whole are shown in
Fig1. Multiple regression analysis showed strong statistically significant
association between CCT and IOP (beta=0.24, p=0.005, 95% CI=0.009-0.05) and
also TOF and IOP (beta=-0.38, p<0.001, 95% CI= -33.15_13.14). This association
remained the same after adjustment for age in all groups. In subgroup analysis, the
association between CCT and IOP remained significant in OHT/POAG group
(beta=0.23, p=0.04, 95%CI=0.001-0.038) and also in healthy individuals
(beta=0.52, p=0.001, 95% CI=0.02-0.06). Overall, there is no significant linear
correlation between CCT and TOF in all groups.
Conclusions: Higher CCT is related to higher IOP, and higher IOP is related to
lower TOF. After accounting for these relationships, however, there was no
observed relationship between CCT and TOF. These data suggest that TOF is an
independent aqueous parameter that can be measured without bias caused by CCT.
Commercial Relationships: Pouya Alaghband, None; Evgenia Kanonidou,

None; Laura Beltran-Agullo, None; Darryl R. Overby, None; K Sheng Lim,
None
Support: None

Variations of Episcleral Venous Pressure with Body Position in Healthy
Subjects
Nitika Arora1, Jay W. McLaren2, Arthur J. Sit2. 1Ophthalmology, Mayo School of
Graduate Medical Education, Rochester, MN; 2Ophthalmology, Mayo Clinic,
Rochester, MN.
Purpose: Intraocular pressure (IOP) varies with body position, and these variations
are assumed to be in part caused by changes in episcleral venous pressure (EVP).
However, the effect of body position on EVP is poorly understood. In this study,
we investigated changes in EVP between two body positions, sitting and prone.
Methods: IOP was measured by using a pneumatonometer in 25 eyes of 13 healthy
volunteers (mean age, 43 years; range 24 to 73 years) in a seated position. EVP was
then measured 4 times in a selected episcleral vein by using an automated, slit-lamp
mounted, venomanometer, based on the pressure chamber method. Venous
pressure was assumed to be equal to the pressure in the chamber when the vein first
began to collapse as the chamber was inflated at a constant rate. After 30 minutes,
the subject was placed in a prone position. After five minutes, IOP was remeasured
and EVP in the same vein was remeasured twice with the neck extended and the
head resting on the chin rest of the slit lamp. EVP in the prone position was
compared to EVP in the seated position by using a paired t-test. The change in IOP
between the seated and prone positions was also compared to the change in EVP by
using a paired t-test.
Results: Mean IOP in the sitting and prone positions were 13.0 2.6 mmHg (mean
SD) and 15.2 2.6 mmHg respectively and were significantly different from
each other (p<0.001). Mean EVP in the sitting and prone positions were 6.4 1.4
mmHg and 7.7 1.7 mmHg and were significantly different from each other
(p<0.001). The rise in IOP in the prone position from IOP in the seated position
(2.1 1.0 mmHg) was greater than the rise in EVP in the prone position from EVP
in the seated position (1.3 1.5 mmHg, p=0.015).
Conclusions: In the prone position IOP and EVP are higher than they are in the
sitting position. The rise in EVP when changing from the upright to prone position
could partly explain the rise in IOP. However, the Goldmann equation predicts that
the change in IOP should be equal to the change in EVP if aqueous humor flow,
outflow facility and uveoscleral flow remain constant. This suggests that other
factors, in addition to the increase in EVP, may contribute to the rise in IOP when
changing from upright to recumbent body position. Understanding the origin of the
positional changes in IOP would advance our understanding of physiologic factors
that determine IOP and the origin of pressure elevation in glaucoma.
Commercial Relationships: Nitika Arora, None; Jay W. McLaren,
None; Arthur J. Sit, None
Support: American Health Assistance Foundation, Mayo Foundation for Research
and Medical Education, Research to Prevent Blindness (unrestricted department
grant)
Effect of Oleoylethanolamide on Aqueous Humor Outflow
Akhilesh Kumar, Zhuanhong Qiao, Pritesh P. Kumar, Zhao-Hui Song.
Pharmacology & Toxicology, University of Louisville, Louisville, KY.
Purpose: To study the effects of oleoylethanolamide (OEA) on aqueous humor
outflow via the trabecular meshwork pathway.
Methods: The effects of OEA on aqueous humor outflow via the trabecular
meshwork pathway were measured using a porcine anterior segment perfused organ
culture model. Different concentrations of OEA were administered to the perfusion
medium and the aqueous humor outflow facility was monitored for 5 hours.
SR141716A, SR144528, O-1918, and GW6471 were administered to anterior
segment to determine the involvement of various receptors on the outflow effects
of OEA. PD98059, an inhibitor of the p42/44 MAP kinase pathway , was used to
examine the involvement of the MAP kinase signal transduction pathway in
regulating OEA induced enhancement of outflow facility. OEA induced activation
of p42/44 MAP kinase was determined by western blot analysis using an antiphospho p42/44 MAP kinase antibody.
Results: Administration of OEA caused a concentration-dependent enhancement of
aqueous humor outflow facility, with the maximum effect (162.4 15.3 % of basal
outflow facility) achieved at 2 hour after the administration of 30 nM of OEA.
Pretreatment with either 1 M SR144528 or 3 M GW6471 produced a partial
antagonism on the OEA-induced increased aqueous humor outflow facility.
However, pretreatment with either 1 M O-1918 or 1 M SR141716A had no
effect on OEA induced enhancement of aqueous humor outflow facility. Treatment
of trabecular meshwork cells with OEA for 10 min activated phosphorylation of
p42/44 MAP kinase, which was blocked by pretreatment with either GW6471 or
SR144528. Furthermore, PD98059, an inhibitor of the p42/44 MAP kinase
pathway, blocked both OEA-induced phosphorylation of p42/44 MAP kinase and
enhancement of aqueous humor outflow facility.
Conclusions: The results from this study demonstrate that OEA increases aqueous
humor outflow through the trabecular meshwork pathway and these effects may be
mediated by PPAR and non-CB1/CB2 cannabinoid receptors through activation
of p42/44 MAP kinase.
Commercial Relationships: Akhilesh Kumar, None; Zhuanhong Qiao,

None; Pritesh P. Kumar, None; Zhao-Hui Song, None
Support: NIH Grants EY13632 and DA11551
Micro RNA in Human Aqueous Humor
Evan P. Lagouros1, Jeffrey Dunmire1, Rachida Bouhenni1, Deepak P. Edward2.
1
Ophthalmology, Summa Health System, Akron, OH; 2Ophthalmology, King
Khaled Eye Specialist Hospital, King Khaled Eye Specialist Hospital, Riyadh,
Kingd, Saudi Arabia.
Purpose: MiRNAs are small non-coding RNA molecules with regulatory function
and marked tissue specificity that can modulate multiple gene targets. They have
been detected in body fluids and are associated with various physiologic and
pathologic processes. We analyzed aqueous humor (AH) from human subjects
undergoing cataract surgery to establish the presence and relative quantities of
known miRNAs.
Methods: AH was collected from three patients without known ocular diseases
other than cataract and a normal systemic history. Quantitative real time PCR was
used to detect the different miRNAs present in the pooled AH in a microarray
platform containing 257 known human miRNAs. Data was normalized and
analyzed using BioRad iQ5 analysis software v2.1. Product melting temperature
dissociation curves were used to validate positive calls and any reaction generating
multiple peaks was excluded as negative.
Results: Ninety-one of 257 tested miRNAs were identified in the pooled AH. The
top 5 abundant miRNAs were miR-412, miR-202, miR-486-5p, miR-671-3p, and
miR-548a-5p. Four miRNAs, previously shown as unique to saliva, compared with
other body fluids, were also detected in AH. In addition, the highly abundant
miRNAs in other body fluids, such as miR335 and miR515, were also detected in
the AH but at lower expression levels.
Conclusions: The presence of miRNAs in AH suggests that they may have
functional roles in regulating target genes in tissues lining the anterior chamber.
Further analysis of the AH miRNA population may identify potential gene targets
and provide insights regarding their role in AH regulation, glaucoma and anterior
segment disease processes.
Commercial Relationships: Evan P. Lagouros, None; Jeffrey Dunmire,
None; Rachida Bouhenni, None; Deepak P. Edward, None
Support: None
Effects Of Bevacizumab On Intraocular Pressure Influenced By The
Trabecular Meshwork
Kai Januschowski, Charlotte Schramm, Oliver Ottmann, Karl Bartz-Schmidt, Peter
Szurman. Department of Ophthalmology, Univ Eye Hospital Tuebingen,
Tuebingen, Germany.
Purpose: To investigate the effect of bevacizumab in the aqueous humor on the
trabecular meshwork using an perfused anterior chamber model and analyze the
ultrastructural effect on trabecular meshwork tissue. Transient and sustained
elevated intraocular pressures can occur after injection with bevacizumab, mainly
in patients with primary open angle glaucoma (POAG).
Methods: Porcine eyes were prepared as described by Johnson et al. Anterior
segments were perfused with DMEM, supplemented with 1.5 mg/mL glucose, 1%
(vol/vol) fetal calf serum (FCS) and antibiotics (100 U/mL penicillin, 0.1 mg/mL
streptomycin, and 17 g/mL gentamicin; all from Invitrogen, Germany) at a constant
flow rate of 4.5 L/min. The anterior eye segments were maintained in an incubator
at 37C in 7% CO2, while the IOP was continuously monitored using 142PC01G
pressure sensors (Honeywell, USA). Data was secured by the Datalogger AD
128 (Valitec, USA).
After an adaptation period of 12 hours, bevacizumab was injected directly into the
anterior chamber. Reflux was prevented by compressing the injection site with
forceps for 20 seconds. The contralateral control received an injection with an
equal volume of normal perfusion medium. Development of intraocular pressure
was followed for 48 hours under constant perfusion. At the end of the perfusion, 1to 2-mm wide specimens of the four eye quadrants were excised. Specimens of 10
eye pairs were fixed in 4% PFA for immunohistochemical investigations (TUNELstain) and in glutaraldehyde fixative for electron microscopy (model 902 A; Carl
Zeiss).
Results: During perfusion studies a reduction in intraocular pressure of 30.17 %
was reduced at the end of 48 hours. No statistical significant difference between
control and eyes treated with bevacizumab was noted (p>0.05). After 72 h TUNELstain showed 8.67 % of dead cells in the region of the trabecular meshwork of eyes
injected with bevacizumab compared to 13.67 % of cell death in the control. In
electron microscopy no ultrastructural change regarding diameter of loop vessels,
extracellular matrix and basalmembrane thickness was noted.
Conclusions: The results could show no effect of bevacizumab on intraocular
pressure influenced by the trabecular meshwork in a porcine model.

Commercial Relationships: Kai Januschowski, None; Charlotte Schramm,
None; Oliver Ottmann, None; Karl Bartz-Schmidt, None; Peter Szurman, None
Support: None
Pilot Study: The Impact Of Advanced Glaucoma On Changes In Aqueous
Outflow After Cataract Surgery
Mark S. Hansen, Kelly W. Muir, Henry Tseng, Anthony N. Kuo, Pratap Challa,
Cecile Santiago-Turla, Alice Ventura, Molly M. Walsh. Department of
Ophthalmology, Duke University Eye Center, Durham, NC.
Purpose: Prior studies have provided evidence for a decrease in intraocular
pressure after cataract surgery in patients with advanced glaucoma. Some have
proposed cataract extraction as a potential therapy in glaucoma to decrease the
intraocular pressure. We hypothesized that the intraocular pressure decreases as a
result of increased aqueous outflow. In this study, we measured aqueous outflow
facility using tonography before and after cataract surgery in patients with
advanced glaucoma and compared them to normal controls. Tonography was
established to measure the patency of the pathway of aqueous humor flow and
determine if changes in intraocular pressure are due to change in facility of outflow
or a change in the net rate of aqueous humor formation.
Methods: Tonography was perfomed by a skilled technician on 6 end-stage
glaucoma patients and 6 control patients both preoperatively and postoperatively
(90 days after surgery). The patient was placed in a comfortable, supine position
and the tonometer was cleaned and calibrated. Proparacaine was placed in the
appropriate eye prior to tonography measurements. The patient was accustomed to
the probe with 3 short trials prior to the beginning of the recorded test. Tonography
was recorded for at least 4 minutes using the appropriate tonometer weights
according to the initial intraocular pressure recording. After tonography
measurements were performed, the Po/C ratios were calculated. A student t-test
was performed to determine whether or not the difference between the 2 groups
were statistically significant.
Results: The initial results from our pilot study revealed that end-stage glaucoma
patients (minus one) had an increase in the Po/C ratios after the surgery, whereas
all control patients had a decrease in the Po/C ratio.
Conclusions: Contrary to our hypothesis, the increase in Po/C ratio in the endstage glaucoma patients indicates a decrease in aqueous outflow after surgery. This
is a significant consideration in management decisions with regards to patients with
glaucoma. Further studies are warranted to determine whether or not these trends
maintain statistical significance with a larger sample size.
Commercial Relationships: Mark S. Hansen, None; Kelly W. Muir,
None; Henry Tseng, None; Anthony N. Kuo, None; Pratap Challa, None; Cecile
Santiago-Turla, None; Alice Ventura, None; Molly M. Walsh, None
Support: NIH P30 EY-005722
Fluid Dynamic Characteristics of Aqueous Humour in a Posterior-Chamber
Phakic Intraocular Lens with a Central Perforation using Computational
Fluid Dynamics
Takushi Kawamorita1, Hiroshi Uozato1, Yuko Shibata2, Maki Shindo2, Masatsugu
Kanazawa2, Masakazu Hirota2, Kimiya Shimizu3. 1Orthoptics and Visual Science,
Kitasato University School of Allied Health Sciences, Sagamihara, Japan;
2
Ophthalmology and Visual Sciences, Kitasato University Graduate School of
Medical Sciences, Sagamihara, Japan; 3Ophthalmology, Kitasato University School
of Medicine, Sagamihara, Japan.
Purpose: A modified implantable collamer lens (ICL) with a central hole
(diameter, 0.36 mm), a KS-AquaPORT (STAAR SURGICAL), was created to
improve aqueous humour circulation. The aim of this study was to investigate the
fluid dynamic characteristics of aqueous humour in a KS-AquaPORT using
computational fluid dynamics, and effect of laser iridotomy (LI).
Methods: Fluid dynamics simulation using an ICL was performed with thermalhydraulic analysis software FloEFD V5 (Mentor Graphics Corp.). For the
simulation, three-dimensional eye models based on a modified Liou-Brennan
model eye with conventional ICL (Model ICM, STAAR SURGICAL) and a KSAquaPORT were used. Both ICLs were -9.0 diopters (D) and 12.0 mm in length,
with an optic of 5.5 mm. The vaulting was 0.50 mm. The quantity of aqueous
humour produced by the ciliary body was set at 2.80 l/min. Flow distribution
between the anterior surface of the crystalline lens and the posterior surface of the
ICL was also calculated, and trajectory analysis was performed.
Results: The flow velocity 0.25 mm in front of the centre of the crystalline lens
was 1.4810-1 mm/sec for the KS-AquaPORT without LI, 1.2010-1 mm/sec for
the KS-AquaPORT with LI 250 m, and 3.6010-4 mm/sec for the conventional
ICL with LI 250 m. Outward flow from the hole in the KS-AquaPORT was
confirmed by trajectory analysis.
Conclusions: These results suggest that use of the KS-AquaPORT improve
aqueous humour circulation, preventing secondary cataracts and eliminating the

need for LI.
Commercial Relationships: Takushi Kawamorita, None; Hiroshi Uozato,
None; Yuko Shibata, None; Maki Shindo, None; Masatsugu Kanazawa,
None; Masakazu Hirota, None; Kimiya Shimizu, STAAR SURGICAL (R)
Support: None
Effect of 0.04% AR-13324 on Aqueous Humor Dynamics in Normotensive
Monkey Eyes
Rong-Fang Wang1, Janet B. Serle1, Casey Kopczynski2. 1Ophthalmology, Mount
Sinai School of Medicine, New York, NY; 2Aerie Pharmaceuticals, Research
Triangle Park, NC.
Purpose: Topical administration of selective inhibitors of rho kinase (Rock) has
been shown to reduce intraocular pressure (IOP) in rabbits, monkeys, healthy
human eyes and patients with ocular hypertension and glaucoma. AR-13324 is a
Rock inhibitor and an inhibitor of the nor-epinephrine transporter (NET). The
purpose of this study is to determine the mechanism by which topically applied
AR-13324 reduces IOP in normotensive monkey eyes.
Methods: Seven normotensive monkeys were used. Tonographic outflow facility
(C) was measured prior to drug administration and repeated 6 hours after
administration of 50 l (25l 2) of 0.04% AR-13324 to one eye and an equal
volume of vehicle to the contralateral control eye. After a washout period of at least
1 week, baseline aqueous humor flow rates (F) were measured hourly for 6 hours
beginning at 10:00 a.m. The following day at 8 a.m., 50 L (25 l 2) of 0.04%
AR-13324 was applied to one eye of each animal and vehicle to the fellow eye.
Aqueous humor flow rates were measured at the same times as on the baseline day
beginning 2 hrs after dosing.
Results: Six hours after a single dose of 0.04% AR-13324 to 7 normal monkey
eyes, C was increased (p<0.05) by 53% in drug-treated eyes compared with either
contralateral vehicle-treated-control eyes or baseline measurements. The IOP
measured by pneumatonometer in treated eyes was reduced (p<0.005) by 25%
when compared with baseline measurements and by 24% when compared with
contralateral vehicle-treated eyes. For 6 hrs after a single-dose of 0.04% AR-13324,
F was reduced (p<0.05) by 20% and 23% when compared with contralateral
vehicle-treated eyes and baseline values, respectively.
Conclusions: AR-13324 reduces IOP in normotensive monkey eyes. A dual
mechanism of action, increase in tonographic outflow facility and decrease of
aqueous humor flow rates, accounts for the IOP reduction in normotensive monkey
eyes.
Commercial Relationships: Rong-Fang Wang, None; Janet B. Serle, None;
Casey Kopczynski, Aerie Pharmaceuticals (E)
Support: Aerie Pharmaceuticals, Inc. and an unrestricted grant from RPB
Biological Feasibility Study for Implantable Continuous Intraocular Pressure
Monitor
Kimberly A. Brown1, Ronald E. Frenkel1,2, Heather B. Seith1, Esdras Arrieta3,
Jean-Marie Parel3, M Jaeger3, R Kline-Schroeder3. 1Ophthalmology, East Florida
Eye Insitute, Stuart, FL; 2Ophthalmology, Bascom Palmer, Miami, FL;
3
Ophthalmology, Ophthalmic Biophysics Center--Bascom Palmer, Miami, FL.
Purpose: To determine the biological feasibility of implantation of an anterior
chamber device to continuously monitor intraocular pressure.
Methods: A round disc of 3.3 mm diameter and 500 micrometer thickness, coated
with Parylene which had been modified in order to facilitate suturing was
implanted in the anterior chamber of one eye of three feline subjects. Follow up
was at the slit lamp or by exam under anesthesia periodically from day 1 to 90.
Results: In two of three subjects device stability and centration was good
throughout the observed period. Focal inflammation of the cornea and fibrin in the
anterior chamber were present in all three subjects. In all subjects iris synechiae
developed between the iris and the device. In subject 1 there was significant diffuse
corneal edema, an iron fragment was found in the endothelium on day 28 and by
day 90 the device was resting on the corneal endothelium, there were significant
iron and rust deposits and significant diffuse corneal pannus and opacification.
Subject 1 developed a cataract. Subjects 1 and 3 developed rubeosis of the iris in
the area surrounding the device. Inflammatory reaction was observed, fibrin
covered the sutures. Mechanical contact between the lens and the device caused
cataract formation. No clinical changes or alterations were observed in the retina.
The feline anterior chamber is not an easy model for implantation due to the eye
and orbit anatomy because the eyelid aperture and nictitating membrane limit globe
exposure and the cul de sac is shallow.
Conclusions: Continuous intraocular sensor placement in the anterior chamber is
potentially feasible if the sensor is reduced in size such that it does not contact the
corneal endothelium, iris, or lens.

Commercial Relationships: Kimberly A. Brown, None; Ronald E. Frenkel,
None; Heather B. Seith, None; Esdras Arrieta, None; Jean-Marie Parel,
None; M. Jaeger, None; R. Kline-Schroeder, None
Support: None
Assessment of Anterior Segment Anatomy Using Anterior Segment Optical
Coherence Tomography and Ultrasound Biomicroscopy Following Argon
Laser Peripheral Iridoplasty
Brian J. Song, Max Forbes, Dana Blumberg, Ronald H. Silverman.
Ophthalmology, Columbia University Medical Center, New York, NY.
Purpose: Argon laser peripheral iridoplasty (ALPI) is one of the few treatment
modalities available for patients with persistent appositional closure following laser
peripheral iridotomy who remain at risk of developing angle-closure glaucoma.
However, there is limited data in the literature to quantify the changes that occur in
the iridocorneal angle to support the efficacy of ALPI in these patients. The
purpose of this study is to evaluate the changes that occur in the anterior segment
using ultrasound biomicroscopy (UBM) and anterior segment optical coherence
tomography (AS-OCT) after ALPI in patients with persistent appositional closure
after laser peripheral iridotomy.
Methods: Three eyes of two patients with persistent narrow angles on gonioscopy
following peripheral laser iridotomy were enrolled in this case series. Gonioscopy,
AS-OCT, and UBM were performed in all cases before ALPI and one week after
the procedure. Gonioscopy was graded from 0 to 4 according to the Schaffer
grading system. AS-OCT was used to assess the anterior chamber, and UBM was
used to image the ciliary sulcus. Anterior chamber depth, angle opening distance at
500 microns (AOD 500), ciliary sulcus conformation grade (grade 0 = absent,
grade 1 = blunted, grade 2 = formed), and iris ciliary body contact length (ICB)
were measured before and after ALPI. These metrics were graded, and the change
in mean value was compared using a paired Student t-test. P-values < 0.05 were
considered statistically significant.
Results: Mean gonioscopic grade increased from 0.38 (prior to ALPI) to 1.38
following ALPI (p = 0.0027). There was no change in anterior chamber depth
following ALPI. The mean change in AOD 500 was 64.5 microns (p = 0.056),
while ciliary sulcus conformation grade increased from 0.16 to 0.57 following
ALPI (p = 0.021). There was a slight decrease in ICB after ALPI (-18.67 microns),
although this difference was not statistically significant (p = 0.592).
Conclusions: Changes in angle morphology can be effectively imaged and
assessed quantitatively using AS-OCT and UBM. In addition, these imaging
modalities can be used to track treatment efficacy following ALPI. Based on these
findings, ALPI may be an effective treatment modality in patients with persistent
appositional closure following laser peripheral iridotomy.
Commercial Relationships: Brian J. Song, None; Max Forbes, None; Dana
Blumberg, None; Ronald H. Silverman, None
Support: None
276 Back of the Eye: Pharmacological Targets
Monday, May 7, 2012, 3:45 PM - 5:30 PM
Palm A Paper Session
Fenofibrate Protects Retinal Mller cells from Oxidative Stress in a PPARalpha and AMPK-Dependent Mechanism
Elizabeth P. moran1A, Yusuke Takahashi1B, Ying Chen1B, Jian-Xing Ma1C. ACell
Biology, BEndocrinology and Diabetes, CPhysiology, 1University of Oklahoma
Health Sciences Center, Oklahoma City, OK.
Purpose: Clinical trials identified that the PPAR agonist fenofibrate inhibits
diabetic retinopathy, but the mechanism is unknown. Preliminary reports suggest
that fenofibrates protective effect is AMPK-dependent and PPAR-independent in
retinal endothelial cells, but evidence are circumstantial, and no downstream target
of AMPK has been proposed. Furthermore, the effect of fenofibrate on retinal
Mller cells, which are critical in the development of DR, is unknown.
Methods: We used 4-Hydroxynonenal to induce oxidative stress in human Mller
cells and quantified fenofibrate protection using an MTT assay. We determined its
dependence on PPAR and AMPK using small molecule inhibition. We used
western blotting to determine expression of UCP2 and activation of AMPK and
used adenoviral PPAR overexpression to determine whether these effects were a
consequence of PPAR activation.
Results: We identified that fenofibrate-mediated cytoprotection is AMPK and
PPAR-dependent (fig 1). Moreover, both fenofibrate and PPAR overexpression
activate AMPK(fig 2). We found that the mitochondrial uncoupling protein UCP2
is upregulated (fig 2).
Conclusions: Taken together, our data suggest that fenofibrates protective effect is
AMPK and PPAR dependent. Furthermore, because both fenofibrate and PPAR
overexpression activate AMPK, this event may be PPAR-specific. We have also
identified for the first time that both fenofibrate and PPAR upregulate UCP2,
which is known to inhibit neurodegeneration and also diabetic complications.
UCP2 is stabilized by active AMPK, and we hypothesize that fenofibrate AMPK
activation may be responsible for UCP2
upregulation.
Commercial Relationships: Elizabeth P. moran, None; Yusuke Takahashi,

None; Ying Chen, None; Jian-Xing Ma, None
Support: None
ASP-634: An Oral Drug Candidate for Diabetic Macular Edema
Tamie J. Chilcote, Sukanto Sinha. ActiveSite Pharmaceuticals Inc, San Francisco,
CA.
Purpose: ASP-440, a small-molecule plasma kallikrein inhibitor, was recently
shown to be effective, administered systemically, in reducing blood-retinal barrier
breakdown in diabetic rats (PMID: 21444925). The goal of this study was to assess
the safety and selectivity of ASP-440, and obtain an orally-active prodrug of ASP440, suitable for oral administration in the clinic for suppressing retinal vascular
leakage in diabetic macular edema (DME).
Methods: In vitro screening against a panel of proteases, receptors and ionchannels, and repeat-dosing studies with ASP-440 in vivo, were used to assess
selectivity and safety. A clinically successful prodrug strategy was utilized to
generate three distinct prodrugs of ASP-440. Oral dosing in rats and dogs was
utilized to select one prodrug that demonstrated sufficient oral bioavailability for
once-daily dosing in humans at a pharmacologically acceptable dose.
Results: Plasma levels of ASP-440 were found to correlate with the extent of
inhibition of diabetes-induced retinal vascular leakage (r2 = 0.99, p = 0.03),
indicating that plasma levels provide a surrogate measure of efficacious exposure.
ASP-440 did not exhibit meaningful interaction with any of the 41 off-targets
tested. Repeat dosing of ASP-440 (i.v.) at 5 mg/kg for 7 days resulted in daily peak
plasma levels >1000x EC90 levels. No adverse effects were noted, including in
hematology, clinical chemistry and renal function measures. All prodrugs were
found to be orally bioavailable, and were converted to ASP-440 in vivo. Of these,
ASP-634 exhibited the highest bioavailability, and oral dosing at 10 mg/kg in rats
resulted in peak plasma levels >25x EC90 levels. Oral dosing in dogs at 20 mg/kg
resulted in peak plasma levels >150x EC90 levels. All doses were well-tolerated.
Conclusions: Administered systemically, ASP-440 is a selective, safe and
efficacious inhibitor of diabetes-induced blood-retinal barrier breakdown. Oral
dosing with the prodrug ASP-634 in rats & dogs resulted in large multiples of
efficacious exposure of ASP-440, and these super-therapeutic doses were welltolerated in both species. Based on these data, qd p.o. dosing at 70 mg in the clinic
is projected to result in 24 h maintenance of >EC90 levels. ASP-634 is a novel oral
clinical candidate for DME.
Commercial Relationships: Tamie J. Chilcote, ActiveSite Pharmaceuticals (E),
Inventor (P); Sukanto Sinha, ActiveSite Pharmaceuticals (E), Inventor (P)
Eye Drops Of Small Molecular Weight Inhibitor SRPIN340, Target SRPK1/2
To Control VEGF Mediated Choroidal Neovascularisation

Melissa V. Gammons1, Masatoshi Hagiwara2, Andrew D. Dick3, David O. Bates1.
1
Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom;
2
Department of Anatomy and Developmental Biology, Graduate School of
Medicine, Kyoto University, Kyoto, Japan; 3Ophthalmology, Univ of BristolBristol Eye Hosp, Bristol, United Kingdom.
Purpose: Choroidal neovascularization (CNV) is the primary cause of vision loss
in patients with exudative age-related macular degeneration (AMD). Current antiVEGF-A therapies have proven both safe and effective in randomized clinical
trials, however, such treatments rely on intraocular injections as often as every
month. VEGF is alternatively spliced to produce pro-angiogenic and antiangiogenic families of sister isoforms. During pro-angiogenic isoform selection,
SRSF1 (ASF/SF2) is phosphorylated by SRPK1, allowing its nuclear localization
where it can bind to the proximal splice site (PSS) on VEGF pre-mRNA. Small
molecule inhibitors of SRPK1 have been shown to switch VEGF splicing, such
molecules are membrane permeable and show low toxicity. We therefore tested the
hypothesis that topical application of a small molecule inhibitor of SRPK1/2,
SRPIN340, could attenuate VEGF mediated CNV through selectively downregulating pro-angiogenic isoform production.
Methods: SRPIN340, an inhibitor of SRPK1 and SRPK2, was tested in a model of
laser-induced choroidal neovascularisation in C57B/6 mice. Subsequent to laser
photocoagulation 12 mice were treated with eye drops (20 drops over 14 days)
containing SRPIN340 (100g/ml, 10g/ml, 1g/ml, 0.1g/ml) or vehicle in the
control eye. The extent of CNV was analysed by flatmount of lectin stained
choroids on day 14.
Results: SRPIN340 eye drops dose dependently inhibited choroidal
neovascularisation in treated eyes with an EC50 of 3.2g/ml. SRPIN340 also
altered the pro-angiogenic to anti-angiogenic VEGF balance in human primary
retinal pigmented epithelial (RPE) cells from VEGF165 to VEGF165b at the RNA
and protein level. Furthermore SRPIN340 reversed IGF-1 induced nuclear
localization of SRSF1.
Conclusions: SRPIN340, when given as eye drops, supressed VEGF mediated
choroidal neovascularization. Furthermore, in addition to maintaining the
production of cytoprotective VEGFxxxb isoforms, SRPIN340 treatment was not
toxic to cells even at concentrations as high as 5mg/ml. This treatment could be an
alternative therapy to anti-VEGF-A intraocular injections in patients with exudative
AMD.
Commercial Relationships: Melissa V. Gammons, None; Masatoshi Hagiwara,

None; Andrew D. Dick, None; David O. Bates, None
Support: Fight for Sight, SCaRF
Anti-Proliferative Actions of the mTOR Inhibitor Temsirolimus in Primary
Human Retinal Pigmentepithelium (RPE) and Vascular Endothelial Cells
Susanna Koenig, Raffael G. Liegl, Aljoscha S. Neubauer, Lukas Reznicek, Christos
Haritoglou, Michael W. Ulbig, Anselm Kampik, Marcus Kernt. Ophathalmology,
Ludwig Maximilians University, Munich, Muenchen, Germany.
Purpose: The mammalian target of rapamycin (mTOR) plays a central role in cell
growth and proliferation. Dysregulation of mTOR has been linked to proliferative
retinal disease such as age-related macular degeneration (AMD) and diabetic
retinopathy (DR). This study investigates potential anti-proliferative effects of the
mTOR inhibitor temsirolimus on primary human retinal pigmentepithelium (RPE)
and vascular endothelial cells
Methods: Human umbilical vein endothelial cells (HUVEC) and RPE cells were
incubated for 24 hours with different concentrations of temsirolimus (0.1 to
25g/mL). Propidium iodide (PI) and Hoechst 33342 staining and tetrazolium dyereduction assay (MTT) were performed to exclude toxic concentrations. To
determine cell proliferation, cells were incubated with temsirolimus at the
maximum slope of the growth curve for 24 hours and MTT test was performed. In
addition, cell attachment was assessed by the MTT test and migration was
determined by a modified Boyden chamber method. 3D-Angiogenesis assay was

performed.
Results: Temsirolimus concentrations from 0.1 to 10g/mL for RPE, and 0.1 to
5g/mL for HUVEC showed no effects on cellular viability. At a concentration of
0.5g/mL, temsirolimus effectively inhibited angiogenesis, proliferation,
attachment, and migration of HUVEC and RPE cells. 0,5g/mL temisirolimus
accounted for the following: inhibition of cell proliferation to 41% for HUVEC and
82% for RPE cells, reduction in cell attachment to 63% (HUVEC) and 81% (RPE),
and inhibition of cell migration to 41%(HUVEC) and 28% (RPE), compared to the
untreated control. All effects were dose dependent. No toxic effects were detected
compared with controls.
Conclusions: In our experimental set-up, temsirolimus showed significant
inhibitory action on RPE and HUVEC angiogenesis, proliferation, migration and
attachment. Anti-proliferative effects were significant stronger in HUVEC,
compared to RPE cells. Our in vitro results provide early evidence that
temsirolimus provides promising properties for treatment of proliferative retinal
disease.
Commercial Relationships: Susanna Koenig, None; Raffael G. Liegl,
None; Aljoscha S. Neubauer, None; Lukas Reznicek, None; Christos
Haritoglou, None; Michael W. Ulbig, None; Anselm Kampik, None; Marcus
Kernt, None
Support: None
Multiple Treatments of the Sustained-Release Dexamethasone Implant in
Retinal Vein Occlusion
Matthew M. Wessel, Donald J. D'Amico, Szilard Kiss. Ophthalmology, Weill
Cornell Medical College, New York, NY.
Purpose: To report our experience with multiple injections of dexamethasone 0.7
mg sustained-release intravitreal implant (Ozurdex(); Allergan, Inc, Irvine, CA)
for the treatment of macular edema (ME) associated with retinal vein occlusion
(RVO).
Methods: A retrospective chart review of patients with RVO treated with at least
two sustained-release dexamethasone 0.7 mg intravitreal implants was performed.
Efficacy of treatment was determined by complete ophthalmic examination
including Snellen visual acuity (VA) and central retinal thickness as measured by
spectral domain ocular coherence tomography (OCT).
Results: Eight eyes of eight patients treated with a total of 23 dexamethasone 0.7
mg sustained-release intravitreal implants were included. Patients were followed at
regular intervals, and retreatment was based on recurrence of ME on OCT and/or
worsening of VA. All patients responded with improvement in VA and decrease in
ME. On average, time to retreatment was 23.2 weeks (median, 20.3 weeks; range,
16.0-41.0 weeks). Patients received an average of 2.9 total injections, with one
patient receiving 5 treatments. One patient developed a visually significant cataract
following multiple injections; no other serious ocular or systemic adverse events
were noted during the follow-up period. No patients in this series required
intraocular pressure lowering therapy.
Conclusions: In patients with recurrent macular edema in the setting of retinal vein
occlusion, multiple sustained-release dexamethasone 0.7 mg intravitreal implants
provide an effective treatment option that is well tolerated.
Commercial Relationships: Matthew M. Wessel, None; Donald J. D'Amico,
None; Szilard Kiss, Allergan, Inc (C)
Support: Research to Prevent Blindness, unrestricted departmental grant

Localisation Of The P2X7 Receptor In The Human Retina Using A Novel
mRNA Profiling Technique
Julie Sanderson1A, Ning Ma1A, Jeremy D. Rhodes1B, David C. Broadway2. ASchool
of Pharmacy, BSchool of Biology, 1University of East Anglia, Norwich, United
Kingdom; 2Department of Ohthalmology, Norfolk and Norwich University
Hospital, Norwich, United Kingdom.
Purpose: We have previously shown that activation of P2X7 receptors in the
human retina causes loss of retinal ganglion cells (RGCs). However, currently the
localization of the P2X7 receptor in the retina is not clear. The purpose of these
experiments was to determine the localisation of the P2X7 receptor in the human
retina using a novel mRNA profiling technique.
Methods: Human retina was obtained from the East Anglian Eye Bank within 24
hours post mortem. The macula region of the retina was dissected, mounted and
cryosectioned (20m sections) in the plane of the retinal nuclear layers. RNA was
extracted and QRT-PCR conducted for retinal markers (RCVRN; photoreceptors,
CALB; horizontal cells, CHAT; amacrine cells and THY-1; RGCs) and P2X7
(P2X7R) mRNA (n=6). Immunohistochemistry was carried out on transverse
sections for P2X7 receptor using an antibody raised to the extracellular domain.
Results: P2X7 receptor mRNA was found to be expressed in the human retina.
There was no significant difference between expression in the macula and

expression in peripheral retina (n=4). Expression was low in the outer retina with
no coincidence with the photoreceptor marker recoverin (RCVRN). The initial peak
of P2X7 expression corresponded with calbindin (CALB) expression and then
remained elevated, including in the retinal ganglion cell layer.
Immunohistochemistry showed labelling in the outer plexiform layer with less
intense labelling evident in the inner plexiform layer.
Conclusions: The P2X7 receptor was located in the outer and inner plexiform
layers of the human retina. There was no evidence for expression in the
photoreceptors, but P2X7R mRNA expression was coincident with the horizontal
cell marker CALB. In the inner retina, P2X7R expression was coincident with both
amacrine and RGC markers. This suggests that RGCs do express the P2X7 receptor
and that RGC death occurring as a result of P2X7 stimulation could be mediated by
direct mechanisms although indirect mechanisms may also play a role.
Commercial Relationships: Julie Sanderson, None; Ning Ma, None; Jeremy D.
Rhodes, None; David C. Broadway, None
Support: The Humane Research Trust, The Norwich Glaucoma Research Fund.
Effects of Pioglitazone on immune modulation in choroidal neovascularization
Anne F. Alex, Sebastian Cordes, Peter Heiduschka, Nicole Eter. Department of
Ophthalmology, University of Muenster Medical Center, Muenster, Germany.
Purpose: Choroidal neovascularization (CNV) is pathognomic in the wet form of
age-related macular degeneration (AMD) and can lead to retinal bleeding and
severe vision loss. The latest hypothesis of pathogenesis is an immune cell
regulated process of CNV. Pioglitazone (PPAR agonist) is an anti-diabetic drug
with known anti-inflammatory properties. The aim of this study was to investigate
the anti-inflammatory and immune modulatory effects of pioglitazone in a mouse
model of laser-induced CNV.
Methods: Human retinal endothelial cells (hREC) were incubated with different
concentrations of pioglitazone. Absolute cell numbers, living and dead cells were
determined and proliferation measured by flow cytometry. Furthermore,
CX3CR1+/GFP mice were fed with either pioglitazone or its solvent cellulose and
treated with an argon laser. Six and 14 days after laser, eyes were enucleated and
retina and choroid separated. One day before, some mice were anaesthetized and
autofluorescence imaging for GFP-positive cells was performed. For flow
cytometric analysis, CD11b staining for determination of microglial cells was
performed and cell characteristics measured. With real-time PCR, the changes of
pro- and anti-inflammatory cytokine levels (TNF, IL-6, IL-10) were evaluated.
Results: In vitro, pioglitazone did not show an effect on the proliferation of hREC.
In vivo, six days after laser, a strong trend in the reduction of GFP-positive cells
could be measured in the pioglitazone treated group. CD11b high cells,
representing microglia, were reduced. GFP-positive cells were hardly reduced 14
days after laser.
A strong anti-inflammatory effect could be seen on cytokine levels. Both, TNF
and IL-6, were reduced in the pioglitazone treated group. The anti-inflammatory
IL-10 was upregulated.
In autofluorescence imaging, markedly less GFP-positive cell were present in the
area of laser under pioglitazone treatment.
Conclusions: Pioglitazone showed a strong anti-inflammatory effect in laserinduced CNV. Repeatedly, a strong trend in the reduction of CX3CR1-positive
immune cells could be demonstrated six days after laser. Additionally, the
immigration of immune cells into the area of laser was obviously reduced in
autofluorescence imaging.
Pioglitazone could be a therapeutic approach, which interacts earlier in the cascade
of CNV pathogenesis.
Commercial Relationships: Anne F. Alex, None; Sebastian Cordes,
None; Peter Heiduschka, None; Nicole Eter, None
Support: None
317 Angiogenesis and Antiangiogenic Agents
Tuesday, May 8, 2012, 8:30 AM - 10:15 AM
Program #/Board # Range: 2939-2971/A384-A416
Program Number: 2939 Poster Board Number: A384
Utility Of Ranibizumab In A Choroidal Macular Nevus Associated With
Subretinal Neovascularization And A Macular Pucker
Marta Nuez, Mari Paz Mendivil. Ophthalmology, Universitary Hospital of Basurto,
Bilbao, Spain.
Purpose: Describe the advantages of Intravitreal Ranibizumab in a case of
choroidal macular nevus associated with subretinal neovascularization and a
macular pucker.
Methods: We present the case report of a 70-year-old man with visual acuity of
20/20 in his right eye in year 2005 in whom the diagnosis of macular nevus was
made ( a pigmented, well defined, not elevated lession of two disk diameter size
with some drusen overlying ). In february of 2008 he referred a decrease in his

visual acuity of the right eye ( 20/50 ). His fundus examination suggested subretinal
neovascularization, so we made an optical coherence tomography and a fluorescein
angiography which confirmed the macular choroidal nevus with subretinal
neovascularization. Our patient was treated with three intravitreal Ranibizumab
injections and two months later he referred important visual acuity improvement
(20/25), the neovascularization had dissapeared and the choroidal nevus had
decreased until being almost imperceptible. In year 2010 he referred again a
decrease in his visual acuity of the right eye ( visual acuity 20/32 ), the fundus
examination and optical coherence tomography revealed a macular pucker, but
there were no signs of choroidal neovascularization .
Results: The treatment with ranibizumab was successful in our patient, achieving
to date the disappearance of the choroidal neovascularization associated with
choroidal macular nevus and improving his visual acuity.
Conclusions: Choroidal neovascularization associated with choroidal macular
nevus is not a frequent complication. Besides, there is very few biblography about
the use of antivascular endotelial grow factor antibodies (anti-VEGF) in these
cases. In our case report, Ranibizumab seems to be an effective treatment for
choroidal neovascularization associated to choroidal macular nevus, achieving
good results to date ( even 3 years after the intravitreal injections
).
Commercial Relationships: Marta Nuez, None; Mari Paz Mendivil, None
Support: None
Treatment of Complicated Retinal Arterial Macroaneurysm with Intravitreal
Bevacizumab
Simona Romano1, Francesco Pichi1, Andrea Lembo1, Mariachiara Morara2,
Antonio P. Ciardella3, Paolo Nucci4. 1University Eye Clinic, San Giuseppe
Hospital, Milan, Italy; 2Ophthalmology, Ospedale S.Orsola Malpighi, Bologna,
Italy; 3Ophthalmology, Policlinico S Orsola Malpighi, Bologna, Italy;
4
Ophthalmology, SAN GIUSEPPE HOSPITAL University of Milan, Milan, Italy.
Purpose: To evaluate the anatomical and functional results of the treatment with
bevacizumab (Avastin) in complicated retinal arterial macroaneurysm (RAM).
Methods: 37 eyes with retinal complications associated with a retinal arterial
macroaneurysm affecting the fovea were evaluated. All patients underwent a
comprehensive ophthalmologic examination, fluorescein angiography, and OCT
examination.
Each patient included underwent 3 monthly injections of bevacizumab (Avastin)
1.25 mg/0.05 ml; three follow-up visits were planned at week 2,6 and 10, and the
last follow-up took place 6 months from the initial visit.
Results: At 2 weeks following the first intravitreal bevacizumab (Avastin)
injection, mean BCVA improved to 0.320.02 logMAR (p = 0.0254). Most eyes
with minimal hemorrhage showed resolution of the exudative changes, along with a
physiologic appearance of the fovea. In eyes with a predominant hemorrhagic
component, the retinal hemorrhage was reduced and mean CRT decreased by 329.6
m to 213.9 m. At 6 weeks of follow-up, 2 weeks after the second injection, FA
showed complete closure of the macroaneurysm in 36/38 cases (94.7%) and the
macular edema surrounding the macroaneurysm resolved further. Two weeks
following the third injection, the macular edema had completely resolved and hard
exudates regressed slowly in 100% of patients.
Conclusions: Anti-VEGF drugs might actively close the involved pathologically
permeabilized retinal artery and normalize the vessel wall formation by localized
inhibition of VEGF.
This is the first case series of patients who have been treated with intravitreal
bevacizumab (Avastin) therapy for retinal macroaneurysm and showed rapid
decrease in CRT and visual improvement.

Commercial Relationships: Simona Romano, None; Francesco Pichi,
None; Andrea Lembo, None; Mariachiara Morara, None; Antonio P. Ciardella,
None; Paolo Nucci, None
Support: None
Comparison of Angiostatic Properties of Pigmented Epithelium Derived
Factor(PEDF) and Infliximab in a Neovascularization Model
Alexei Lukashev1, Natalia Gavrilova2, Irina Saburina3, Vasilii Shirokov2, Olga
Tischenko2. 1Stemedica Cell Technologies, San Diego, CA; 2Ophthalmology,
Moscow University of Medicine and Dentistry, Moscow, Russian Federation;
3
Institute of General Pathology, Moscow, Russian Federation.
Purpose: To analyze and compare the use of Infliximab alone and in combination
with PEDF on retina in a neovascularization model.
Methods: A neonatal neovascularization model in Wistar rats, exposed to
hyperoxic then to normal environment was used in experiments. Two blocks of
experiments were performed.
Block 1-Establishing a baseline: Three tests (3x N=11) and one healthy control
groups were used to define the level of tumor necrotic factor (TNF-alpha) and
vascular endothelial growth factor (VEGF 121,165) at day 13, 15 and 18 post
natal(PN) in relation to the density of vascularization(DOV) of retina.
Block 2-Angiostatic experiment: Six (6x N=11) groups were injected intravitreally
on day 13-15 PN by Infleximab; PEDF and a combination of Infleximab and
PEDF. Additional two groups(2x N=11) were injected with Bevacizumab and
Ranibizumab for comparison. Four days after injection eyes were removed. Indirect
immunofluorescence staining with biotinylated Griffonia Simplicifolia Lectin and
Streptavidin Fluorescein was used to visualize retina vascular system. The level of
TNF-alpha and VEGF 121,165 in the sensory retina and retinal pigmented
epithelial (RPE) cells layer were measured by ELISA.
Results: The increase of the TNF-alpha level is significant at day 15 PN, and
VEGF-165 at day 18 PN. Both trends correlate to each other(r=0.78; p<0.05) as
well as with DOV (r= 0.72; 0.82; p<0.05). All test groups in the block 2 express
significant decrease of DOV along with a significant decrease of TNF-alpha and
VEGF165. Most notably this effect was observed in the group injected with
Infliximab at day 13 PN and PEDF at day 15 PN.
Conclusions: Injection of Infliximab and PEDF resulted in a strong angiostatic
effect by decreasing the density of vascularization. Angiostatic effect of injection of
Infliximab and PEDF in combination is higher than Infleximab, PEDF,
Bevacizumab, Ranibizumab just alone.
Commercial Relationships: Alexei Lukashev, Stemedica (E); Natalia
Gavrilova, None; Irina Saburina, None; Vasilii Shirokov, None; Olga
Tischenko, None
Support: None
Effect of Intravitreal Bevacizumab (Avastin ) on the Foveal Retinal
Thickness in the Injected and Contralateral Eye in a Diverse Clinic Population
Jonathan Naysan, Khurram M. Chaudhary, Ronni M. Lieberman. Ophthalmology,
North Shore - Long Island Jewish, Great Neck, NY.
Purpose: There has been speculation on the systemic absorption of intravitreal
bevacizumab (Avastin ) in treated patients. This study examines the effect of a
single intravitreal injection of bevacizumab (1.25mg/0.05mL) on the foveal retinal
thickness (FRT) in both the injected and the contralateral eye in a diverse clinic
population.
Methods: A retrospective review of 11 patients (22 eyes) who received a single
intravitreal injection of bevacizumab was conducted. The foveal retinal thickness
was measured using the Zeiss OCT software both before and one month after each
patient was treated with a single dose of bevacizumab. Additional data included
demographics, diagnoses at time of injection, best corrected visual acuities
(BCVA) (converted to logMar) at the time of injection and at 1 month postinjection, and past medical history. Significance was calculated using the Z test.
Results: Of the 11 patients, there were 8 Asians, 2 Caucasians, and 1 African
American. All of the 11 patients had been diagnosed with clinically significant
macular edema (CSME) associated with diabetes. The mean FRT of the injected
eyes prior to injection was 321.6m 177.5m (range, 197 to 552m). The mean
FRT of the contralateral eyes was 335.9m 515.5m (range, 121 to 1152m). At
one month post injection, the mean FRT of the injected eye showed an 18.6%
decrease from baseline to a mean of 261.6m 172m (p=0.6317). The
contralateral eye group showed a decrease of 0.7% to a mean of 333.6m 508m
(p=0.6064).
Conclusions: We found an expected decrease in overall retinal thickness in the
injected eye at one month post injection. In the contralateral eye there was an
overall decrease in FRT which was not found to be significant. Although minimal
systemic absorption of bevacizumab has been documented, which may effect the
contralateral eye initially, there does not appear to be an effect at one month.
Further study is needed investigating the effects of multiple bevacizumab injections

with a longer period of follow up, in addition to early (1 to 7 days) effects on the
contralateral eye. These findings may further contribute to the safety profile of
intravitreal bevacizumab.
Commercial Relationships: Jonathan Naysan, None; Khurram M.
Chaudhary, None; Ronni M. Lieberman, None
Support: None
Effects of VEGF Trap and Bevacizumab on Neovascularization and Retinal
Revascularization in Murine Model of Oxygen Induced Retinopathy (OIR)
Eunice Cheung, Ivan B. Lobov, George D. Yancopoulos, Stanley J. Wiegand.
Ophthalmology, Regeneron Pharmaceuticals, Inc., Tarrytown, NY.
Purpose: Vascular Endothelial Growth Factor (VEGF) plays an essential role in
normal and pathological angiogenesis. VEGF Trap (aflibercept), is a potent VEGF
inhibitor that binds all isoforms of VEGF-A as well as the VEGF R1 ligands,
placental growth factor (PlGF) and VEGF-B. When administered systemically,
VEGF Trap effectively blocks pathological neovascularization in OIR, while
promoting normative revascularization of the retina (IOVS 2006; 47:E-Abstract
1750; IOVS 2011:E-Abstract 3210). In this preclinical study, we evaluated the
effects of VEGF Trap and bevacizumab, a selective blocker of VEGF-A, in the
murine model of OIR.
Methods: Because bevacizumab effectively binds and blocks only human, and not
murine VEGF-A, these experiments employed transgenic mice in which the gene
for murine VEGF-A was replaced by the gene encoding human VEGF-A, exons 27. (huVEGF mice). huVEGF mice were place in a hyperoxic environment (75%
O2) at postnatal day 6 (P6) and returned to room air at P11. VEGF Trap,
bevacizumab or a control protein (Fc region of human IgG 1) were administered i.p.
on P12 at the same dose (6.25mg/kg), one day following return to room air.
Hypoxyprobe (Hypoxyprobe, Inc.) was injected at P16, eyes were harvested one
hour later, and retinal flatmounts were stained with anti-hypoxyprobe FITC-labeled
antibody (Hypoxyprobe, Inc.) and with biotinylated Isolectin (Sigma) and
streptavidin Alexa Fluor 594 (Invitrogen) to visualize blood vessels.
Results: Compared to controls, both VEGF Trap and bevacizumab treatments
tended to accelerate the regrowth of retinal blood vessels in huVEGF mice.
However, VEGF Trap treated eyes had both the smallest residual avascular areas as
well as the greatest reduction in neovascularization compared to both bevacizumab
treated mice and controls. Similarly, although both treatments produced a marked,
concomitant reduction in retinal hypoxia, as determined by hypoxyprobe staining,
VEGF Trap treated eyes had smaller areas of retinal hypoxia compared to
bevacizumab treated eyes.
Conclusions: In this preclinical study, systemic administration of anti-VEGF
agents shortly after return to room air not only prevented pathological
neovascularization, but also promoted normal vascular regrowth. VEGF Trap
markedly reduced hypoxic areas and reduced vascular abnormalities, to a greater
extent than seen with bevacizumab. Whether this difference is attributable to the
higher binding affinity of VEGF Trap, or its ability to neutralize PlGF and VEGFB, as well as VEGF-A, remains to be determined.
Commercial Relationships: Eunice Cheung, Regeneron Pharmaceuticals, Inc.
(E); Ivan B. Lobov, Regeneron Pharmaceuticals, Inc. (E); George D.
Yancopoulos, Regeneron Pharmaceuticals, Inc. (E); Stanley J. Wiegand,
Regeneron Pharmaceuticals, Inc. (E)
Support: None
Bevacizumab Eye Drops: A Pilot Study On Penetration Of The Anti-vegf In
The Aqueous Humor
Tommaso Micelli Ferrari, saba ciani, massimo lorusso, marco leozappa, alessia
dipietro, luisa micelli ferrari. UOC Oculistica, Ente Ecclesiatico, Bari, Italy.
Purpose: PURPOSE.Vascular endotelial growth factor is essential for
neovascularization.The use of anti VEGF therapies has largely shown to inhibit this
process.Here, the penetration of topical bevacizumab in anterior chamber and its
effects on VEGF levels in humor acqueous
Methods: METHODS.VEGF humour levels were measured after application of
bevacizumab topical eye drops, four times a day in 20 patients who were going to
undergo to chataract surgery.The study was based on standard sandwich enzymelinked immuno sorbent assay technology and an undirect method. Each Humour
sample and specific detection polyclonal antibody were added to the wells and then
washed away with PBS buffer.an enzymatic complex was used to visualize
enzymatic reaction.The catalyzed product produce a blue color that changed in
yellow after adding acidic stop solution.the density of yellow is proportional to the
human VEGF amount of sample captured in plate.The results were compared with
case control
Results: RESULTS.The application of bevacizumab topical eye drops determine
acqueous VEGF levels reduction compared with case control with a standard

deviation of 0,036 and a medium value of 0,140, respectly instead of 0,039 and
0,036.No negative effects were reported.
Conclusions: CONCLUSION.Topical application of Bevacizumab, can penetrate
anterior chamber and can inhibit VEGF cytokines by reducing their levels.We need
to examine many more patients but,in future, Avastin eye drops would mean the
same anti VEGF effects with less complicance than intravitreal use.
Commercial Relationships: Tommaso Micelli Ferrari, None; saba ciani,
None; massimo lorusso, None; marco leozappa, None; alessia dipietro,
None; luisa micelli ferrari, None
Support: None
Anti-VEGF Therapy Reduces the Rate of Neovascular Glaucoma Due to
Central Retinal Vein Occlusion
Christina L. Ryu, Adrian Elfersy, Paul Edwards, Uday Desai, Thomas Hessburg,
Hua Gao. Department of Ophthalmology, Henry Ford Hospital, Detroit, MI.
Purpose: Ischemic central retinal vein occlusion (CRVO) is known to cause
neovascular glaucoma (NVG). Incidence of NVG in previous reports ranges from
23% to 60%, with a recent study of CRVO natural history from Hayreh reporting
NVG incidence of 35%. It is well known that neovascularization is caused by
vascular endothelial growth factor (VEGF) from retinal ischemia. Recently, antiVEGF therapy has become widely used to treat macular edema due to CRVO. This
study is to determine if anti-VEGF therapy can reduce the rate of NVG.
Methods: This is a retrospective study of ischemic and nonischemic CRVO
patients who received anti-VEGF therapy for macular edema. Patient charts were
reviewed from November, 2004 to July, 2011. Patients with presenting
neovascularization, prior panretinal photocoagulation (PRP), and prior anti-VEGF
treatments were excluded. Patients with open angle glaucoma and nonproliferative
diabetic retinopathy were included. The primary outcome is the rate of NVG.
Results: Thirty eyes of 30 patients with CRVO were included in the study. 17 of
30 eyes were non-ischemic CRVO, and 13 were ischemic CRVO. Of the 17 nonischemic CRVO eyes, mean presenting visual acuity was 20/80. They received an
average of 4.6 anti-VEGF injections (bevacizumab or ranibizumab) over a period
of 9.2 months (one injection every 1.9 months) for treatment of cystoid macular
edema. Total mean follow up time was 16.9 months. No eye developed NVG,
which is consistent with prior reports of nonischemic CRVO. However 1 eye
(5.9%) required PRP for neovascularization. Mean visual acuity at last follow up
was 20/50. Mean pre-treatment central retinal thickness (CRT) on OCT was 481
microns, and mean post-treatment CRT, 313 microns. Of the 13 ischemic CRVO
eyes, mean presenting visual acuity was 20/320. They received an average of 3.5
anti-VEGF injections over a period of 7.4 months (one injection every 2.8 months).
Total mean follow up time was 12.2 months. No eye developed NVG, but 3 eyes
(23.1%) developed neovascularization requiring PRP. Mean presenting IOP was
15.8 mmHg, and mean final follow up IOP, 14.8. Mean visual acuity at last follow
up was 20/800. Mean pre-treatment CRT was 615 microns, and mean posttreatment CRT, 438 microns.
Conclusions: Our study shows that anti-VEGF therapy significantly reduced the
rate of NVG in ischemic CRVO compared to the previously reported rate in the
literature. Thus anti-VEGF therapy may improve the natural course of CRVO,
especially in ischemic patients. A larger clinical trial is needed to confirm the
results of our study.
Commercial Relationships: Christina L. Ryu, None; Adrian Elfersy,
None; Paul Edwards, None; Uday Desai, None; Thomas Hessburg, None; Hua
Gao, None
Support: Alliance for Vision Research, Inc.
Anti-angiogenic Effects Of Nov C-ter: Down-regulation Of Pro-inflammatory
Cytokines Through VEGF-independent Pathways
Chadi Mehanna1,2, Norbert Minet2, Marie-Christine Naud3, Laurence Leconte2,
Jean-Louis Bourges1, Francine Behar-Cohen1. 1CRC - UMRS 872 - Hotel Dieu
Hospital - Ophthalmology, University of Paris Descartes, Paris, France; 2SISENE,
Paris, France; 3Team 17, CRC, UMRS 872, INSERM, Paris, France.
Purpose: to study the possible mechanisms of action involved in the antiangiogenic effects observed with NOV C-ter (Sisne, France) in the corneal
micropocket assay.
Methods: The corneal micropocket assay was performed on 37 Lewis male rats of
6-8 weeks old. LPS pellets (Sigma-Aldrich, France) were implanted into the
corneal stroma followed by sub-conjunctival injections every other day for 8 days.
Rats were randomly assigned to the following groups: no treatment, PBS,
bevacizumab 250g, NOV C-ter 5 g, NOV C-ter 10 g and NOV 2g. At day 9,
angiogenesis was assessed clinically using a grading score, animals were
euthanized and corneas and conjunctivas were extracted. Real-time PCR analysis
was performed to evaluate the expression of several genes: VEGF, VEGF-R1,
VEGF-R2, CD31, iNOS, IL-1, IL-6, TNF-, MCP-1, desmin, STAT3 and
connexin-43.
Results: NOV C-ter 10g and NOV 2g significantly reduced LPS induced
angiogenesis (P < 0.05). No significant effect was observed for bevacizumab. Realtime PCR analysis of the corneas showed no significant effects on the expression of
VEGF, VEGF-R1, VEGF-R2, iNOS, IL-1, MCP-1, desmin, and STAT3. IL-6 was
downregulated by NOV 2g and NOV C-ter 10g (P < 0.0001), CD31, TNF- and
connexin-43 were down-regulated by NOV 2g (P < 0.05). In the conjunctivas,
NOV C-ter 10g down-regulated iNOS, MCP-1, connexin-43 and STAT3 (P <
0.05).
Conclusions: Anti-angiogenic effects observed for NOV C-ter, in the rat corneal
micropocket assay, seem to be independent of the VEGF pathway and may linked
to down-regulation of inflammation mediators.
Commercial Relationships: Chadi Mehanna, SISENE (E); Norbert Minet,
SISENE (E); Marie-Christine Naud, None; Laurence Leconte, SISENE
(E); Jean-Louis Bourges, None; Francine Behar-Cohen, None
Support: SISENE
Lymphocytes-derived Microparticles Modulate Angiostatic Factors In Retinal
Pigment Epithelium Cells
Pierre Hardy1A, Houda Tahiri1A, Chun Yang1A, Francois Duhamel1B, Carmen
Gagnon1B. APediatrics & Pharmacology, BPharmacology, 1University of Montreal,
Montreal, QC, Canada.
Purpose: The retinal pigment epithelium cells (RPE) play a central role in retinal
vascularisation. RPE cells produce a variety of growth factors helping to maintain
the structural integrity of choriocapillaris endothelium. We have previously
reported that Human T-lymphocyte-derived microparticles (LMPs) significantly
inhibit pathological angiogenesis interfering through VEGF/VEGFR2 signalling
pathway in vitro and vivo experiments. In addition, we recently observed the strong
antiangiogenic effects of LMPs on choroidal neovascularization and
overexpression of the neurotrophins low-affinity p75NTR receptor in cultured
choroidal explants. This study is designed to determine how RPE cells mediate the
anti-angiogenic effects of LMPs in the choroidal neovascularisation.
Methods: LMPs were produced by treatment of human T-lymphocytes with
actinomycin D. The rat model of choroidal explants was used to determine the
antiangiogenic effects of LMPs. RPE-removed choroidal tissues were prepared
using dispase enzyme solution. Expression of angiostatic factors from choroidal
and RPE-removed choroidal tissues were assessed by RT- PCR. Caspase-3 was
used to determine cell apoptosis in choroidal explants. Primary cultured RPE cells
were used in in vitro experiments.
Results: LMPs time-dependently inhibited choroidal neovascularisation (NV).
Removing RPE cells from the choroidal explants resulted in a strong abolishment
of the antiangiogenic effects of LMPs. Accordingly, LMPs significantly increased
the expression of angiostatic factors in choroidal explants such as pigment
epithelial-derived factor (PEDF), neurotrophin growth factor (NGF) but not in
RPE-removed choroids. Interestingly, the culture medium from RPE cells was able
to rescue the anti-angiogenic effect of LMPs on choroidal NV. Using specific
antibodies against PEDF, p75NTR or shRNA against p75NTR significantly
blocked the anti-angiogenic effect of LMPs. Consequently, the LMPs-induced NGF
caused a significantly apoptosis of choroidal endothelial cells.
Conclusions: The strong antiangiogenic effects of LMPs on choroidal NV are
largely dependent on targeting both RPE and choroidal endothelial cells_which
play important roles in the choroidal angiogenesis. More specifically, PEDF and
NGF, the anti-angiogenic factors produced from RPE cells, are important mediators
of LMPs on choroidal NV. Our data suggested that LMPs may be of therapeutic
value in treating ocular neovascular diseases.
Commercial Relationships: Pierre Hardy, None; Houda Tahiri, None; Chun
Yang, None; Francois Duhamel, None; Carmen Gagnon, None
Support: CIHR GRANT MOP - 86631
Intravitreal Injection Of Bevacizumab For Naive Myopic Choroidal
Neovascularization: Results Obtained After 18 Months Of Treatment
flore DE BATS, Jean Daniel Grange, Philippe Denis, Laurent Kodjikian. Croix
Rousse Hospital, Lyon, LYON, France.
Purpose: Choroidal neovascularization is a complication of high myopia which
can reduce the visual prognosis in young patients. The purpose of this study was to
evaluate the efficacity and safety of bevacizumab in the first-line treatment of
myopic choroidal neovascularization.
Methods: We report a retrospective study of patients with subfoveal or juxtafoveal
choroidal neovascularization associated with pathologic myopia treated with
intravitreal injection of bevacizumab in Lyon, France, from January 2009 to June
2010. Best-corrected visual acuity, ocular pressure, fundus examination, optical
coherence tomography and fluorescein angiography were performed for each
patient at baseline and monthly. Indications for retreatment were persistent on

recurrence of exsudative activity.
Results: The study included 7 eyes of 7 patients with a mean of age of 44 and
spherical equivalent refractive error of -12 diopters. The mean follow-up time was
18 months. The mean number of intravitreal injections was 3 at the end of the first
year. 6 patients maintained or improved their vision. No injection complications or
drug-related side effects were noted during the follow-up period.
Conclusions: In this study, intravitreal injection of bevacizumab seems to be a safe
and effective treatment for myopic choroidal neovascularization resulting in
functionnal and anatomic improvement.
Commercial Relationships: flore De bats, None; Jean Daniel Grange,
None; Philippe Denis, None; Laurent Kodjikian, None
Support: None
Arresten Regulates Choroidal Angiogenesis By Fasl, Caspase-3 And Parp
Mediated Apoptosis In-vitro And In-vivo
Raj K. Verma1, Venugopal Gunda2, Chandra S. Boosani2, Sudhakar A. Yakkanti3.
1
Cell Signalling Retinal angiogenesis, Boys Town National Research Hospital,
Omaha, NE; 2Genetics, Boys Town Nt'l Research Hosp, Omaha, NE; 3Genetics/
Retinal Cell Signaling, Boys Town Natl Res Hospital, Omaha, NE.
Purpose: Type IV collagen plays a crucial role in regulation of angiogenesis.
Proteolytic degradation of type IV collagen in the vascular basement membrane
(VBM) generates antiangiogenic molecules. We previously reported that the
antiangiogenic non-collagenous (NC1) domain derived from 1 chain of type IV
collagen, 1(IV)NC1 (arresten) induces apoptosis in endothelial cells. However,
active site of arresten responsible for its antiangiogenic properties is not yet
identified. In the present study, the N- and C-terminal regions of arresten were
cloned as two individual subunits and studied their anti-angiogenic and proapoptotic properties.
Methods: Both the N- and C-terminal subunits of arresten were cloned in the
pET22b (+) expression vector and expressed in Escherichia coli. Expressed
subunits were purified using affinity and size exclusion chromatographies with
simultaneous renaturation.
Results: Our preliminary in-vitro results with mouse choroidal endothelial cells
(MCECs) have shown inhibition of VEGF and bFGF induced endothelial cells
proliferation, migration and tube formation by both the subunits. In addition,
arresten promoted FasL mediated apoptosis through caspase-3 and PARP both invitro and in-vivo.
Conclusions: Both N- and C-terminal subunits of arresten exhibited antiangiogenic
activity in MCECs by showing inhibitory effects on VEGF and bFGF induced
proliferation, migration and tube formation. The FasL mediated apoptosis
activation by arresten is also identified.
Commercial Relationships: Raj K. Verma, None; Venugopal Gunda,
None; Chandra S. Boosani, None; Sudhakar A. Yakkanti, None
Support: 5R01CA143128-02
Effect of Morpholino Targeting Flt-1 in Mice Corneal Keratoplasty Model
Yang Kyung Cho1,2, Hironori Uehara2, Jason R. Young2, Wei Huang2, Ling Luo2,3,
Xiaohui Zhang2, Bonnie Archer2, Jacquelyn Simonis2, Thomas Olson2, Bala
Ambati2. 1Ophthalmology, St.Vincent's hospital, The Catholic University of Korea,
Suwon, Republic of Korea; 2Moran Eye Center, University of Utah, Salt Lake City,
UT; 3Ophthalmology, The 306 Hospital, Beijing, China.
Purpose: Morpholinos are synthetic molecules similar to DNA oligonucleotides
which can bind mRNA and pre-mRNA and sterically block the molecular
machinery of translation or alternative splicing. We sought to determine whether
morpholinos can target the alternative splicing of Flt-1 pre-mRNA to upregulate the
production of sFlt, limiting neovascularization and lymphangiogenesis resulting in
increased graft survival in a murine corneal penetrating keratoplasty(PK) model.
Methods: We used a murine corneal keratoplasty model to investigate the effect of
a morpholino that promotes sFlt to suppress neovascularization and
lymphangiogenesis to increase graft survival. We performed mice corneal
penetrating keratoplasty in one eye of each Balb/C mouse (C57BL6 mice as
donors) and injected 40ng/ul of Flt morpholino(n=16), nonspecific (STD)
morpholino(n=18), and PBS(n=11) into the subconjucntival space on the day of PK
and weekly until postoperative 4 weeks. Corneal opacity was graded to evaluate
graft survival weekly through postoperative week 8, at which point the corneas
were harvested. After flat mounting the harvested corneas, we performed
immunohistochemistry and fluorescent microscopy to digitally quantify
neovascularization. We compared the area of neovascularization and
lymphangiogenesis to the total corneal area and graft area
Results: Flt morpholino increased graft survival compared to the PBS group
(p=0.043).Flt morpholino group showed less neovascularized area in both total
cornea (0.22670.0139) and graft (0.10050.0173) cornea when compared with
STD the morpholino group (0.27710.0257 in total cornea, 0.21900.0328 in graft
cornea, respectively) and PBS group (0.27770.0220 in total cornea,

0.18830.0384 in graft cornea, respectively) (all ps<0.05). The Flt morpholino
group showed less lymphangiogensis in both total cornea (0.07800.0095) and
graft cornea (0.07190.0109) compared with the STD morpholino group
(0.10050.0114 in total cornea, 0.12580.0198 in graft cornea) and PBS group
(0.11990.0078 in total cornea, 0.12310.0165 in graft cornea) (all ps<0.05).
Conclusions: We found that morpholinos promoting sFlt reduced angiogenesis and
lymphangiogenesis compared with STD morpholino and PBS in a murine corneal
penetrating keratoplasty model and increased graft survival compared with PBS.
Commercial Relationships: Yang Kyung Cho, None; Hironori Uehara,
None; Jason R. Young, None; Wei Huang, None; Ling Luo, None; Xiaohui
Zhang, None; Bonnie Archer, None; Jacquelyn Simonis, None; Thomas Olson,
None; Bala Ambati, None
Support: NIHEY017950
Preliminary Results of Combined Anti-VEGF Therapy and Laser
Photocoagulation (LPC) in Patients with Retinal Vein Occlusion (RVO): Pilot
Study
Olga Y. Truneva, Irina V. Davydova, Natalia N. Olunina. Eye Laser Surgery
Center, Krasnoyarsk, Russian Federation.
Purpose: One of the severe complications of RVO is cystoid macular edema
(CME), which challenges the performance of grid LPC. We study the efficacy of
combined treatment including retinal LPC and intravitreal injections of
ranibizumab on visual acuity (VA) and retinal morphology in patients with RVOassociated CME.
Methods: Study included 7 patients (7 eyes) with RVO (2 central RVO and 5
branch RVO), aged 44-54 years. Mean VA was 20/100. MeanSt.D. optical
coherent tomography (OCT)-measured central retinal thickness (CRT) was
562.596.0m. Patients were treated with monthly intravitreal injections of
ranibizumab 0.5mg followed by panretinal or grid LPC, wavelength 532 nm.
Number of injections varied from 2 to 3. LPC was added in 3-4 weeks after the first
injection, as the CRT reduced and non-perfusion areas appeared on the fluorescent
angiography (FA). Ranibizumab was discontinued when OCT showed resolution of
macular cysts and normalization of retinal topography. Patients are followed up
with VA, fundoscopy, FA and OCT for 10 months.
Results: In all cases, CME significantly regressed within 2 weeks after the first
injection of ranibizumab. CRT at 2-week time point reduced by (MeanSt.D.)
332.633.0m. Mean VA improved to 20/50. All patients noted decreased
metamorphopsias and central scotomas. Two patients received LPC 2 weeks after
the first injection. Both developed recurrent CME 6-8 weeks after the LPC. Their
CME resolved after additional injections of ranibizumab and LPC. The rest 5
patients received three monthly injections of ranibizumab. Their CME improved
gradually after each injection and fully resolved after the third injection. LPC in
these 5 patients was performed step-by-step as both the CME and the hemorrhages
along the main arcades resolved, according to the OCT and FA. At 4-month time
point after the last treatment, none of the patients had recurrence of macular edema.
Conclusions: Use of ranibizumab in central and branch RVO results in rapid
resolution of CME and recovery of macular topography and provides optimized
conditions for the sufficient extent of LPC. In combination, ranibizumab and LPC
are effective in achieving better outcomes in VA than LPC alone. Follow up with
the patients and new patient recruitment is in progress.
Commercial Relationships: Olga Y. Truneva, None; Irina V. Davydova,
None; Natalia N. Olunina, None
Support: None
Intravitreal Bevacizumab versus Ranibizumab for Rubeosis Iridis
Joao J. Nassaralla, Jr.1, Belquiz A. Nassaralla2. 1Retina and Vitreous, Instituto de
Olhos de Goiania and UnB, Goiania, Brazil; 2Cataract Cornea & Refractive
Surgery, Instituto de Olhos de Goiania, Goiania, Brazil.
Purpose: Bevacizumab (Avastin) and Ranibzumab (Lucentis) are an anti-VEGF
recombinant humanized monoclonal IgG1 antibody (both are produced by the same
company _Genentech, based in San Francisco - CA - USA). But there are
differences between the two drugs. Lucentis is administered in the form of smaller
molecules, which is thought to give Lucentis an advantage over Avastin in its
ability to penetrate the eye's retina and halt abnormal blood vessel growth
contributing to advanced macular degeneration and scarring that causes blindness.
They may have a role in treating ocular disorders involving fibrovascular
proliferation. To determine and compare whether intraocular bevacizumab and
ranibzumab decreases RI in patients with neovascular glaucoma (NVG).
Methods: The study included 72 eyes of 64 patients with secondary NVG due to
proliferative diabetic retinopathy (n=60) or ischemic vessel occlusion (n=12). All
patients received an intraocular injection (IOI) of 1.25 mg of bevacizumab (IB) or
ranibizumab (IR), from October 2005 to October 2010 at the Goiania Eye Institute.

Intraocular injection was performed in topical anaesthesia either in the anterior
chamber (n=58) or via pars plana intravitreally (n=14). RI was investigated
prospectively by iris biomicroscopy. An expanded informed consent with an offlabel use waiver and explanation letter was reviewed with all patients. The IOI was
performed under sterile conditions in an operating theatre and was performed with
proximetacaine (Anestalcon - Alcon - Brazil) topical anesthesia and the injection
did via pars plana.
Results: Degree of RI decreased significantly (p<0.01) within 1 week after
application of both drugs. The improvement was maintained for at least 4 weeks.
The two drugs are about equal in their effectiveness. An inflammatory response
with fibrinous reaction and pseudohypopyon was observed in three cases (2
bevacizumab and 1 with ranibizumab) one day after surgery. The reaction persisted
only 2 days. However, that cost differences for those receiving treatment are major
at about $40 per injection for Avastin and $2,000 per injection for Lucentis.
Conclusions: The preliminary conclusion is that the two drugs are about equal in
their effectiveness, but. IB or IR may provide an additional strategy to the treatment
of RI. Its long term effect and impact on NVG has to be determined. The number of
patients in this pilot study was limited and the follow-up is too short to make any
specific treatment recommendations, but the favorable short-term results suggest
further study is needed. More studies are needed to evaluate the effect of this
medication prior to clinical use.
Commercial Relationships: Joao J. Nassaralla, Jr., None; Belquiz A.
Nassaralla, None
Support: None
Perfused but not leaking
David A. Salz1, Gary Shienbaum2, Jonathan Prenner3, Howard Fine3, Richard
Kaiser1. 1Wills Eye Institute, Philadelphia, PA; 2Bascom Palmer Eye Institute,
Miami, FL; 3Robert Wood Johnson University Hospital, New Brunswick, NJ.
Purpose: Perfused but not leaking (PNL) describes the angiographic state that
develops after cessation of leakage following anti-vascular endothelial growth
factor (VEGF) therapy. PNL describes an identifiable, persistent choroidal
neovascular complex that has stopped leaking but has not regressed. Herein, we
present 11 cases exhibiting this clinical phenomenon.
Methods: Retrospective, interventional, consecutive case series. Eleven eyes of 11
patients with neovascular age-related macular degeneration (AMD) who achieved
PNL status were examined. PNL was defined based on characteristic fluorescein
angiographic features, i.e. early hyperfluorescence and absence of late leakage,
combined with normalization of intra- and sub-retinal fluid on optical coherence
tomography (OCT) following anti-VEGF therapy. Baseline characteristics,
treatment course, and outcomes data were collected and analyzed.
Results: The mean age was 76 years old (range, 59 - 87). Baseline mean Snellen
visual acuity and OCT central subfield thickness (CST) were 20/79 (range, 20/25 2/200) and 301 m (range, 190 - 477), respectively. Four eyes received
bevacizumab and 7 eyes received ranibizumab. The mean number of injections
until PNL was recognized was 4.7 (range, 2 - 10). Mean Snellen visual acuity and
OCT CST at the time of PNL recognition were 20/50 (range, 20/20 - 6/200) and
258 m (range, 180 - 303).
Conclusions: PNL is a common but frequently overlooked angiographic finding
that demonstrates the antimitogenic and antipermeability effects of anti-VEGF
therapy, but also highlights that blocking VEGF alone does not lead to vascular
regression. Clinical implications of PNL need to be defined but it could explain the
high recurrence rate appreciated when anti-VEGF therapy is discontinued.
Commercial Relationships: David A. Salz, None; Gary Shienbaum, None;
Jonathan Prenner, equity owner of Ophthotech and Neovista (I); Howard Fine,
None; Richard Kaiser, equity owner of Ophthotech and Neovista (I)
Support: None
Binding and Neutralization of Vascular Endothelial Growth Factor and
Related Ligands by VEGF Trap, Ranibizumab and Bevacizumab
Ashique Rafique1A, Ergang Shi1A, Qin Ruan1A, Joel Martin1A, Michael P. Rosconi1B,
Erica Pyles1B, Nick Papadopoulos Papadopoulos1A, George D. Yancopoulos1, Neil
Stahl1, Stanley J. Wiegand1C. ATherapeutic Protein, BPreclinical Development and
Protein Chemistry, COpthalmology and Cardiovascular Research, 1Regeneron
Pharmaceutical Inc, Tarrytown, NY.
Purpose: To evaluate the in vitro activity and binding characteristics of VEGF
Trap (aflibercept), bevacizumab (BEV) and ranibizumab (RAN) to vascular
endothelial growth factor-A (VEGF-A121 ,VEGF-A165), VEGF-B10-118 and placental
growth factor (PlGF-1 and PlGF-2).
Methods: Kinetic binding parameters were measured using Surface Plasmon
Resonance (Biacore) and equilibrium binding constant in solution was determined
using (Kinexa) technology. VEGF-A121, VEGF-A165 and PlGF-2 induced activation
of VEGF receptors (VEGFR1, VEGFR2) were investigated using HEK293 cells

stably transfected with an NFB-luciferase plasmid and either human VEGFR1 or
VEGFR2. VEGF-A165 calcium mobilization and migration were investigated using
human umbilical vein endothelial cells (HUVEC).
Results: VEGF Trap bound with high affinity to human VEGF-A121 (KD=0.36pM)
and VEGF-A165 (KD=0.49pM) and to the equivalent proteins from rabbit, rat and
mouse. High affinity binding also was observed between VEGF Trap and human
PlGF-1 (KD=392pM) and PlGF-2 (KD=38.8pM). BEV and RAN bound to VEGFA165 with 118- and 94-fold lower affinity than VEGF Trap, respectively as
determined by Surface Plasmon Resonance. In the VEGFR1 cell line, VEGF Trap
effectively blocked luciferase activity induced by 20 pM VEGF-A121 (IC50=15pM)
or VEGF-A165 (IC50=16pM) and was ~50 to 90 times more potent than BEV or
RAN. Only VEGF Trap was able to block PlGF-2-induced (40 pM) luciferase
activity in this cell line (IC50=2.89 nM). In the VEGFR2 cell line, VEGF Trap
effectively blocked luciferase activity induced by 20 pM VEGF-A121 (IC50=16pM)
or 20 pM VEGF-A165 (IC50=26pM) and was 30-50 times more potent than BEV or
RAN.
Conclusions: These data differentiate VEGF Trap from bevacizumab and
ranibizumab in terms of binding affinity for VEGF-A isoforms, as well as its ability
to bind PlGF and VEGF-B. In addition, VEGF Trap demonstrates increased
potency relative to bevacizumab and ranibizumab in blocking VEGF induced
receptor(s) activation and HUVEC migration.
Commercial Relationships: Ashique Rafique, Regeneron Pharmaceutical Inc
(F, E); Ergang Shi, Regeneron Pharmaceutical Inc (F, E); Qin Ruan, Regeneron
Pharmaceutical Inc (F, E); Joel Martin, Regeneron Pharmaceutical Inc (F, E);
Michael P. Rosconi, Regeneron Pharmaceutical Inc (F, E); Erica Pyles,
Regeneron Pharmaceutical Inc (F, E); Nick Papadopoulos Papadopoulos,
Regeneron Pharmaceutical Inc (F, E); George D. Yancopoulos, Regeneron
Pharmaceutical Inc (F, E); Neil Stahl, Regeneron Pharmaceutical Inc (F, E);
Stanley J. Wiegand, Regeneron Pharmaceutical Inc (F, E)
Support: None
Effect of Combined Topical Heparin and Steroid on Corneal
Neovascularization in Children
Stephan Michels1, Stephan L. Kaminski2, Rike Michels3. 1Ophthalmology, Triemli
Hospital Zurich, Zurich, Switzerland; 2Ophthalmology, University of Vienna,
Vienna, Austria; 3Ophthalmology, University Hospital Zurich, Zurich, Switzerland.
Purpose: To demonstrate the effect of topical heparin combined with topical
steroid on corneal neovascularizations (CN) in children.
Methods: Four children (5 eyes) with new onset progressive CN in at least one eye
received topical rimexolone or dexamethasone in combination with heparin until
complete regression of the CN is obtained. The regression of CN is documented by
slit-lamp/anterior segment photography.
Results: All 5 eyes showed complete regression of the CN within 5 months. An
anti-angiogenic effect was found as early as one week following start of topical
combination treatment. No ocular and systemic side effects were detected and
treatment was well tolerated by all children. In the 3 eyes with involvement of the
optical axis symmetrical visual acuity was obtained by amblyopia treatment.
Recurrence of the CN was detectable in 2 eyes at one and six months after ending
combination therapy. Both eyes responded favourably to retreatment.
Conclusions: Combination of topical heparin and steroid leads to rapid regression
and complete inactivity of CN. This therapeutic approach is promising especially in
children with limited therapeutic alternatives and a high risk for amblyopia.
Commercial Relationships: Stephan Michels, None; Stephan L. Kaminski,
None; Rike Michels, None
Support: None
Intracorneal Bevacizumab (Avastin) For The Treatment of Corneal
Neovascularization
Stephan L. Kaminski1, Isabella Baumgartner2, Stephan Michels3. 1Ophthalmology,
University of Vienna, Vienna, Austria; 2Ophthalmology, Medical University of
Vienna, Vienna, Austria; 3Ophthalmology, Triemli Hospital, Zuerich, Switzerland.
Purpose: To evaluate the effect of repeated intracorneal bevacizumab on
inflammatory corneal neovascularization.
Methods: Six patients with corneal neovascularization were treated with
intracorneal injections of 5mg bevacizumab. All patients had persistent corneal
neovascularization for at least 10 months unresponsive to other treatments. Patients
were monitored by ophthalmic exam, anterior segment photography and corneal
fluorescein angiography (FA). Each patient completed a minimum follow-up of 12
months.
Results: All eyes showed at least partial regression of the corneal
neovascularization. Mean size of the neovascularization as measured by corneal FA
regressed from 3.07 0.87 mm from baseline to 1.740.79 mm at 12 months

follow-up. Eyes being without intracorneal bevacizumab injections for more than 6
weeks showed partial regression of the corneal neovascularization. There was no
evidence of any severe corneal or systemic adverse events.
Conclusions: Intracorneal bevacizumab showed promising effects in inflammatory
neovascularization of the cornea unresponsive to other treatment. Anti-VEGF
therapy might become a valuable treatment option for corneal neovascularization.
Commercial Relationships: Stephan L. Kaminski, None; Isabella
Baumgartner, None; Stephan Michels, None
Support: None
Iris Neovascularisation Remodelling Under Intravitreal Vegf Suppression
Leonidas Zografos1, Aude Ambresin2, Ann SCHALENBOURG1. 1Ophthalmology,
Jules-Gonin Eye Hospital, Lausanne, Switzerland; 2Ophthalmology, Jules Gonin
Eye Hospital, Lausanne, Switzerland.
Purpose: The aim of this prospective study was to analyse the benefit of
intravitreal (IV) anti VEGF injection on iris neovascularisation (NVI) secondary to
proton beam irradiation of posterior uveal malignant melanomas (MM)
Methods: All eyes presenting secondary NVI were consecutively included. Iris
fluorescein angiography (FA) was performed at baseline and at each follow up
(FU) visit. Based on FA, NVI was classified as diffuse, sectorial, pupillary and
tufts. The degree of dye leakage was quantified. Retreatment was applied in case of
persistent leaking new vessels.
Results: From 2006 to 2010, 920 eyes received proton therapy for MM. 69 eyes
presented secondary NVI. At baseline, 43% were diffuse and dye leakage was
marked in 55%. Sectorial iris ischemia was present in 64% of cases. During the
follow up period, the numbers of injections ranged from 1-6. Regression of NVI
was observed in 68% of eyes and 67% of eyes showed reduction of dye diffusion.
Over time, remodelling of the ischemic iris with progressive revascularisation
occurred in 32% of eyes and 50% of eyes showed maturation of the NVI. Eye
retention probability was 96%.
Conclusions: In growth of well shaped new vessels in the ischemic zone and
maturation of the NVI suggest that organised angiogenesis can occur under
pharmacologically modulated VEGF expression.Anti VEGF injections is a
promising therapeutic approach of NVI secondary to proton beam irradiation
achieving a high rate of eye retention
Commercial Relationships: Leonidas Zografos, None; Aude Ambresin,
None; Ann Schalenbourg, None
Support: None
Alpha 6 Type IV Collagen Non-Collagenous Domain Regulates Elastin and
VGVAPG Activated Choroidal Endothelial Cell Migration and Tube
Formation
Venugopal Gunda1, Raj K. Verma1, Chandra S. Boosani1, Sudhakar A. Yakkanti1,2.
1
Genetics, Boys Town Nt'l Research Hosp, Omaha, NE; 2Department of
Biomedical Sciences, Creighton University School of Medicine, Omaha, NE.
Purpose: Choroidal neovascularization evident in age related macular degeneration
involves the migration and tube formation by choroidal endothelial cells, which are
regulated through the extracellular matrix components. Elastins and their derivative
bioactive peptide, VGVAPG play pathological roles by promoting endothelial cell
(EC) migration and tube formation in choroidal neovascularization. The type IV
collagen derived non-collagenous domains (NC1) are known to exhibit antiangiogenic properties and inhibit neovascularization. However, the effect of any of
the type IV collagen NC1 domain on elastin mediated angiogenesis was not
studied. We have evaluated the effect of type IV collagen alpha 6 chain derived
non-collagenous domain [6(IV)NC1] on elastin and VGVAPG mediated
angiogenic effects in-vitro.
Methods: Recombinant human 6(IV)NC1 was cloned and purified from
transformed E.coli for in vitro evaluations. Effect of purified 6(IV)NC1 on kappa
elastin, mouse elastin and VGVAPG mediated mouse choroidal endothelial cell
(MCEC) migration and tube formation were studied using Boyden chamber and
Matrigel methods respectively. MMP-14 levels from cell culture supernatants and
cell lysates of elastin-, VGVAPG- and 6(IV)NC1- treated MCEC cultures were
also evaluated using western blot analyses.
Results: Recombianat 6(IV)NC1 inhibited mouse choroidal endothelial cell
migration and tube formation activated by kappa elastin, mouse elastin and the
bioactive peptide VGVAPG. 6(IV)NC1 also showed effect on elastin and
VGVAPG mediated MMP-14 secretion by MCECs.
Conclusions: Recombinant 6(IV)NC1 inhibits elastin and VGVAPG stimulated
angiogenesis in vitro. Elastin and VGVAPG mediated MCEC migration and tube
formation are inhibited by 6(IV)NC1. Elastin and VGVAPG mediated MMP-14
secretion by MCEC are inhibited by 6(IV)NC1. The mechanism of action of
6(IV)NC1 and its in-vivo potential are yet to be deciphered.
Commercial Relationships: Venugopal Gunda, None; Raj K. Verma,

None; Chandra S. Boosani, None; Sudhakar A. Yakkanti, None
Support: NIH/NCI grant RO1CA143128, Dobleman Head and Neck Cancer
Institute grant DHNCI-61905
Response Of Choroidal Neovascularization In Animal Models With Use Of
Amniotic Membrane
ARACELI ROJAS DAZ1, Rojas Diaz Joel1, Ramirez Estudillo Juan Abel1, Levine
Berebichez Arthur1, Lpez Espinosa Nadia L.2, Bautista de Lucio Victor Manuel2.
1
Retina, Fundacion Hospital Nuesta Sra de la Luz, Mexico Distrito Federal,
Mexico; 2Investigacion, Instituto Oftalmologico Conde de Valenciana, mexico
Distrito Federal, Mexico.
Purpose: o Identify the response of choroidal neovascularization with the use of
amniotic membrane. establishing a reproducible model of laser choroidal
neovasculariacin, identifying in each group and variable expression of
proangiogenic factors.
Methods: A prospective, observational, longitudinal, experimental, interventional,
analytical and descriptive study was done. Four groups were created a market
consisting of three rabbits, the first with placebo intravitreal injection, the second
intravitreal injection of lyophilized amniotic membrane, the third was a model of
choroidal neovacularizacin created with laser, and the fourth a model of choroidal
neovascularization treated with intravitreal amniotic membrane, studies clinical,
fluorangiographyc and determination of pro and anti-angiogenic factors by
chemiluminescence.
Results: A total of 15 rabbits 3 of which were used to create the model of choroidal
neovascularization where the average laser power used was 2 W with exposure of
500 ms and spot diameter of 100 microns and the remaining 12 were divided into 4
groups described above, the rabbits in group 4 who had choroidal
neovascularization with application of amniotic membrane had better clinical
response and fluorangiography compared to group 3 received no treatment with
amniotic membrane, the expression of proangiogenic factors are overexpressed in
the group untreated and decreased in the group treated with amniotic membrane.
Conclusions: There were clinical differences between the group not treated with
amniotic membrane and treated with the same, being better in the group treated
with amniotic membrane, There are over expression of VEGF and proangiogenic
factors in the group with choroidal neovascularization, decreasing in the group
treated with amniotic membrane. Model was obtained reproducible
neovascularization with laser.
Commercial Relationships: ARACELI Rojas daz, None; Rojas Diaz Joel,
None; Ramirez Estudillo Juan Abel, None; Levine Berebichez Arthur,
None; Lpez Espinosa Nadia L., None; Bautista de Lucio Victor Manuel, None
Support: None
Comparative Evaluation of the New Validated In Vitro Assay of Human
Angiogenesis and Clinical Data of Anti-Angiogenic Compounds Used in
Retinal Diseases
Ulla Aapola1A, Jertta-Riina Sarkanen1B, Hannele Uusitalo-Jrvinen1A, Tuula
Heinonen1B, Hannu Uusitalo1A. AOphthal/SILK R&D Ctr Ophthal Inno, BFICAM
Finnish Center for Alternative Methods, 1University of Tampere, Tampere,
Finland.
Purpose: The purpose of the study was to evaluate the correlation of the effective
doses of clinically used anti-angiogenic ophthalmic drugs and those doses
inhibiting angiogenesis in a novel in vitro assay. The assay is an intra-laboratory
validated human cell culture assay with current applicability domain containing so
far more than 30 different chemicals from clinical drugs and chemicals. The tested
compounds included bevacizumab, ranibizumab and triamcinolone from which the
effective dose data was available from controlled randomized clinical studies.
Methods: The angiogenesis fibroblast endothelial cell co-culture assay was
established under Good Laboratory Practice. In the assay, the pre-vascular like
tubule formation was induced with 10 ng/ml vascular endothelial growth factor
(VEGF) and 1 ng/ml basic fibroblast growth factor. The cell assay was exposed to
test drugs for six days, drugs applied to assay twice during the study. The tubule
length and tubule branching were evaluated semiquantitatively under microscope
by comparing to positive and negative controls at a predetermined scale.
Results: The VEGF-A antibody bevacizumab inhibited tubule formation
significantly at 0.05 g/ml (50% inhibition at 0.1 g/ml), the high binding
monoclonal VEGF-A antibody ranibizumab at 0.01 g/ml (50% inhibition at 0.05
g/ml), and triamcinolone acetonaide at 0.01 g/ml (50% inhibition at 10 g/ml).
Conclusions: The results obtained in this study show good concordance with the
therapeutic vitreal concentrations reported in clinical trials of various retinal
diseases, indicating that the effective concentrations of several angiogenesisaffecting chemicals can be reliably screened in this human cell culture angiogenesis
assay. This is not only cost-effective and ethical step for the non-clinical phase of

the development of new treatments for retinal disease but also accelerates this
phase and drug discovery.
Commercial Relationships: Ulla Aapola, None; Jertta-Riina Sarkanen,
None; Hannele Uusitalo-Jrvinen, None; Tuula Heinonen, None; Hannu
Uusitalo, None
Support: European Regional Development Fund, Elsemay Bjrn Fund
Tissue Plasminogen Activator As An Anti-angiogenic Agent
Aki Kato, Tsutomu Yasukawa, Kon-ichi Arai, Ayae Kubota, Yuichiro Ogura.
Ophthalmology, Nagoya City Univ Med School, Nagoya, Japan.
Purpose: Anti-vascular endothelial growth factor (VEGF) drugs such as
ranibizumab has been effectively utilized in the treatment of age-related macular
degeneration. However, once-developed fibrovascular tissues often remain in the
subretinal space and mechanically damage the retina. Tissue plasminogen activator
(tPA) is a fibrinolytic compound, utilized originally to treat embolic or thrombotic
stroke and as an adjuvant for displacement of submacular hemorrhage. The purpose
of this study is to investigate possible complimentary anti-angiogenic effects of
tPA.
Methods: Human umbilical vein endothelial cells (HUVECs) were cultured and
seeded onto collagen I-coated 96-well culture plates at a density of 3x103
cells/well. On the following day, tPA was added to the medium at various
concentrations. After 24 h after incubation, cell growth was assessed by using XTT
assay. A basic fibroblast growth factor (bFGF)-impregnated gelatin hydrogel sheet
was implanted into a corneal pocket in rabbits to induce corneal neovascularization.
Then intravitreal injection of tPA (4x104 IU/100 l) (n=6) or saline as a control
(n=4) was performed. The maximal length of induced corneal neovascularization
was measured at 1, 2 and 4 week.
Results: Neither growth nor viability of HUVECs was affected by tPA at
concentrations of 5x105 IU/ml or lower. In a rabbit corneal neovascularization
model, limbal injections were followed by sprouting of new vessels from the
limbus toward bFGF-impregnated hydrogel. The mean length of new vessels was
1.00.4 mm at week 1, 1.60.7 mm at week 2, and 3.11.9 mm at week 4 in
control eyes. In contrast, neovascularization was highly suppressed in eyes with
intravitreal tPA. The mean length of new vessels was 0.170.4 mm at week 1, 00
mm at week 2, and 00 mm at week 4.
Conclusions: In vitro experiment showed that tPA did not affect the growth and
viability of endothelial cells. Nevertheless, tPA remarkably suppressed the
development of neovascularization in a rabbit corneal neovascularization model.
These findings suggested that fibrin might be essential for angiogenesis and that
tPA might be a possible adjuvant in the treatment of ocular angiogenesis itself as
well as submacular hemorrhage.
Commercial Relationships: Aki Kato, None; Tsutomu Yasukawa, None; Konichi Arai, None; Ayae Kubota, None; Yuichiro Ogura, None
Support: H23-116 Grant-in Aid for Scientific Research from Ministry of Health,
Labor and Welfare of Japan
In Vitro And In Vivo Response Of VEGFR-2 To VEGF165 With/Without
Ranibizumab
Amber Woolfenden, Elizabeth Fassbender, Siyuan Shen, Kathyrn McAllister, Lisa
Baker, Bruce D. Jaffee, Stephen H. Poor, Yubin Qiu. Ophthamology
Pharmacology, Novartis Institutes for Biomedical Research, Cambridge, MA.
Purpose: Establish and validate an in vitro and in vivo pharmacodynamic assay for
VEGFR-2.
Methods: Human Umbilical Vein Embryonic Cells (HUVECs) were seeded in 96
well plates at 1X106 and allowed to adhere for 2 hours. Recombinant Human
VEGF165 (rhVEGF165) in saline was added to cells at a concentration range of
0.025ng/ml to 1ug/ml and incubated at 37C for 10 minutes. Plates were placed on
ice, medium aspirated and cell lysis buffer added for 1 hour. Cell lysates were
centrifuged and supernatant analyzed by enzyme-linked immunosorbent assay
(ELISA) detecting VEGFR-2 autophosphorylation (p-VEGFR-2).
Dutch Belted rabbits (n = 3 or 4 rabbits/group) were challenged with an intravitreal
injection of 5, 10 or 20 g rhVEGF165 in saline at a volume of 50 l to both eyes.
Rabbit eyes were harvested on ice 20 minutes post rhVEGF challenge, retina and
retinal pigment epithelium (RPE)/choroid isolated and snap frozen. Tissues were
later homogenized in the presence of protease and phosphatase inhibitors and
retinal p-VEGFR-2 levels were analyzed by ELISA. 500 g of Ranibizumab or
vehicle control in a volume of 50 l were injected into the vitreous of both eyes of
rabbits (n = 3 or 4 rabbits/group). Rabbit eyes were then challenged with an
intravitreal injection of 10 g of rhVEGF165 in saline at a volume of 50 l in one
study 3 days after injection and in a second study at 5 days after injection of
ranibizumab. Rabbit eyes were harvested after 20 minutes and retinal and
RPE/choroid p-VEGFR-2 levels were analyzed by ELISA as described above.
Results: Addition of rhVEGF165 to HUVEC cells demonstrated a dose dependant
increase in levels of activated phospho-VEGFR-2 (rhVEGF EC50=62ng/ml).

Intravitreal injection of rhVEGF165 at 5, 10, and 20 g/eye resulted in
approximately a 2 fold increase of retinal p-VEGFR-2 compared with saline
injected controls, a dose response was not observed. Autophosphorylation of
VEGFR-2 in the RPE/choroid was minimal under this experimental condition.
Intravitreal ranibizumab injected at a dose of 500 g/eye on day 3 or day 5 prior to
10 g rhVEGF intravitreal challenge reduced p-VEGFR-2 in the retina by an
average of 57% as compared to saline controls.
Conclusions: This model system may be used to assess pharmacodynamics effects
of anti-VEGF agents and VEGFR-2 inhibitors.
Commercial Relationships: Amber Woolfenden, None; Elizabeth Fassbender,
None; Siyuan Shen, None; Kathyrn McAllister, None; Lisa Baker, None; Bruce
D. Jaffee, None; Stephen H. Poor, None; Yubin Qiu, None
Support: None
A Quantitative In Vivo Assay For Measuring Tnf- Induced Retinal Vascular
Permeability
Mike Lin. Retina Research, Alcon Research Ltd, Fort Worth, TX.
Purpose: Purpose: Blood-retinal barrier breakdown occurs in many ocular
diseases associated with inflammation. The effect of inflammatory cytokine Tumor
necrosis factor (TNF)- on retinal vascular permeability was investigated in adult
rats, and a quantitative in vivo assay was established.
Methods: Methods: Adult Sprague Dawley rats were intravitreally challenged
with a range of doses (10-1000 ng) of recombinant human (rh) TNF-. At several
time points post TNF- stimulation, retinal vascular permeability was evaluated by
measuring the extravasated vascular tracer Evans Blue dye in retinal tissue
normalized with plasma Evans Blue concentration and retina weight. The optimum
amount and duration of rhTNF- stimulation were used in later inhibitor study, in
which two TNF- specific inhibitors, AL-50738 and AL-88974, were given
intravitreally 24 hrs prior to TNF- injection. In addition, the retinal protein level of
Vascular Endothelial Growth Factor (VEGF) after TNF- treatment was measured
by Elisa.
Results: Results: rh TNF- dose-dependently induced retinal vascular leakage in
rats, with robust leakage observed at 48-72 hours after 300-1000 ng TNF-
intravitreal injection. Two specific TNF- inhibitors, ESBA105 and ESBA1622
efficaciously prevented TNF- induced retinal vascular leakage. No significant
change in retinal VEGF protein level was observed after recombinant human or rat
TNF- treatment.
Conclusions: Conclusions: The rat retinal vasculature reproducibly responded to
human TNF- stimulation in a VEGF-independent manner. Evans Blue
measurement can quantitatively measure TNF- induced retinal vascular leakage.
This in vivo model can be further used for evaluating anti-TNF- agents.
Commercial Relationships: Mike Lin, None
Support: None
Serum Levels Of Intravitreally Placed I-124 Bevacizumab And I-124
Ranibizumab In A Rabbit Model Following Lensectomy, Vitrectomy And No
Surgery
Angela Jiang1A, Jillian Wang1A, Cedric Pratt1A, Michelle Carlton1B, George
Hinkle1B, Michael V. Knopp1B, John Christoforidis1A. AOphthalmology,
B
Radiology, 1The Ohio State University College of Medicine, Columbus, OH.
Purpose: To determine the serum levels of I-124 bevacizumab and I-124
ranibizumab after intravitreal injection following lensectomy and vitrectomy and to
compare these with non-operated eyes in a rabbit model.
Methods: Six Dutch-belted rabbits underwent pars plana vitrectomy (PPV), 6
rabbits underwent pars plana lensectomy (PPL) and 6 rabbits served as non-surgical
controls. Twelve days following the surgical procedures, each operated eye
underwent an intravitreal injection consisting of 0.5 mg/0.05 ml I-124 labeled
ranibizumab or 1.25 mg/0.05 ml I-124 labeled bevacizumab. Serum levels from
each rabbit were drawn on days 2, 5, 7, 14, 21, 28 and 35. Serum radioactivity
levels in counts/min for I-124 bevacizumab and I-124 ranibizumab were measured
with a gamma counter (Perkin Elmer-Wizard 2).
Results: Serum radioactivity measurements with standard deviations on day 2 for
I-124 bevacizumab were 51,314 (+/-9,425) counts/min for non-surgical controls,
175,248 (+/-19,724) counts/min after PPV and 230,330 (+/-6523) counts/min after
PPL. For I-124 ranibizumab these values were 6,531 (+/- 5,619) counts/min for
non-surgical controls, 2,474 (+/-486) counts/min after PPV and 4,722 (+/-2,957)
counts/min after PPL. The serum half-lives with standard deviations of elimination
for I-124 bevacizumab were 16.84 (+/-7.23) days for non-surgical controls, 2.49
(+/- 0.07) days after PPV and 3.91 (+/-1.39) days after PPL. For I-124 ranibizumab
the half-lives were 3.81 (+/-1.65) days for non-surgical controls, 3.56 (+/-0.30)
days after PPV and 2.78 (+/-0.52) days after PPL.
Conclusions: Serum levels were higher for I-124 bevacizumab than I-124

ranibizumab at all time points for all three study groups. The serum levels of I-124
bevacizumab were elevated following lensectomy and vitrectomy compared to nonsurgical eyes following intravitreal injection, while no such pattern was present for
I-124 ranibizumab. The half-life of I-124 bevacizumab was prolonged in nonsurgical eyes presumably due to a slower release from the vitreous cavity, while the
half-lives for I-124 ranibizumab did not differ between treatment groups.
Measurement of serum radioactivity levels of intravitreally placed radiolabeled
intravitreal agents represents a novel technique for quantitating agent serum levels.
Commercial Relationships: Angela Jiang, None; Jillian Wang, None; Cedric
Pratt, None; Michelle Carlton, None; George Hinkle, None; Michael V. Knopp,
None; John Christoforidis, None
Support: Central Ohio Lions Fund
Effect Of Anti-vegf Drugs And Steroids On The Inner Blood-retinal Barrier,
After Experimental BRVO In Rats
Peggy Bouzika1, Nicole Gilodi1, Alain Conti1, Miltiadis K. Tsilimbaris2, Constantin
J. Pournaras1. 1Ophthalmology, Geneva University Hospitals, Geneva,
Switzerland; 2Ophthalmology-Research Acct, University of Crete, Heraklion,
Greece.
Purpose: To evaluate the effect of anti-VEGF drugs and steroids on the inner
blood-retinal barrier (BRB) function after experimental branch retinal vein
occlusion (BRVO) in rats. The integrity of the barrier is related to the expression of
occludins by retinal endothelial cells.
Methods: Laser photocoagulation was applied to retinal veins of 12 Long-Evans
rats, in order to induce BRVO. The occlusion was performed on the right eye of
each animal, while the left eye was used as control. One day after the occlusion, the
right eye of each animal was injected intravitreally with either artificial aqueous
humor (n=4), bevacizumab (n=4) or triamcinolone (n=4). The animals were
sacrificed 24 hours later; all retinas were harvested and prepared for indirect
immunohistochemistry, using an anti-occluding primary antibody. The specimens
were examined using a confocal microscope and images were acquired.
Results: In normal retinas, occludins appeared as a dense, well-organised,
fluorescent reticulum along the vascular wall. In eyes with BRVO, the structure of
occludins was disrupted; irregular linear fluorescence appeared along occluded
veins, while absence of fluorescence suggested down-regulation of occludin
expression. Occludin distribution was much improved in animals having received
treatment post BRVO.
Conclusions: The administration of bevacizumab and triamcinolone post BRVO
seems to improve the function of the inner BRB. This could explain macular edema
reduction after the use of these drugs. These results are part of an ongoing
experiment, which will include the use of combination therapy in the management
of BRVO.
Commercial Relationships: Peggy Bouzika, None; Nicole Gilodi, None; Alain
Conti, None; Miltiadis K. Tsilimbaris, None; Constantin J. Pournaras, None
Support: None
Vascular Endothelial Growth Factor in Plasma of Patients with ARMD after
Intravitreal Injection of Bevacizumab, Ranibizumab and Pegaptanib
Gerhard F. Kieselbach1, Claus Zehtner2A, Martina T. Kralinger2A, Rudolf
Kirchmair2B. 1Ophthalmology, Medical University of Innsbruck, Telfs, Austria;
A
Ophthalmology, BInternal Medicine, 2Medical University of Innsbruck, Innsbruck,
Austria.
Purpose: To investigate plasma vascular endothelial growth factor (VEGF) levels
after intravitreal Bevacizumab, Ranibizumab and Pegaptanib injection (IVI) into
eyes with age related macular degeneration (ARMD)
Methods: Thirty eyes of 30 patients with ARMD and 11 eyes of 11 patients
without ocular diseases (wod) were randomized after diagnosis of wet ARMD.
Each group consisted of ten eyes, identified for treatment with one of the three
study agents. Analysis included evaluation of basic clinical conditions and
measurement of plasma VEGF concentrations using enzyme-linked immunosorbent
assays before intravitreal Injection of Bevacizumab, Ranibizumab and Pegaptanib.
Measurement of plasma VEGF concentrations one week and four weeks after
intravitreal Injection of Bevacizumab, Ranibizumab and Pegaptanib was continued.
Results: ARMD eyes without IVI had normal plasma VEGF levels compared with
the eyes in the wod group. Mean concentration before IVI was 99.5 and 112.2 one
week after IVI. Four weeks after IVI mean concentration was 25.4 with a high
deviation. This was a statistically significant decrease compared to plasma VEGF
levels before and the wod group. The difference between level before and after one
week was not significant. Comparing Bevacizumab with Ranibizumab and both
with Pegaptanib no statistically significant difference could be observed. Overall
there was no plasma level difference observed between Bevacizumab, Ranibizumab
and Pegaptanib
Conclusions: Plasma VEGF levels in patients with ARMD were at the same level
like in patients without ocular diseases. Four weeks after IVI the VEGF plasma
concentrations were significantly decreased in all groups. Our findings confirm
recent studies referring to comparable SAE after IVI with Bevacizumab and
Ranibizumab.
Commercial Relationships: Gerhard F. Kieselbach, None; Claus Zehtner,
None; Martina T. Kralinger, None; Rudolf Kirchmair, None
Support: None
Clinical Trial: https://eudract.ema.europa.eu, 2010024654-11
Effect Of A Single Intravitreal Injection Of Anti-VEGF Agents On Retinal
Arteriolar Caliber In Mini Pig Eyes
Georgios Mangioris, Ioannis K. Petropoulos, Efstratios Mendrinos, Constantin J.
Pournaras. Department of Ophthalmology, Geneva University Hospitals, Geneva,
Switzerland.
Purpose: The aim is to investigate the short-term effect of a single intravitreal
bevacizumab, ranibizumab or pegaptanib sodium injection on the retinal arteriolar
caliber in minipigs.
Methods: Eight eyes received an intravitreal injection: bevacizumab 1,25mg (n=3),
ranibizumab 0,5mg (n=4) and pegaptanib sodium 0,3mg (n=1). The diameter of the
retinal arterioles was measured in vivo with a Retinal Vessel Analyzer (RVA)
every 15 minutes for 2 hours.
Results: For the bevacizumab group, mean retinal arteriolar diameter was 198.9m
at baseline, 190.6m after 30min, 183.5m after 45min, 169.9m after 1h,
182.2m after 1h and 30min and 169.6m after 2h. For the ranibizumab group,
mean retinal arteriolar diameter was 191.16m at baseline, 176.5m after 30min,
169.5m after 45min, 167.7m after 1h, 162.5m after 1h and 30min and 163.4m
after 2h. The eye that received pegaptanib sodium showed no modification of the
arteriolar diameter. There was no significant change in MAP during the follow-up
period (p > 0.05).
Conclusions: After the injection of bevacizumab statistical significant
vasoconstriction was reached 1h after injection (p< 0.01) and persisted until the end
of the measurements (2h). Ranibizumab injection induces a transient
vasoconstriction of the retinal arterioles. Statistical significant (p < 0.05) arteriolar
vasoconstriction is reached only 45min following the injection. Preliminary results
after pegaptanib sodium injection revealed no vasomotor effect of the substance.
The results suggest that intravitreal injection of anti VEGF induces retinal
vasoconstriction. Further studies with a larger number of subjects would be helpful
in establishing more clearly the effect of intravitreal anti-vascular endothelial
growth factor treatment on retinal vessel diameters.
Commercial Relationships: Georgios Mangioris, None; Ioannis K.
Petropoulos, None; Efstratios Mendrinos, None; Constantin J. Pournaras,
None
Support: None
Effect of anti-VEGF Therapy on Cellular Markers during the Proliferative
Phase of Capillary Hemangioma Growth
Brett W. Davies. Ophthalmology, Wilford Hall Medical Center, San Antonio, TX.
Purpose: The proliferative phase of capillary hemangioma growth is characterized
by high expression of vascular endothelial growth factor (VEGF), proliferating cell
nuclear antigen (PCNA), CD 31, and von Willebrand factor (vWF). The purpose of
this study is to determine the effect of anti-VEGF therapy on capillary hemangioma
growth, and to specifically look at the effect on these signaling markers.
Methods: A baseline litter of Wistar R rat pups (n=10) were injected with 109.7
plaque forming units of murine polyomavirus biweekly. Capillary hemangioma
growth was confirmed in 100% of the pups after 3 weeks of life. Rat pups were
then separated into a control group (n=12) or a treatment group (n=15). The control
group received biweekly doses of the polyomavirus until week 3.5, and then four
pups were sacrificed each week thereafter. The treatment group also received
biweekly doses of the polyomavirus until week 3, and then received biweekly doses
of bevacizumab (Avastin, Genentech) 5mg/kg. A percentage of the pups in the
treatment group were also sacrificed each week. At the time of sacrifice, each rat
underwent gross and microscopic evaluation to determine the presence of
hemangiomas. When found, the hemangiomas were tested for the presence of
VEGF, PCNA, CD31, and vWF.
Results: Hemangiomas were found in 12/12 (100%) pups in the control group, and
in 10/15 (66%) pups in the treatment group. This was a statistically significant
reduction in incidence (p<0.05). Only 44% of the hemangiomas from the control
group tested positive for VEGF, while 80% were positive from the treatment group.
Hemangiomas from the control group were more likely to stain positive in the 1st
week compared to the treatment group (75% vs 37%). There was no significant
difference between the groups with respect to the PCNA, CD 31, and vWF.
Conclusions: While anti-VEGF therapy reduced the incidence of capillary
hemangiomas in the treatment group, it did not reduce VEGF production in the

lesions when compared to the control group. It is hypothesized up regulation of
VEGF may have occurred at the treatment dose and route given. VEGF remains a
potential target to disrupt capillary hemangioma growth.
Commercial Relationships: Brett W. Davies, None
Support: None
In Vitro Effect Of Bevacizumab Treatment On Cell Proliferation, Death And
Differentiation In The Developing Retina Of Rats
Monique Matsuda1, Paloma G. Krempel1, Thiago Puntar2A, Alfred SchollFranco2B, Andr Luis F. Portes3, Silvana Allodi2C, Ndia Campos O. Miguel2A,
Mario Luiz R. Monteiro1. 1Laboratory of Ophthalmology, University of Sao Paulo,
Sao Paulo, Brazil; AProgram of Cell Biology and Development, BNeurobiology
Program, CInstitute of Biophysics Carlos Chagas Filho, 2Federal University of Rio
de Janeiro, Rio de Janeiro, Brazil; 3Retina and Vitreous Service, Bonsucesso
General Hospital, Rio de Janeiro, Brazil.
Purpose: The vascular endothelial growth factor (VEGF) is essential of the
development central nervous system of vertebrates by promoting neurogenesis,
neuronal patterning, neuroprotection and gliogenesis. Bevacizumab (BVZ) has
been extensively used as an anti-VEGF agent for controlling pathological
neovascularization in the retina of both adult patients with a wide array of retinal
diseases and more recently, in newborn infants with retinopathy of prematurity.
However, our hypothesis is that BVZ may also interfere with essential cellular
processes in the development of retina since VEGF is important in protecting and
maintenance of neurons. The aim of our study was to investigate the effects of BVZ
in the differentiation, cell death and proliferation in the developing retina of rats.
Methods: Sixty retinas of 2-day-old Lister Hooded rats were dissected after
enucleation of the eyes and the retinal explants were raised in culture medium with
(experimental group) or without (controls) use of 0.5 mg/mL of BVZ during 48
hours. After the treatment, retinal explants were separated for analyses by
immunohistochemistry (IHC) and gene expression by real-time polymerase chain
reaction (RT-PCR) for the markers of proliferating cell nuclear antigen (PCNA) (to
detect retinal cell proliferation), caspase-3 (a marker of cell death by apoptosis),
beclin-1 (a marker of cell death by autophagy), vimentin (for labeling
undifferentiated glia) and glial fibrillary acidic protein (GFAP) (for labeling
astrocytes and Mller cells). Photographs of the retina for observation and
quantification were taken using a confocal microscope (Leica) TCS-SP5 and the
mRNA content was measured by SYBR Green fluorescence. Comparisons between
the groups were performed using the Students unpaired or Mann-Whitney.
Results: No abnormalities were observed both in the IHC material and in the real
time RT-PCR analysis of retina explants regarding programmed cell death, as well
as proliferating cells. However, a significant increase in the vimentin protein
content and a decrease in the GFAP mRNA were observed in the BVZ-treated
group compared to controls.
Conclusions: Our study indicates that BVZ leads to abnormalities in the content of
vimentin filament protein and mRNA expression of GFAP. BVZ can therefore
interfere with the processes of differentiation of retinal cells. These data show the
importance of further studies to evaluate the effects of BVZ and suggest caution in
its use, particularly in the developing retina, as currently used in children with
retinopathy of prematurity.
Commercial Relationships: Monique Matsuda, None; Paloma G. Krempel,
None; Thiago Puntar, None; Alfred Scholl-Franco, None; Andr Luis F. Portes,
None; Silvana Allodi, None; Ndia Campos O. Miguel, None; Mario Luiz R.
Monteiro, None
Support: FAPESP 2011/12271-3
Impact of Intravitreal Bevacizumab on Intraocular Pressure
Ghulam Dastgir, Sara Ferri, John Danias, Eric Shrier. SUNY Downstate Medical
Center, Brooklyn, NY.
Purpose: Bevacizumab is an anti-vascular endothelial growth factor agent used in
the management of various neovascular ocular conditions. Case studies in the
literature have reported significant increases in intraocular pressure (IOP) following
intravitreal injection of bevacizumab. This study explores the long-term effect of
intravitreal bevacizumab on IOP in glaucomatous eyes, nonglaucomatous eyes, and
eyes at high risk for glaucoma.
Methods: A retrospective chart review of 87 eyes of 87 patients who received
intravitreal bevacizumab injections by one surgeon (ES) for various ophthalmic
indications was performed. The study population consisted of patients with and
without a history of glaucoma in the injected eye. Patients with active neovascular
glaucoma, history of glaucoma filtration surgery, and cataract surgery following
bevacizumab administration were excluded. Data included two-year average
intraocular pressures prior to administration of bevacizumab and regular follow-up
of IOP beginning 4-6 weeks after first bevacizumab injection. Endpoint was the
addition of at least one IOP-lowering medication over the course of the treatment
period. Chi-square analyses were performed to compare IOP changes over various
time points.
Results: Of the 87 eyes receiving intravitreal bevacizumab, 60 eyes were
nonglaucomatous, 22 eyes were glaucomatous, and 5 eyes were at high risk for
glaucoma. The median follow-up time was 14 months with a range of 2 months to
57 months. There was no difference between pre-avastin average IOP and 4-6 week
follow-up IOP (=0.317) or the average of the final three IOPs at follow-up
(=0.892). There was a significant difference between the pre-injection IOP and
the maximum measured IOP at follow up (=0.049). A total of 5 eyes (5.7%) in
the study population required the addition of an IOP-lowering medication; two
were glaucomatous, two were nonglaucomatous, and one was at high risk for
glaucoma.
Conclusions: Intravitreal bevacizumab may cause a significant elevation of IOP
over the course of follow-up in both glaucomatous and nonglaucomatous eyes.
However, this increase may be transient and clinically may not require initiation of
IOP-lowering medication. We recommend regular close monitoring of IOP in
patients receiving intravitreal bevacizumab irrespective of a history of glaucoma.
Commercial Relationships: Ghulam Dastgir, None; Sara Ferri, None; John
Danias, None; Eric Shrier, None
Support: None
The Effect Of Intravitreal Bevacizumab And Ranibizumab On Cutaneous
Tensile Strength During Wound Healing
Jillian Wang1A, Angela Jiang1A, James Willard2, Cedric Pratt1A, Sashwati Roy1B,
Heather Powell2, John Christoforidis1A. AOphthalmology, BSurgery, 1Ohio State
University College of Medicine, Columbus, OH; 2Engineering, Ohio State
University, Columbus, OH.
Purpose: To investigate the effect of intravitreal bevacizumab and ranibizumab on
wound tension during cutaneous wound healing in a rabbit model and to compare
this effect to placebo intravitreal saline controls at two time points.
Methods: One hundred and twenty New Zealand white rabbits underwent two full
thickness cutaneous wounds using standard 6mm dermatologic punch biopsies.
Following this, the rabbits were randomly assigned to one of three treatment groups
each consisting of forty rabbits. Each group received intravitreal injections in the
left eye consisting of 1.25 mg/0.05 ml bevacizumab, 0.5 mg/0.05 ml ranibizumab,
or 0.05 ml of normal saline. Twenty rabbits from each agent group underwent
wound harvesting on day 7 and twenty on day 14. The tensile maximal load for
each harvested wound specimen was measured using wound tensiometry by
mounting each specimen into the grips of a TestResources mechanical tester. A
linear mixed model with random intercepts was fit to compare the difference in
max load between treatment groups and the control group. Dunnett-Hsus method
for multiple comparisons was used to adjust for multiple hypothesis tests.
Results: On day 7 the following wound tension reading means with standard error
and 95% confidence intervals in dynes were found: saline placebos 8.25 +/- 0.86
(6.52, 9.97) , bevacizumab 7.80 +/- 1.01 (5.79, 9.81) (p=0.922), and ranibizumab
5.46 +/- 0.85 (3.74, 7.18) (p=0.048). On day 14 the following wound tension
reading means with standard error and 95% confidence intervals in dynes were
found: saline placebos 7.34 +/- 0.55 (6.24, 8.44), bevacizumab 6.05 +/- 0.54 (4.97,
7.14) (p=0.18), and ranibizumab 7.99 +/- 0.54 (6.91, 9.08) (p=0.60).
Conclusions: In our study only intravitreal ranibizumab was found to exert a
statistically significant inhibition of cutaneous wound tensile strength at day 7
compared to placebo controls. At day 14 neither agent produced any significant
effect on tensile wound strength compared to placebo controls. Since angiogenesis
is an integral component of the proliferative phase of wound healing, we encourage
clinicians to be observant of their patients' recent surgical history and to consider
refraining from the use of intravitreal ranibizumab therapy during the peri-operative
period.
Commercial Relationships: Jillian Wang, None; Angela Jiang, None; James
Willard, None; Cedric Pratt, None; Sashwati Roy, None; Heather Powell,
None; John Christoforidis, None
Support: National Center for Research Resources UL1RR025755
348 Diabetic Retinopathy
Tuesday, May 8, 2012, 1:45 PM - 3:30 PM
Effect Of Intraretinal Bevacizumab Before Vitrectomy On Proliferative
Diabetic Retinopathy
Hayato Mitamura, Takayuki Baba, Toshiyuki Oshitari, Eiju Sato, Shuichi
Yamamoto. Opthalmology, Chiba university, Chiba, Japan.

Purpose: To evaluate the postoperative BCVA and surgical complications after
pars plana vitrectomy (PPV) for proliferative diabetic retinopathy (PDR) using
intravitreal bevacizumab (IVB) as an adjunct.
Methods: A retrospective, consecutive, non-comparative, interventional case
series. One hundred seventeen eyes of 106 PDR patients that received 1.25 mg IVB
3 days before PPV were studied. PPV was carried out with 20G or 23G vitrectomy
instruments with phacoemulsification and implantation of an intraocular lens. The
main outcome measures were the BCVA and postoperative surgical complications.
All cases were followed for 6 months.
Results: The average age of the patients was 52.7 12.6 years. Preoperatively,
vitreous hemorrhage (112 eyes, 95.7%), tractional retinal detachment (52 eyes,
48.7%), and neovascular glaucoma (7 eyes, 6.0%) were present. The BCVA
improved from 1.65 0.81 to 0.60 0.63 logMAR units at six months
postoperatively (P < 0.001). The average intraocular pressure was not changed
significantly during the postoperative period of 6 months (15.3 3.6 vs 16.3 6.1
mmHg, P=0.162). Postoperatively, vitreous hemorrhage was presented in 4 eyes
(1.4%), rhegmatogenous retinal detachment in 2 eyes (1.7%), and neovascular
glaucoma in 9 eyes (7.7%). Eyes with vitreous hemorrhage and rhegmatogenous
retinal detachment were successfully treated by a second vitrectomy. In the nine
eyes with postoperative neovascular glaucoma, 2 eyes had neovascular glaucoma
and 4 eyes had a severe tractional retinal detachment preoperatively. Two -thirds of
the cases with postoperative neovascular glaucoma had BCVA 20/200 at the final
visit.
Conclusions: PPV with IVB as an adjunct had relatively good BCVA with fewer
recurrent vitreous hemorrhages. However, eyes that developed neovascular
glaucoma postoperatively had poor final BCVA.
Commercial Relationships: Hayato Mitamura, None; Takayuki Baba,
None; Toshiyuki Oshitari, None; Eiju Sato, None; Shuichi Yamamoto, None
Support: None
Comparative efficacy of combined treatment including intravitreal injection of
0.5 mg of Lucentis (ranibizumab) and laser photocoagulation for patients with
Proliferative Diabetic Retinopathy (PDR): Short Term Results
Daniel Ferraz, Sr., Lisa Vasquez, Sr., Augusto Motta, Sr., Rony Preti, Sr., Celso
Morita, Sr., Otacilio O. Maia Jnior, Sr., Walter Takahashi, Sr.. Ophthalmology,
University of Sao Paulo, Sao Paulo, Brazil.
Purpose: Evaluate the efficacy of combined treatment with ranibizumab and laser
photocoagulation versus laser photocoagulation alone in patients with severe PDR
by the mean change in BCVA and differences in Optic Coherence Tomography
(OCT) retinal thickness and total macular volume at week 4 and 12 compared to
baseline.
Methods: Prospective, randomized, comparative and interventional study. At the
baseline and follow up visits (week 4 and 12), the patients underwent complete
ophthalmic examination (including ETDRS Best Corrected Visual Acuity (BCVA)
measurements, applanation tonometry, non dilated and dilated slit-lamp
examinations, indirect fundus examination, and OCT evaluation). All patients were
treated with bilateral laser photocoagulation consisting of full scatter PRP treatment
performed in three episodes according to ETDRS guideline. An intravitreal
injection of ranibizumab were performed in one of the eyes - as defined per a
randomization list at week 1 and 3 (group 1 - PRP and group 2 - RPR + Intravitreal
Ranibizumab) . The fellow eye received a sham injection. The x2 and Student t test
were applied to compare the differences between two groups to categorical and
continuous variables, respectively. The null hypothesis were rejected for P values
of <.05.
Results: Thirty patients (n= 60 eyes) completed the 12-week study follow-up
period. At the baseline the Best Corrected Visual Acuity (letters) was 36 16,93
and 39 12,52 (p=0.603); and central macular thickness CTM (m) was 20420,3
and 19231,1 (p=0,777), in the PRP and PRP + IVR groups, respectively. Total
Macular Volume mm3 (TMV) was 7.254 244,52 in group 1 and 6974 292,47 in
Group 2 (p=0,122). The BCVA at week 4 was 40 11,64 letters in group 1 and 42
13,45 in group 2 (p=0,053); at week 12 was 39 10,35 in group 1 and 37 12,47
in group 2 (p=0,455). The CMT and TMV observed in the PRP group at week 4
were 220 27,09 (p=0,172) and 8.463 301,33 (p=0,039), respectively. In PRP +
IVR group, the CMT and TMV were 224 26,87 (p=0,02) e 7.662 312,32
(p=0,03). At week 12, the CMT was 222 31,43 (p=0,172) and TVM was 7.761
307,34 (p=0,502) in group 1 and 200 29,07 (p=0,03) and 7.572 334,75 (p=0,05)
in group 2.
Conclusions: Intravitreal ranibizumab plus PRP were associated with not OCT
worsening at week 4 and 12 compared with PRP alone in eyes with high-risk PDR,
but did not appear to influence the visual acuity in eyes treated with PRP.
Commercial Relationships: Daniel Ferraz, Sr., None; Lisa Vasquez, Sr.,
None; Augusto Motta, Sr., None; Rony Preti, Sr., None; Celso Morita, Sr.,
None; Otacilio O. Maia Jnior, Sr., None; Walter Takahashi, Sr., None
Support: None

The Phenomenon of Metabolic Memory in Diabetic Retinopathy and
Treatment Effects of Captopril on Non-Proliferative Diabetic Retinopathy
Xun Xu, Xiao lu Yang, Ning Wang, Zhi Zheng, Hui yi Jin. Department of
Ophthalmology, Shanghai First People's Hospital, Shanghai, China.
Purpose: Diabetic retinopathy (DR) is a leading cause of blindness in people of
working age in developed countries. Our previous study results showed that
protective effect of an angiotensin-converting enzyme inhibitor (ACEI) on DR
correlates with the inhibition of "metabolic memory" phenomenon. Here, we
assessed the efficacy and safety of an ACEI, captopril as compared with Vitamin C
in patients with non-proliferative diabetic retinopathy (NPDR).
Methods: In this 24-month trial, 317 type 2 diabetic patients with moderate-tosevere NPDR were randomly divided into captopril group with a dose of 12.5 mg
twice a day at first 3 months and three times per day 3 months thereafter (202
patients), and Vitamin C group with a dose of 100 mg once daily (115 patients). At
the end point of the trial, general clinic examinations, including blood pressure,
glycated hemoglobin, and comprehensive standardized ophthalmic examinations
were performed. Color fundus photography was used to grade diabetic retinopathy
and optical coherence tomography (OCT) was used to detect macular edema.
Results: A total of 83.66% of the patients in the captopril group versus 73.04% in
the Vitamin C group remained unchanged in DR grading (p=0.024), and 55.45%
versus 37.39% had an improved effect on the macular edema (P=0.002). The levels
of blood pressure and glycated hemoglobin in the two groups of patients remained
within the normal range during the entire follow-up and no significant difference
was found between the baseline and last visits, suggesting that ACEI drugs play a
protective effect on the diabetic retinopathy patients independent of its anti-blood
pressure role.
Conclusions: Captopril can improve or delay the development of diabetic
retinopathy and macular edema, which can be used in the early treatment of type 2
diabetic patients with
NPDR.
Commercial Relationships: Xun Xu, None; Xiao lu Yang, None; Ning Wang,
None; Zhi Zheng, None; Hui yi Jin, None
Support: National Natural Science Foundation of China No. 30872827
Effects Of Fenofibrate On Modified-ldl-induced Retinal Cell Injury And
Retinopathy In Stz-diabetic Mice
Jing Zhang, Shihe Yang, Dongxu Fu, Mingyuan Wu, Mei Du, Kenneth Wilson,
Timothy Lyons. Diabetes and Endocrinology, OUHSC, Oklahoma City, OK.
Purpose: Fenofibrate, a peroxisome proliferator-activated receptor PPAR-a
agonist, is used to treat hypertriglyceridemia. Recently, two large clinical studies
independently demonstrated beneficial effects of fenofibrate in diabetic retinopathy
(DR), but the mechanisms for such an effect are unknown. The aim of this study is
to evaluate the mechanisms of fenofibrate action in DR using a novel animal
model: modified LDL-enhanced DR in STZ-diabetic mice.
Methods: In vitro: Human Retinal Pigment Epithelium (RPE) cells were exposed
to Native (N-) LDL or highly oxidized glycated (HOG-) LDL (200 mg/L) for 24
h with/without pre-treatment with fenofibrate (10, 50, 100 M; 1h). RPE cell
survival was determined by cell viability (CCK-8) assay. In vivo: STZ-induced
diabetic and non-diabetic male C57BL/6J mice were fed different doses of
fenofibrate (0, 100, 300mg/kg.d) for two months; then, to enhance DR, received
intra-vitreal injection of N-LDL or HOG-LDL (5 g/l, 1l/eye) or PBS (as
control). Seven days later, electroretinograms (ERG), H&E, flat mount, retinal
vascular permeability assay were performed; expression of inflammation (VEGF,
GFAP), apoptosis (BAX) and ER stress (KDEL) markers were conducted by
western blot analysis.
Results: In vitro: Viability data for RPE cells were as follows (control (SFM) =

100%); HOG-LDL only: 60.96.5%; HOG-LDL pretreated with 10, 50, 100 M
fenofibrate: 65.73.9%, 75.92.5%*, 84.42.7%** respectively (meanSE, *
p<0.05, ** p<0.001 vs. HOG-LDL only, n=3). In vivo: In non-diabetic mice,
neither intra-vitreal injection of HOG-LDL nor fenofibrate diets altered retinal
function, structure (ERG and H&E staining), or vascular integrity, and no
significant changes were noted in markers of inflammation, apoptosis or ER stress.
In contrast, in diabetic mice, seven days after intra-vitreal injection, HOG-LDL
disrupted retinal histology, impaired retinal function, and increased blood-retinal
barrier breakdown. High but not low dose dietary fenofibrate improved retinal
function and structure; significantly attenuated retinal vascular leakage and
inflammation, and blocked the activation of apoptosis and ER stress.
Conclusions: Fenofibrate had beneficial effects on retinopathy in this STZ-diabetic
mouse model. It countered the effects of intra-vitreal administration of LDL,
suggesting that its protective effects operate directly within the retina.
Commercial Relationships: Jing Zhang, None; Shihe Yang, None; Dongxu Fu,
None; Mingyuan Wu, None; Mei Du, None; Kenneth Wilson, None; Timothy
Lyons, None
Support: OCAST HR08-67
Correlation between photoreceptors status using Spectral Domain Optical
Coherence Tomography(SD-OCT) and Visual outcome after injection of
intravitreal Bevacizumab in Diabetic Macular oedema(DMO)
Subashree Dhananjayan, Manju Chandran, Gulrez Ansari, Lorraine North, Aman
Narang, Nitin Jain, Leena Bhat, Geeta Menon. Ophthalmology Department,
Frimley Park Hospital NHS Foundation Trust, Surrey, United Kingdom.
Purpose: To investigate the correlation between photoreceptor status and final
visual acuity (VA) following intravitreal injection of Bevacizumab in eyes with
Diabetic Macular oedema (DMO).
Methods: We retrospectively reviewed 35 eyes of 33 patients with Diabetic
Macular oedema (DMO) who were treated with intravitreal injection of
Bevacizumab. Using Spectral Domain Optical Coherence Tomography(SD-OCT)
the following specific features were assessed :
1. Condition of external limiting membrane (ELM) at the fovea.
2. Integrity of photoreceptor inner segment/outer segment (IS/OS) junction.
The main outcome measures were percentage of disrupted ELM and IS/OS lines
and correlation between visual acuity (VA) and photoreceptor integrity. All patients
had modified macular grid laser treatment and two of them had triamcinolone
injection before intravitreal injection of Bevacizumab. Central Macular Thickness
(CMT) was also assessed with SD-OCT.
Treatment consisted of intravitreal injection of Bevacizumab 1.25mg/0.05ml once
every 4 weeks for the first three months and retreatment was given depending on
OCT findings at monthly follow up visit. Minimum follow up was for a period of
12 months.
Results: Mean improvement in visual acuity was 4.86 letters with 55 microns
reduction in central macular thickness. 29.45% had no disruption in IS/OS junction
and ELM lines achieving more than 10 letter gain. 53.10% had stability in vision
showed disruption of the IS/OS junction and ELM. 17.45% had loss of more than
15 letters and showed absence of ELM and IS/OS lines. Mean number of injections
during this period was 6.4. No ocular or systemic adverse events were noted.
Conclusions: Integrity of external limiting membrane and the inner segment/outer
segment line is a reliable indicator of favourable visual outcome following
treatment of Diabetic Macular oedema with intravitreal injection of Bevacizumab.
Commercial Relationships: Subashree Dhananjayan, None; Manju Chandran,
None; Gulrez Ansari, None; Lorraine North, None; Aman Narang, None; Nitin
Jain, None; Leena Bhat, None; Geeta Menon, None
Support: None
Suramin and PPADS Downregulate Proinflammatory Citokines in the Retina
of a Rat Model of Type 1 Diabetes
Juan E. Gallo1,2, Jorge E. Mancini1, Juan O. Croxatto3. 1Ophthalmology, Hospital
Universitario Austral, Pilar, Argentina; 2Nanomedicine & Vision Group,
Universidad Austral, Pilar, Argentina; 3Eye Pathology, Fundacion Oftalmol
Argentina J Malbran, Ciudad de Buenos Aires, Argentina.
Purpose: To evaluate the expression of TNF-a, IL-17, c-PLA2, GFAP and P2X2 in
the retina of type 1 diabetic rat treated with PPADS and Suramin
Methods: Sixteen male Wistar rats of 250gr were treated with an intraperitoneal
injection of 45mg/kg of streptozotocin. Only animals with glycemia levels above
200mg/dl were included in the study. The treated animals were divided into three
groups of 4 rats. These were intraperitoneally injected with: 1) Suramin, 2) PPADS,
3) Suramin + PPADS at 9 and 26 weeks of diabetes in each group. Four remaining
diabetic animals received no treatment. Twelve non-diabetic rats with the same age
of diabetics were used as a control group. Animals were sacrificed after 38 weeks
of diabetes. Cross sections of retinas were analyzed by immunohistochemistry
using primary antibodies against GFAP, TNF-alfa, IL-17, cPLA2 and P2X2
receptor.
Results: Immunostaining of the analyzed antibodies were clearly up-regulated in
diabetic animals without treatment. Diabetic rats treated with Suramin, PPADS and
the combined compound showed up a pattern and immunostaining similar to that
seen in the control group. Staining was much lower in animals treated with suramin
than that found in those treated with PPADS
Conclusions: Suramin and PPADS may be good therapeutical candidates for the
treatment of diabetic retinopathy during the inflammatory stage of the disease.
Further studies are being conducted.
Commercial Relationships: Juan E. Gallo, None; Jorge E. Mancini,
None; Juan O. Croxatto, None
Support: None
Arginase Inhibitors As Potential Therapy For Diabetic Retinopathy
Shawn C. Elms1A, William Caldwell1B, Haroldo A. Toque1B, Ruth Caldwell2.
A
Vision Discovery Institute, BPharmacology Department, 1Georgia Health Science
University, Augusta, GA; 2Charlie Norwood VA Medical Center, Augusta, GA.
Purpose: Ischemic injury and endothelial dysfunction are prominent features of
diabetic retinopathy (DR), but the molecular mechanisms of the damage are not
understood. In healthy individuals, nitric oxide (NO) produced by NO synthase
(NOS) maintains normal blood flow and endothelial function. The urea cycle
enzyme arginase (Arg) can limit NO formation by competing for L-arginine, a cosubstrate for both enzymes. Our objective is to investigate the role of Arg in the
diabetes-induced retinal vascular dysfunction of DR.
Methods: Using a novel mouse funduscope capable of imaging the retinal
microcirculation down to 2.25 microns, we measured retinal vessel diameter
change to endothelial dependent and independent vasodilators in streptozotocin
(STZ)-induced diabetic and normoglycemic control mice treated with an arginase
inhibitor or lacking one copy of the Arg 1 gene (Arg1+/-). We also examined Arg
protein expression/activity in diabetic and control retinas.
Results: Our studies showed that endothelial dependent retinal artery relaxation is
markedly impaired in diabetic mice (60% of normoglycemic control), while Arg
activity and expression are increased. The diabetes-induced vascular dysfunction
was largely prevented in mice treated with specific Arg inhibitors (BEC and ABH)
or in Arg1+/- mice (85% of control), thus indicating Arg as a mediator of diabetesinduced retinal endothelial dependent vascular dysfunction.
Conclusions: Our data indicate that increases in Arg protein/activity are involved
in diabetic retinal vascular dysfunction. The specific Arg inhibitors BEC and ABH
cause an improvement in vascular function in response to diabetic insult. Using this
novel imaging technology to measure vascular function in vivo in conjunction with
Arg inhibitors offers a novel methodology for pharmacological research on retinal
microvascular disease.
Commercial Relationships: Shawn C. Elms, None; William Caldwell,
None; Haroldo A. Toque, None; Ruth Caldwell, None
One-year Follow-up Of Serum Vegf Levels In Patients Treated With
Intravitreal Bevacizumab For Diabetic Retinopathy
Andreas Pollreisz1, Matthias Bolz1, Markus Ritter1, Gerald Schmidinger1, Noemi
Maar1, Christoph D. Scholda2, Ursula Schmidt-Erfurth1, DRRG - Diabetic
Retinopathy Research Group Vienna. 1Department of Ophthalmology, Medical
University Vienna, Vienna, Austria; 2Department of Ophthalmology, University of
Vienna, Vienna, Austria.
Purpose: To evaluate levels of vascular endothelial growth factor (VEGF) in the
serum of patients with diabetic retinopathy treated with intravitreal bevacizumab
over the course of 1 year.
Methods: 11 patients with diabetic macula edema (DME) and 7 patients with
proliferative diabetic retinopathy (PDRP) were included in the study. Patients were
treated with intravitreal bevacizumab (1 mg, 0.04 ml) at each monthly visit when
there was persistent macula edema in the DME group and persistence or recurrence
of new vessels in the PDRP group. Serum samples were collected at baseline (prior
to first intravitreal injection) and at month 1, 3, 6, 9 and 12. Concentration of
VEGF-A was measured with a commercially available enzyme-linked
immunosorbent assay (ELISA).
Results: The mean baseline VEGF concentration was 96.16 pg/ml (36.10) (SEM)
in the DME group and 59.85 pg/ml (34.85) (SEM) in the PDRP group. At month 1,
3 and 6 mean VEGF levels decreased to 72.34 pg/ml (30.14), 62.20 pg/ml (20.20)
and 71.9 pg/ml (22.73) in DME patients and to 49.91 pg/ml (28.48), 44.42 pg/ml
(16.97) and 46.18 pg/ml (14.25) in PDRP patients, respectively. 5.3 intravitreal
bevacizumab injections were required until month 6 in the DME and 2.6 injections
in the PDRP group. From month 6 to month 12 the number of injection-free periods
increased, which resulted in 3.1 injections in the DME and 1.3 injections in the

PDRP group. During this time period mean serum VEGF levels increased to 80.56
pg/ml (26.41) (DME) and 60.32 pg/ml (23.21) (PDRP) at month 9 and to 85.57
pg/ml (26.11) (DME) and 58.33 pg/ml (33.59) (PDRP) at month 12.
Conclusions: Our study shows the effect of intravitreally-applied bevacizumab on
VEGF levels in the peripheral blood over the course of 1 year in patients with
diabetic retinopathy treated according to PRN regimens. In the majority of patients
intravitreally-applied bevacizumab lead to decreased systemic VEGF levels that
increased after prolonged injection-free periods.
Commercial Relationships: Andreas Pollreisz, None; Matthias Bolz,
None; Markus Ritter, None; Gerald Schmidinger, None; Noemi Maar,
None; Christoph D. Scholda, None; Ursula Schmidt-Erfurth, None
Support: None
Intravitreal Ranibizumab Injection Combined With Laser Photocoagulation
In Proliferative Diabetic Retinopathy With Macular Edema
Rafael A. Bueno-Garcia, Janet Monrroy-Herrera. Retina Service, Instituto de
Seguro Social del Estado de Mexico y Municipios, Mexico City, Mexico.
Purpose: To evaluate therapeutic effects and usefulness of treatment of intravitreal
injection of Anti-VGF (Lucentis 0.5 mg) after panretinal photocoagulation (PRP) in
patients with clinically significant macular edema secondary to proliferative
diabetic retinopathy (PDR).
Methods: We review the charts of 59 patients (66 eyes) with macular edema and
PDR with full PRP who were treated with Lucentis 0.5mg between October 2010
and October 2011. We analyzed best-corrected visual acuity (BCVA), fluorescein
angiography, and OCT.
Results: All patient received full PRP at least 1 moth before star Anti-VGF
therapy. The total injections was 239. The average injection performed to treat
DME was 3.5; BCVA (log MAR) improved from preoperative 0.870.36 to
0.830.46 at 3 months and it was maintained until at least 2 months. No one patient
required additional laser PRP during the studied period. The thickness of fovea
decreased from average 306.0873.57 m to average 250.057.92 m at 3 months,
which was significantly maintained during 2 months.
Conclusions: Therapy with Intravitral Ranibizumab after PRP might be an
effective treatment modality in the treatment of macular edema and PDR, and
might prevent the subsequent PRP need in PDR patients, Intravitreal Ranibizumab
might be effective for reducing diabetic macular edema and preventing aggravation
of PDR with stabilization of the vision
Commercial Relationships: Rafael A. Bueno-Garcia, None; Janet MonrroyHerrera, None
Support: None
Effect Of Systemic Angiotensin Receptor Blocker To Intravitreal Vascular
Endothelial Growth Factor In Proliferative Diabetic Retinopathy
Hye Shin Jeon1A, Hyun Woong Kim2, Sang-Joon Lee3, Ji Eun E. Lee4, Boo Sup
Oum1A, Eun Sook Jun1B. AOphthalmology, BMedical Research Institute, 1Pusan
National University Hospital, Busan, Republic of Korea; 2Ophthalmology,
Busanpaik Hospital, Inje University College of Medicine, Busan, Republic of
Korea; 3Ophthalmology, College of Med, Kosin Univ, Busan, Republic of Korea;
4
Ophthalmology, Pusan National Univ Hospital, Busan, Republic of Korea.
Purpose: Renin-angiotensin system is reported to be associated with vitreous level
of vascular endothelial growth factor (VEGF) in proliferative diabetic retinopathy
(PDR). This study is conducted to investigate if systemic angiotensin receptor
blocker (ARB) medication may reduce intravitreal VEGF concentration in PDR.
Methods: Patients who underwent vitrectomy for tractional retinal detachment or
vitreous hemorrhage due to PDR were included in the study. Group A had patients
who had been taking ARB for more than 3 months before vitrectomy, and group B
had patients without hypertension. Patients with epiretinal membrane or macular
hole were also included as control, or group C. The vitreous fluid was collected
during vitrectomy and the venous blood was sampled in the same day. The serum
was separated by centrifuge, and the sampled vitreous and serum were stored in 80C. The VEGF level in vitreous fluid and serum was measured using enzyme
linked immune-sorbent assay.
Results: Among 52 patients, 19 eyes were in the group A, 30 eyes in the group B,
and 30 eyes in the group C. Vitreous VEGF levels were 488.2, 559.0 and 4.5 pg/ml
respectively. Serum VEGF levels were 384.8, 372.8, and 388.1. The ratio of
vitreous VEGF to serum was 4.01, 3.32 and 0.03. Although vitreous VEGF levels
and its vitreous to serum ratio in the PDR groups were significantly higher than in
the control group (P<0.0001), there was no difference between the group A and the
group B.
Conclusions: Systemic medication of ARB did not alter vitreous level of VEGF in
PDR patients. The results of the current study suggest that systemic ARB has a
limitation to improve diabetic retinopathy by modifying intraocular renninangiotensin system.

Commercial Relationships: Hye Shin Jeon, None; Hyun Woong Kim,
None; Sang-Joon Lee, None; Ji Eun E. Lee, None; Boo Sup Oum, None; Eun
Sook Jun, None
Support: None
Chronic Rapamycin Treatment Causes a Deficit in Optokinetic Tracking in
Male Mice and Does Not Prevent Tracking Deficits in the Ins2Akita Mouse
Model of Diabetic Retinopathy
Rene C. Renteria, Asta Vasalauskaite, Nikolay P. Akimov, Songqing Lu.
Department of Physiology, Univ Texas Health Sci Ctr San Antonio, San Antonio,
TX.
Purpose: Diabetic retinopathy (DR) is a complex, multifactorial disease that
affects both neuronal and vascular function. Mechanistic links between the
pathologies are not clearly understood, but the retinal injury in DR includes vessel
hyper-permeability resulting from high vascular endothelial growth factor (VEGF)
expression. Endothelial cells, pericytes, and retinal neurons are progressively lost.
The Ins2Akita model of diabetes (Akita) recapitulates these aspects of DR, and
Akita mice have deficits in optokinetic tracking (OKT; Akimov & Rentera, 2011).
Because VEGF-induced hyper-permeability is known to be suppressed by the
mTOR inhibitor rapamycin, here we tested the hypothesis that chronic rapamycin
treatment (Rapa) would improve OKT visual performance of the Akita mouse.
Methods: Diabetic Akita mice were fed control chow or chow containing microencapsulated rapamycin from 1 until 7 months (mos) of age. Two groups of nondiabetic mice were fed the control or Rapa diet from 3 until 18 mos. Spatial
frequency threshold at maximum contrast (SPFT) and contrast sensitivity (CS) at
0.103 cyc/deg of OKT were determined (OptoMotry; Cerebral Mechanics, Inc.). A
third group of non-diabetic mice was fed from 4 until 25 mos. Retinas were stained
with cell type-specific markers in order to count neuron numbers.
Results: Rapa neither protected against nor worsened OKT decline in diabetic male
Akita mice. Rapa, however, was detrimental to OKT performance in normal mice,
causing decreased SPFT in male, but not female, mice at 18 mos (male Ctrl: 0.379
cyc/deg ctrl; 0.344 cyc/deg Rapa.; p<0.0001). Rapa did not slow normal, agerelated OKT decline. Non-diabetic, 25 mos, Rapa-fed male mice had decreased
inner plexiform layer (IPL) thickness in the retinal periphery, but we detected no
change in numbers of retinal ganglion cells, dopaminergic amacrine cells, and
cholinergic amacrine cells compared to age- and sex-matched, control-fed mice.
Conclusions: Rapa does not prevent either diabetes-induced or age-related declines
in OKT visual function and retinal neuron number. Rapa was detrimental, causing
an OKT SPFT deficit in non-diabetic male mice. Rapa, which increases lifespan in
mice, may thus decrease vision healthspan during normal male aging.
Commercial Relationships: Rene C. Renteria, None; Asta Vasalauskaite,
None; Nikolay P. Akimov, None; Songqing Lu, None
Support: None
Constitutive Signaling Through the TGF- Receptor I ALK5 is Required for
Pericyte Survival in Adult Retinal Vessels
Zeina Dagher1,2, Joseph Vaz1, Michael Goodridge1, Leona E. Ling3, Mara
Lorenzi1,2, Chiara Gerhardinger1,2. 1Schepens Eye Research Institute,
Massachusetts Eye and Ear, Boston, MA; 2Harvard Medical School, Boston, MA;
3
Biogen Idec, Cambridge, MA.
Purpose: To learn about mechanisms and consequences of constitutive TGF-
signaling in the homeostasis of adult retinal vessels. Undisturbed TGF- signaling
is required for normal cardiovascular development, as well as for the functional
integrity of the adult microvasculature. The signaling events involved in the
stabilizing role of TGF- in the adult vasculature are not known, but are becoming
of translational importance because anti-TGF- interventions are being evaluated as
adjunct therapies to prevent diabetic retinopathy and nephropathy.
Methods: We administered to adult rats for 3 weeks SM16 (Biogen Idec), an orally
active compound that selectively inhibits the classical type I TGF- receptor
ALK5 and the phosphorylation of its effectors Smad2/3. SM16 was mixed with the
chow to deliver a dose of 6 mg/Kg bw/day, which had shown efficacy and safety in
rodent models of pathologies involving excess TGF-. The measurement of
Smad2/3 phosphorylation in the whole retina after intravitreal injection of
exogenous TGF- provided evidence of drug efficacy on targets beyond the bloodretinal barrier. We thus measured effects of SM16 on gene expression (Rat
Endothelial Cell Biology RT2 ProfilerTM PCR Array) and apoptosis (TUNEL assay)
in the cells of retinal microvessels. Fresh or fixed retinal microvascular networks
were isolated to a high degree of purity, similar in treated and untreated rats.
Results: In the retinal vessels of SM16-treated rats, three genes changed their
expression level more than 2 fold with P < 0.05 when compared to untreated rats.
Monocyte chemoattractant protein-1 increased, a pro-inflammatory change

consistent with the immunosuppressive role of TGF-. In contrast, interleukin7 and
angiopoietin1 decreased. Angiopoietin1 is produced mostly or solely by pericytes.
Another pericyte product, VEGFa, was also downregulated in the retinal vessels of
SM16-treated rats (1.4-fold, P=0.05). The trypsin digests of SM16-treated rats
showed an increase in the number of TUNEL+ pericytes [median 9 (range 0-27) vs
3 (range 2-11) in untreated rats, P=0.05). There was no increase in TUNEL+
endothelial cells.
Conclusions: Pericytes are endowed with only one type of TGF-RI, ALK5, while
the endothelium also carry ALK1. Pericytes are thus especially susceptible to
ALK5 inhibitors such as SM16; an even minor deprivation of TGF- signaling
through ALK5 as induced by our dose of SM16 compromises their functionality
and survival. It is reasonable to hypothesize that longer duration of SM16 treatment
would have resulted also in an increased rate of endothelial apoptosis.
Commercial Relationships: Zeina Dagher, None; Joseph Vaz, None; Michael
Goodridge, None; Leona E. Ling, Biogen Idec (E); Mara Lorenzi, None; Chiara
Gerhardinger, None
Support: EY017637
Melatonin Protects Against Diabetic Retinopathy By Attenuating Oxidative
Stress And Inflammatory Response
Tingting Jiang, Qing Chang, Gezhi Xu. EENT hospital Shanghai China, Shanghai,
China.
Purpose: The aim of this study was to evaluate the protective effects of melatonin
on diabetic retinopathy(DR).
Methods: Three groups of Sprague-Dawley (SD) rats were enrolled in this
study:(1) a control group , (2) a diabetic group and (3) a diabetic group treated with
melatonin(10mg/kg/day intraperitoneal). Diabetes was induced with a single dose
of 60mg/kg i.p.streptozotocin (STZ). Rats were sacrificed separately at 4,8,12
weeks after diabetes induction. Retinas were extracted and were used for
biochemical studies. Ten animals were included in each subgroup. Akt
phosphorylation,heme oxygenase-1(HO-1),nuclear erythroid 2-related factor 2
(Nrf2), nuclear factor-kappa B (NF-B) and inducible nitric oxide synthase (iNOS)
mRNA and protein expressions were measured by Real-time PCR and Western
Blot.
Results: Nrf2 expression was significantly reduced 8 and 12 weeks after diabetes
was induced compared with the control group (P<0.05) and melatonin upregulated
its expression (P<0.05). The capacity of melatonin to modulate Nrf2 pathway was
associated with increased HO-1 expression (P<0.05), which strengthens antioxidant
defense. Further, melatonin also activated the reduced level of phosphorylated Akt
and downregulated the elevated level of NF-B and proinflammatory cytokine
iNOS (P<0.05).
Conclusions: Melatonin attenuated oxidative stress via the PI3K/Akt-Nrf2
signaling pathway and inflammation by decreasing NF-B activation cascade,
which might be responsible at least in part, for its protective effects in
DR.
Commercial Relationships: Tingting Jiang, None; Qing Chang, None; Gezhi

Xu, None
Support: None
Photobiomodulation Inhibits Early Stages of Diabetic Retinopathy
Johnny Tang1, Yun P. Du2, Jie Tang2, Timothy S. Kern2. 1Ophthalmology and
Visual Sciences, UH Eye Institute Case Medical Center, Cleveland, OH;
2
Endocrinology, Case Western Medical Center, Cleveland, OH.
Purpose: Photobiomodulation has been studied in the past as a method to treat
certain diseases. We evaluated the effects of photobiomodulation on the
development of early stages of diabetic retinopathy in rats and cell culture.
Methods: Streptozotocin-diabetic rats were assigned to 2 groups, treated with or
without light exposure (near-infrared for 3 minutes a day), and compared to agematched nondiabetic control animals. Cell cultures grown in diabetic and nondiabetic simulating media were also treated with or without near-infrared light
exposure.
Results: Control diabetic rats exhibited ERG signal loss, increased inflammatory
markers, leukostasis and oxidative stress. Light therapy for 3 minutes significantly
improved these paramenters. Cells were used to elucidate the mechanism.
Conclusions: Photobiomodulation offers a novel therapeutic approach to inhibit
the development of early stages of diabetic retinopathy and other complications of
diabetes.
Commercial Relationships: Johnny Tang, None; Yun P. Du, None; Jie Tang,
None; Timothy S. Kern, None
Support: VA Foundation Award
Antiangiogenic effect of human apolipoprotein(a) kringle V on retinal
neovascularization
Dong Hyun Jo1,2, Yangmi Lim3, Jin Hyoung Kim2, Jin-Hyung Ahn3, Yu Kyeong
Hwang3, Dong-Ku Kang4, Soo-Ik Chang4, Young Suk Yu1,2, Yeup Yoon3, Jeong Hun
Kim1,2. 1Department of Ophthalmology, College of Medicine, Seoul National
Univeristy, Seoul, Republic of Korea; 2Fight against Angiogenesis-Related
Blindness (FARB) Laboratory, Clinical Research Institute, Seoul National
University Hospital, Seoul, Republic of Korea; 3Mogam Biotechnology Research
Institute, Yongin, Republic of Korea; 4Department of Biochemistry, Chungbuk
National University, Cheongju, Republic of Korea.
Purpose: To investigate antiangiogenic effect of human apolipoprotein(a) kringle
V (rhLK8) on retinal neovascularization
Methods: Cell viability assay was performed to find out cellular toxicity of rhLK8.
We injected rhLK8 intravitreally, and histologic analysis and TUNEL assay were
performed to demonstrate retinal toxicity of rhLK8. Oxygen-induced retinopathy
(OIR) was induced in mice, and to assess the antiangiogenic effect of rhLK8, we
intravitreally injected rhLK8 at postnatal day 14. As in vitro angiogenesis assays,
tube formation and wound migration assays were performed. We assessed the
degree of cell adhesion and migration on different types of extracellular matrix
proteins according to the treatment with rhLK8, antibodies to subtypes of integrins,
or inhibitors of ERK, AKT, and JNK pathways. Protein microarray was performed
to screen with which subunits of integrins rhLK8 exerted its action. Western
blotting was used to investigate downstream signalling of interaction between
rhLK8 and 31 integrin.
Results: rhLK8 inhibited retinal neovascularization in the mouse model of OIR
without definite toxicity on endothelial cells and retinal tissues at the therapeutic
dosage. rhLK8 showed inhibitory effect on angiogenic processes in 2 in vitro
angiogenesis assays: tube formation and wound migration assays. Interestingly,
rhLK8 reduced particulary fibronectin-mediated migration of endothelial cells.
Further experiments showed high binding affinity of rhLK8 to 31 integrin.
Furthermore, rhLK8 inhibited phosphorylation of focal adhesion kinase, resulting
in suppression of activation of JNK through action on p130CAS.
Conclusions: Our data suggested the possible application of rhLK8 in the
treatment of retinal neovascularization through its inhibitory action on fibronectinmediated angiogenesis.
Commercial Relationships: Dong Hyun Jo, None; Yangmi Lim, None; Jin
Hyoung Kim, None; Jin-Hyung Ahn, None; Yu Kyeong Hwang, None; DongKu Kang, None; Soo-Ik Chang, None; Young Suk Yu, None; Yeup Yoon,
None; Jeong Hun Kim, None
Support: None
Novel Curcumin Analogs for Treatment of Diabetic Retinopathy
Danyang Chen, Erica Little, Yan Zhang. Charlesson, LLC, Oklahoma City, OK.
Purpose: Diabetic retinopathy (DR) is a common complication of diabetes mellitus
and the most common cause of visual impairment. Although the exact mechanism

involved in the development of DR is not known, several biochemical pathways,
such as oxidative stress, over-expression of vascular endothelial growth factor
(VEGF) and inflammation, are implicated. Curcumin has a wide range of
pharmacologic effects. However, due to its poor bioavailability and unstability, the
clinical application of curcumin is limited. To develop novel antioxidant and antiangiogenic agents for the treatment of diabetic retinopathy, a series of curcumin
analogs were designed and screened.
Methods: MTT assay was used to determine the cell viability. The intracellular
generation of reactive oxygen species (ROS) stimulated by tumor necrosis factoralpha (TNF-) was used to determine the antioxidant activity. The expression of
VEGF was detected by ELISA.
Results: Among a series of compounds screened, three analogs CLT010-01,
CLT010-07 and CLT010-12 showed a more potent inhibition of the proliferation of
cycling retinal capillary endothelial cells than curcumin and aminoguanidine (an
inhibitor of inducible nitric oxide synthase). These analogs did not significantly
inhibit the growth of pericytes, suggesting potential selectivity of endothelial
growth inhibition. CLT010-01, CLT010-07 and CLT010-12 inhibited TNF-stimulated ROS production and down-regulated the expression of VEGF in
cultured cells.
Conclusions: Several novel curcumin analogs have anti-oxidant and antiangiogenic activities. The in vivo efficacy of these compounds deserves to be
evaluated in diabetic animal models.
Commercial Relationships: Danyang Chen, Charlesson, LLC (E); Erica Little,
Charlesson, LLC (E); Yan Zhang, Charlesson, LLC (E)
Support: NIH 1R43EY019417-01
Inhibitors of HIF-mediated Epigenetic Expression of Histone Demethylases
for Diabetic Retinopathy Treatment
Mridul Mukherji, VK Chaithanya Ponnaluri, Ramya Krishna Vadlapatla, Ashim K
Mitra, Divya Teja Vavilala. Pharmacy - Pharmaceutical Sciences, Univ of Missouri
- Kansas City, Kansas City, MO.
Purpose: We have recently shown that hypoxia induces the expression of a number
of histone lysine demethylases (KDMs) in retinal pigment epithelial cells. The
objective of this study was to identify phytochemicals which inhibit the expression
of these KDMs and therefore, can be used to treat a number of retinal diseases
caused by hypoxia induces neovascularization.
Methods: Since retinal pigment epithelial cells are one of the major cell
constituents and secretors of VEGF in the retina, a human retinal pigment epithelial
cell line (D407) was used for these studies. The expressions of KDMs in D407 cells
were compared using qPCR under the normoxic and hypoxic conditions. Using this
assay a number of phytochemicals were screened to evaluate their efficacy as
expression inhibitors of KDMs and pro-angiogenic factors. Further molecular
characterizations of these phytochemicals were performed using immunoblot and
cytotoxicity assays.
Results: Using the above methods two phytochemicals, MM-1225 and MM-1226,
were identified which inhibited HIF pathway and reduce the expression of KDMs
and other pro-angiogenic genes in retinal pigment epithelial cells in a dosedependent manner. The cytotoxicity assays suggest that these phytochemicals were
nontoxic up to 20 microM and 50 microM concentrations, respectively.
Conclusions: This study demonstrates for the first time that MM-1225 and MM1226 inhibit the epigenetic regulation mediated by HIF pathway. These findings
suggest that therapeutic use of these phytochemicals can be a new avenue to treat
retinal diseases caused by hypoxia induces neovascularization such as diabetic
retinopathy.
Commercial Relationships: Mridul Mukherji, None; VK Chaithanya
Ponnaluri, None; Ramya Krishna Vadlapatla, None; Ashim K Mitra,
None; Divya Teja Vavilala, None
Support: None
Prospective Study Comparing Ultra-wide-field versus Conventional Imaging
for the Detection and Management of Diabetic Retinopathy
Shlomit F. Sandler1, Steven D. Schwartz2, Umar Mian1, Irena Tsui3,2.
1
Ophthalmology, Montefiore Medical Center, Bronx, NY; 2Ophthalmology, Jules
Stein Eye Inst/UCLA, Los Angeles, CA; 3Ophthalmology, Montefiore Medical
Center, New York, NY.
Purpose: To compare clinical exam, conventional color fundus photos (CCFP),
ultra-wide-field color fundus photos (UWFCFP), conventional fluorescein
angiography (CFA), and ultra-wide-field fluorescein angiography (UWFFA) in the
imaging, detection and management of diabetic retinopathy (DR).
Methods: Patients with DR detected on clinical exam at the Montefiore Medical
Center in the Bronx, NY were prospectively enrolled. All patients underwent
dilated fundus exam (DFE) by a vitreoretinal specialist, as well as conventional and
ultra-wide-field color fundus photos and fluorescein angiography by both CFA
(Topcon, Paramus, NJ) and UWFFA (Optos, Marlborough, MA) on different days
but within the same week. All images were reviewed in a masked fashion by a
vitreoretinal specialist who graded the severity of retinopathy using the
International Classification of DR.
Results: Twenty-nine (29) eyes of 15 diabetic patients were included. One
thousand fifteen (1015) images were obtained and reviewed. Significantly fewer
color fundus photos were obtained using the ultra-wide-field technology (mean:
UWFCFP 2.14 vs. CCFP 8.41, p<0.0001), however there was no significant
difference between the number of images obtained during FA between the two
devices (mean: UWFFA 11.52 vs. CFA 12.93, p=0.11). There was limited
agreement in DR staging across all 5 diagnostic modalities (Table). Ultra-widefield color fundus photographs detected no retinopathy in 17.2% of patients, thus
underestimating the severity of DR compared to clinical exam and other imaging
modalities. DME was noted more frequently by CFA (75.9%) or UWFFA (69.0%)
than CCFP (20.7%), UWFCFP (24.1%) or clinical exam (10.3%). Peripheral
vascular leakage was significantly more visible on UWFFA than CFA (8 vs.1 eye).
Conclusions: This is the first study directly comparing UWFFA and CFA
performed on the same eyes within the same week. Information available from
ultra-wide-field imaging and conventional imaging had different advantages and
disadvantages. Therefore, physicians should choose which modality to use based
on patient
pathology.
Commercial Relationships: Shlomit F. Sandler, None; Steven D. Schwartz,

None; Umar Mian, None; Irena Tsui, None
Support: Research to Prevent Blindness
363 IOP Modulation and Retinal Protection
Tuesday, May 8, 2012, 3:45 PM - 5:30 PM
Palm A Paper Session
Dopamine-3 Receptor Modulates Intraocular Pressure
Claudio Bucolo1A, Gian Marco Leggio1A, Adriana Maltese1A, Alessandro
Castorina1B, Velia D'Agata1B, Filippo Drago1A. AClinical and Molecular
Biomedicine, BBio-Medical Sciences, 1University of Catania, Catania, Italy.
Purpose:
The aim of the present study was to investigate the role of D3 receptor on
intraocular pressure regulation using WT and KO D3R-/- mice
Methods:
All experiments were carried out on KO D3R-/- and WT mice. Both mice were used
with normal eye pressure or steroid-induced ocular hypertension. All the animals
were treated according to the ARVO Statement for the Use of Animals in
Ophthalmic and Vision Research. Identification of dopamine receptors transcripts
in iris-ciliary body (ICB) tissue samples was carried out by RT-PCR. Mouse ICB
levels of 7-OH-DPAT, a dopamine D3-preferring receptor agonist, topically
instilled in the mouses eye were determined by HPLC analysis
Results:
As measured by tonometry, the topical application of 7-OH-DPAT, significantly
decreased, in a dose-dependent manner (0.01, 0.1, 1 and 5% w/v), the intraocular
pressure in WT mice both in an ocular normotensive group and an ocular
hypertensive group. Pretreatment with U-99194A, a D3 receptor antagonist,
reverted 7-OH-DPAT induced ocular hypotension in WT mice. No change of
intraocular pressure was observed after topical application of 7-OH-DPAT in KO
D3R-/- mice. PCR analysis demonstrated the presence of all dopamine receptor
genes in eye tissues obtained from WT mice, and the lack of D3R mRNAs in KO
mice. Levels of 7-OH-DPAT (0.147g/mg) were detected, by HPLC analysis, in
the iris-ciliary body 30 min after single topical administration of 0.1% eye drops
Conclusions:
The present study identified the D3R subtype as the most important receptor of the
dopaminergic system to modulate intraocular pressure with relevant implications
for glaucomaCommercial Relationships: Claudio Bucolo, None; Gian Marco

Leggio, None; Adriana Maltese, None; Alessandro Castorina, None; Velia
D'Agata, None; Filippo Drago, None
Support: PON 01_00110
Ciliary Muscle (CM) Bradykinin B2-Receptors: Protein Expression,
Pharmacology of Biochemical Signaling Processes and Relation to IOP
Modulation
Naj Sharif1, Parvaneh Katoli1, Linya Li1, Shouxi Xu1, Curtis Kelly1, Richard
Ornberg1, Yu Wang1, Laura Klekar1, Daniel Scott1, Shahid Husain2. 1Alcon
Research Ltd, Fort Worth, TX; 2Ophthalmology, Medical Univ of South Carolina,
Charleston, SC.
Purpose: Studies were conducted to examine the bradykinin (BK) B2-receptor
system in ciliary muscle (CM) using immunohistochemical techniques, and to
pharmacologically characterize the associated biochemical signal transduction
systems in human CM (h-CM) cells. Some of the latter elements were compared
with pharmacological aspects of human cloned B2-receptor expressed in Chinese
hamster ovary cells (CHO-B2). BK-induced modulation of intraocular pressure
(IOP) of Dutch-Belt rabbits and Cynomolgus monkeys was also studied.
Methods: Previously published procedures were used throughout these studies.
Results: Human and monkey CB (ciliary muscle [CM] and non-pigmented ciliary
epithelium) expressed a high level of B2-receptor protein immunoreactivity. BK
and its analogs differentially stimulated mobilization of [Ca2+]i in primary cultured
h-CM cells: BK EC50 = 2.4 0.2 nM = Hyp3-BK > Lys-BK EC50 = 3.2 nM =
Hyp3,-(2-thienyl)-Ala5,Tyr(Me)8-()-Arg9)-BK (RMP-7) > Met-Lys-BK EC50 =
16.1 nM >> Des-Arg9-BK EC50 = 4.2 M (n = 3-6). BK-induced [Ca2+]i
mobilization was blocked by B2-receptor-selective antagonists, HOE-140 (IC50 =
1.4 0.1nM) and WIN-63448 (IC50 = 174 18 nM) (n = 3-7). A similar rank order
of potency of agonists and antagonists was observed for [Ca2+]i mobilization in
CHO-B2 cells. In h-CM cells, BK-induced [Ca2+]i mobilization response was
abolished when all extracellular Ca2+ was chelated with 1 - 2.5 mM EGTA, and
similarly abolished after a phospholipase C inhibitor (U73122; 10-30 M) was
added to the cells. A similar rank order of potency of the peptides was observed for
the synthesis and release of endogenous total PGs from h-CM cells as noted above
for the [Ca2+]i mobilization response. Total PGs release stimulated by BK in h-CM
and CHO-B2 cells was attenuated in a concentration-dependent manner by the
cyclooxygenase inhibitors, bromfenac and flurbiprofen, and also by the B2antagonists. BK (100 nM) induced a 2-fold increase in extracellular signalregulated kinase-1/2 phosphorylation, and RMP-7 (1-10 M; at 24 hrs) caused a
1.4-2.1-fold increase in pro-MMP-1 and pro-MMP-2 levels above baseline in hCM cells. Topical ocular dosing of BK (50 g) to Cynomolgus monkeys did not
alter IOP. However, intravitreal injection of 50 g of BK (B2-agonist), but not DesArg9-BK or Sar-[D-Phe9]-Des-Arg9-BK (B1-agonists), resulted in a maximum 37.0
5.6% reduction in IOP in rabbit eyes 8 hrs post-injection, relative to vehicleinjected eyes (n = 10/group).
Conclusions: These results support a role for endogenous kallikrein/kinin
components associated with CM, in particular the B2-receptor, in aqueous humor
modulation and thus IOP regulation.
Commercial Relationships: Naj Sharif, Alcon Research Ltd, A Novartis
Company (E); Parvaneh Katoli, Alcon Research Ltd, A Novartis Company (E);
Linya Li, Alcon Research Ltd, A Novartis Company (E); Shouxi Xu, Alcon
Research Ltd, A Novartis Company (E); Curtis Kelly, Alcon Research Ltd, A
Novartis Company (E); Richard Ornberg, Alcon Research Ltd, A Novartis
Company (E); Yu Wang, Alcon Research Ltd, A Novartis Company (E); Laura
Klekar, Alcon Research Ltd, A Novartis Company (E); Daniel Scott, Alcon
Research Ltd, A Novartis Company (E); Shahid Husain, Alcon Research Ltd, A
Novartis Company (F)
Support: None
Protein Acetylation as a Predictor of Retinal Injury
Oday Alsarraf, Phillip W. Yates, Jie Fan, Craig E. Crosson. Ophthalmology Storm Eye Institute, Medical University of South Carolina, Charleston, SC.
Purpose: The dysregulation of protein acetylation is an integral event in numerous
inflammatory and neurodegenerative diseases, including those of the retina. The
current studies investigate whether early changes in acetylation, following ischemic
and ocular hypertensive insult, play a role in retinal injury and survival.
Methods: Changes in the acetylation state of histone-H3 and histone deacetylase
(HDAC) activity were measured using rat retinal lysates at 2, 8 and 24 hours
following 45 minutes of retinal ischemia, and at 3, 7 and 14 days of ocular
hypertensive stress. In addition, caspase-3 activity was measured at 2, 8 and 24
hours in ischemic eyes. To investigate if increases in acetylation can influence
retinal injury, the HDAC inhibitor valproic acid (VPA) (100mg/kg; i.p.) or vehicle
was administered twice daily on study day 0, 1, 2 and 3, to rats that received 45
minutes retinal ischemia, or twice daily for 28 days following the development of
ocular hypertension. The evaluation of retinal structure and function utilized
microscopic and electroretinogram analysis.

Results: In vehicle-treated rats, retinal ischemia decreased acetyl histone-H3 levels
by 15%, 44% and 69% at 2, 8 and 24 hours, compared to contralateral control eyes.
Class I HDAC activity was increased at 2, 8 and 24 hours post ischemia by 14.2
5.3%, 15.9 3.1% and 29.6 5.9%, respectively. Active caspase-3 levels were not
significantly altered at 2 and 8 hours, but were significantly elevated (229.4
51.1%) at 24 hours post ischemic injury. In ocular hypertensive eyes, acetyl
histone-H3 levels were decreased by 5%, 26% and 28% after 3, 7 and 14 days,
compared to contralateral control eyes. Class I HDAC activity was increased at 3, 7
and 14 days by 5.1 4.8%, 12.7 2.3% and 13.6 3.2%. The administration of
VPA provided a six fold increase in retinal histone-H3 acetylation and a reduction
of caspase-3 activity to control levels, at 24 hours post injury. In ischemic and
ocular hypertensive models, VPA administration produced significant functional
and structural protection.
Conclusions: These studies provide evidence that changes in acetylation are early
events post ischemic and ocular hypertensive injury, and precede elevations in
caspase activity. In addition, these changes can be reversed by hyperacetylation via
HDAC inhibition, providing a basis for the development of HDAC inhibitors in the
treatment of retinal degeneration and optic neuropathies.
Commercial Relationships: Oday Alsarraf, None; Phillip W. Yates, None; Jie
Fan, None; Craig E. Crosson, None
Support: EY021368
Alpha2-Adrenergic Receptor Agonists Prevent Neurodegeneration,
Microgliosis, Macrophage Accumulation, and Global Gene Expression
Changes Following Retinal Ischemia-Reperfusion Injury
Steven F. Abcouwer1, Sumathi Shanmugam1, Cheng-mao Lin1, Alistair J. Barber2,
David A. Antonetti1. 1Ophthalmology & Visual Science, Univ of Michigan Kellog
Eye Ctr, Ann Arbor, MI; 2Milton S Hershey Medical Ctr, Penn State College of
Medicine, Hershey, PA.
Purpose: Ischemic retinal diseases, such as retinal vein and arterial occlusions,
presently have limited therapeutic options. Retinal ischemia-reperfusion (IR) is an
acute model of retinal degeneration demonstrating neuronal death, vascular
hyperpermeability, microgliosis and macrophage infiltration. The alpha2adrenergic receptor (2-AR) agonist brimonidine has demonstrated neuroprotective
potential. We hypothesized that 2-AR agonists would prevent IR induced neural
degeneration and vascular permeability, resulting in diminished innate and
inflammatory immune responses.
Methods: Rats were pretreated with the 2-AR agonists, brimonidine and
guanfacine, by intraperitoneal injection prior to IR. Retinas were made ischemic by
transient elevation of intraocular pressure for 45 min and reperfused for up to 48 h.
Cell death was evaluated by TUNEL and by measuring internucleosomal DNA
cleavage. Extravascular albumin was measured using the Evans blue dye
technique. Microgliosis and monocyte populations were evaluated by flow
cytometry of enzymatically-dissociated retinal cells. Global changes in whole retina
gene expression were identified in repeated microarray analyses, with selective
confirmation by qRT-PCR. Retinal monocytes were enriched by density gradient
centrifugation prior to mRNA expression analysis by qRT-PCR.
Results: In addition to neuronal death and vascular hyperpermeability, IR
significantly (p0.01) increased CD45 expression and granularity of microglia,
indicative of activation and phagocytotic activity. IR also significantly (p0.01)
increased numbers of macrophages, including cells with high MHC class II
expression, indicative of an inflammatory antigen-presenting phenotype.
Pretreatment with 2-AR agonists greatly (p0.01) diminished cell death, albumin
leakage, microglial activation and macrophage accumulation in response to IR. 2AR agonist pretreatment significantly (p0.5) inhibited a majority of gene
expression changes caused by IR. Twenty-four of these response-inhibited genes
were validated and 22 of these were highly expressed by enriched retinal
monocytes.
Conclusions: Pretreatment with 2-AR agonists effectively prevented neural,
vascular and innate immune responses to IR injury. This class of drugs may
represent an effective means to prevent ischemic damage and treat retinal vein and
arterial occlusions.
Commercial Relationships: Steven F. Abcouwer, None; Sumathi Shanmugam,
None; Cheng-mao Lin, None; Alistair J. Barber, None; David A. Antonetti,
None
Support: JDRF
Myocilins Coiled-Coil: pH-Dependent Protein Binding and Intracellular
Targeting
William M. Dismuke1, Brian S. McKay1, W Daniel Stamer2. 1Ophthalmology,
University of Arizona, Tucson, AZ; 2Ophthalmology, Duke University, Durham,
NC.

Purpose: Myocilin is a widely expressed protein in which mutations result
uniquely in ocular hypertension, glaucoma and blindness. We previously identified
a SNARE-homology region in myocilin and showed that myocilin is a component
of a membrane-associated protein complex. Here we test the hypothesis that the Nterminal, coiled-coil containing portion of myocilin (lacking the olfactomedin
domain) is sufficient for targeting to the membrane-associated protein complex and
examine conditions that mediate binding.
Methods: Membrane preparations from COS-7 cells stably expressing the Nterminal, coiled-coil-containing portion of myocilin (hth-CC-GFP) that lacks the
olfactomedin domain were analyzed by velocity gradient sedimentation and
western blot. A pull-down assay was developed using the coiled-coil portion of
myocilin as bait for proteins from solubilized retina membrane preparation at two
different pHs (7.4 and 5.5). Captured proteins were analyzed by SDS-PAGE and
silver stain.
Results: Fluorescent microscopy shows the hth-CC-GFP constructs associates with
intracellular vesicles. Western blots show that membrane-associated hth-CC-GFP
constructs separated on linear glycerol gradients (10-40%) into two distinct pools, a
8.6S fraction and a 16.6S fraction, similar to our previous findings with
endogenous myocilin from human tissue. In our pull-down assay, the coiled-coil
portion of myocilin associated with seven additional proteins at pH 5.5 compared to
pH 7.4.
Conclusions: Our data demonstrates that the N-terminal portion of myocilin by
itself is sufficient for targeting myocilin to a membrane-associated protein complex
similar to the full-length protein. In this portion of myocilin we previously
identified a region of the coiled-coil with homology to SNARE proteins. Myocilin's
coiled-coil differs at a few key positions where acidic residues are present instead
of neutral polar or hydrophobic residues. In low pH cellular compartments such as
endosomes, vesicles, multivesicular bodies or Golgi Apparatus, these acidic
residues become more neutral, possibly favoring protein-protein interactions
consistent with our pull-down results.
Commercial Relationships: William M. Dismuke, None; Brian S. McKay,
None; W Daniel Stamer, None
Differential Effects Of Trabecular Meshwork Stiffness On Outflow Facility
And Outflow Obstruction In Normal Human And Porcine Eyes
Lucinda J. Camras1A, W Daniel Stamer1B, David L. Epstein1B, Pedro Gonzalez1B,
Fan Yuan1A. ABiomedical Engineering, BOphthalmology, 1Duke University,
Durham, NC.
Purpose: Trabecular meshwork (TM) stiffness has been postulated to affect
outflow facility (C) and outflow obstruction coefficient (Q), but has yet to be
demonstrated experimentally; we tested this hypothesis in the present study using
perfused normal human and porcine eyes.
Methods: Seven human globes were perfused within 48 hours postmortem (PMT)
at pressures of 10, 20, 30, and 40 mmHg to determine C and Q. The TM was then
isolated and cut into 1-2 cm segments. Optical coherence tomography was used to
determine cross-sectional area of the TM segments. Uniaxial tensile stress was
applied longitudinally to TM segments at a rate of 0.1 % stretch per second to
generate stress-strain curves. The bulk Youngs modulus (E) was calculated at
0.0% and 0.5% strain to represent the circumferential stiffness of the TM at a
relaxed state and when Schlemms canal (SC) is collapsed. Results from the human
data were compared to eleven porcine eyes (perfused within 6 hours PMT) tested
under identical experimental conditions. Regression analysis was used to determine
statistical significance of the relationships. Normally distributed data were analyzed
with two-tailed Student t-test, while non-normally distributed data were analyzed
with Mann-Whitney U tests.
Results: In human eyes (n = 7), a larger E correlated with a higher C at 0.0% (p <
0.05) and 0.5% (p < 0.1) strain. Additionally, a larger C correlated to a lower Q (p
< 0.05). In human eyes lower Q correlated to a thicker and stiffer TM at 0.0% (p <
0.07) and 0.5% (p < 0.05) strain. There was no significant correlation between
PMT and age. Porcine eyes (n = 11) showed a similar trend between Q and E at
0.0% and 0.5% strain (p < 0.06) and a significant correlation between a larger
cross-sectional area and a lower E at 0.0% and 0.5% strain (p < 0.005). E at 0.0%
(p < 0.01) and 0.5% (p < 0.005) strain were more than 15-fold higher in human
(515 136 kPa and 571 129; mean SE) than porcine (24.9 13.1 kPa and 29.5
15.2) eyes, respectively. C and Q were not significantly different between species.
Conclusions: Bulk stiffness of soft-tissue is dependent upon the magnitude,
location, direction, and velocity of the applied forces. We hypothesize that a
circumferentially stiffer TM prevents outflow obstruction by reducing the collapse
of SC and facilitates proper separation in the JCT to aid outflow in normal human
eyes. Based on the organization of the aqueous plexus in porcine eyes, stiffer TM
may only prevent collapse and obstruction, but not aid JCT separation unlike
human eyes. Future studies investigating the TM stiffness under different stress
conditions and in glaucoma eyes may provide new insight into TM function and
IOP homeostasis.
Commercial Relationships: Lucinda J. Camras, None; W Daniel Stamer,

None; David L. Epstein, None; Pedro Gonzalez, None; Fan Yuan, None
Support: Duke Eye Center Small Grant, RPB
Quantitative Differences Among Novel Glaucoma Devices in a Human
Perfusion Model
Carol B. Toris1, Vikas Gulati1, Shan Fan1, Lucinda J. Camras2, Ike Ahmed3,
Thomas W. Samuelson4. 1Ophthalmology, Univ of Nebraska Medical Ctr, Omaha,
NE; 2Biomedical Engineering, Duke University, Durham, NC; 3Ophthalmology,
University of Toronto, Toronto, ON, Canada; 4Phillips Eye Institute, Minneapolis,
MN.
Purpose: This study compares changes in outflow facility of novel glaucoma
devices in an ex vivo model of perfused human anterior segments.
Methods: Human eyes with no history of ocular problems were studied within 48
hours of death. Anterior segments were mounted on fixtures connected to a
perfusion system. Baseline measurements of outflow facility were followed by
measurements after device implantation or sham procedure. Statistical tests were
paired and unpaired two-tailed t-tests. Implants studied were 8mm and 15 mm
intracanalicular scaffolds (Hydrus Aqueous Implant, Ivantis, Irvine, CA) and
trabecular micro-bypass stents (iStent Glaukos, Laguna Hills, CA). Eyes with one
8 mm Hydrus Implant were compared with paired controls and with eyes
containing two iStent implants.
Results: All implants significantly increased the outflow facility compared to
baseline and paired controls (p<0.04). The 8mm Hydrus exhibited a significantly
(p=0.04) greater percent increase in outflow facility than two iStent implants.
Conclusions: Outflow facility increased by 72 and 90% with the 8 mm Hydrus

Implant, 92% with the 15 mm Hydrus Implant and 36% with two iStent implants.
Bypassing the trabecular meshwork and scaffolding Schlemms canal allows fluid
to flow directly into collector channels thus reducing outflow resistance and
decreasing intraocular pressure. While the Hydrus Aqueous Implant showed a
greater percent increase in outflow facility than two iStent Implants, both provide
effective ways to increase outflow facility in human eyes ex vivo.
Commercial Relationships: Carol B. Toris, Glaukos (R), Ivantis (F); Vikas
Gulati, None; Shan Fan, None; Lucinda J. Camras, None; Ike Ahmed, Glaukos
(C), Ivantis (C); Thomas W. Samuelson, Glaukos (C), Ivantis (C)
Support: Ivantis, RPB
407 Retinal Connections
Wednesday, May 9, 2012, 8:30 AM - 10:15 AM
Room 124/125 Paper Session
Vascular Pericytes Are Involved In ATP And PGE2 Induced Relaxation In
Porcine Retinal Arterioles In Vitro
Mikkel Misfeldt, Toke Bek, Simon M. Pedersen. Ophthalmology, Aarhus University
Hospital, Aarhus, Denmark.
Purpose: A recent study has identified a cellular bouton structure external to
vascular smooth muscle cells, which is activated when ATP induces vasorelaxation
in porcine retinal arterioles. The purpose of the present study was to identify
possible pericyte characteristics of this perivascular cell structure, and to study its
involvement in adenosine induced vasorelaxation stimulated by NMDA, ATP and
PGE2.
Methods: The cellular components of porcine retinal arterioles were studied by
immunohistochemistry using primary antibodies against the pericyte markers NG2,
desmin and CD13. Additionally, porcine retinal arterioles ( 150 m) were
mounted in a myograph and placed in a confocal microscope. After loading with a
calcium sensitive fluorophore and pre-contraction with 10-6 M U46619,
concentration-response experiments were performed with ATP (10-8 M to 10-4 M)
and prostaglandin E2 (10-9 M to 10-5 M) during recording of retinal vascular tone
and intracellular fluorescence in the vascular wall (n=6 for all experimental
conditions).

Results: The perivascular cell structure showed immunoreactivity to the pericyte
marker NG2, but not to desmin and CD13. ATP and PGE2, but not NMDA,
induced a concentration-dependent relaxation of the retinal arterioles (p<0.01)
simultaneously with the generation of calcium activity in the perivascular
structures.
Conclusions: ATP and PGE2 induced relaxation of porcine retinal arterioles are
mediated by cellular elements with pericyte characteristics external to the vascular
smooth muscle cells. The function of of these pericytes may be a potential target
for future pharmacological intervention on vascular disturbances in retinal disease.
Commercial Relationships: Mikkel Misfeldt, None; Toke Bek, None; Simon
M. Pedersen, None
Support: None
Complement Factor H Gene Polymorphism Plays A Role In The Regulation
Of Vascular Tone In The Choroid
Reinhard Told1A, Doreen Schmidl1A, Agnes Boltz1B, Stefan Palkovits1B, Semira
Kaya1A, Gerhard Garhofer1A, Leopold Schmetterer1B. ADepartment of Clinical
Pharmacology, BClinical Pharmacology, 1Medical University of Vienna, Vienna,
Austria.
Purpose: We have previously shown that choroidal blood flow in healthy subjects
shows some degree of regulation during isometric exercise. In the recent years
there is increasing evidence that polymorphisms in the complement factor H gene
are significantly associated with age-related macular degeneration. Interestingly,
complement factor H deficient mice show abnormal ocular vasculature
characterized by severe endothelial damage. In the present study we hypothesized
that the carriers of CC at rs1061170, which have an increased risk of AMD, already
show abnormal choroidal blood flow regulation at ages below 35 years.
Methods: A total of 99 healthy subjects (aged between 19 and 35 years) were
included in this study. The effect of 6 minutes of isometric exercise on choroidal
blood flow was investigated and genotyping at rs1061170 was performed. Ocular
perfusion pressure (OPP) was calculated as 2/3*mean arterial pressure-intraocular
pressure. ChBF was measured using laser Doppler flowmetry.
Results: Out of the 99 subjects 18 were homocygous for the CC allele, 50 were
homocygous for the TT allele and 31 subjects were heterocygous. In 3 carriers no
adequate laser Doppler signal could be obtained, so the data arise from the
remaining 96 subjects. The response in OPP was similar between the three studied
groups (p=0.263). By contrast, the increase in ChBF was more pronounced in the
carriers of the CC allele, than in the carriers of the TT allele or the heterozygous
subjects (p = 0.043). This difference is also visible when looking into the
pressure/flow relationship during isometric exercise. ChBF started to increase at an
OPP increase of 64.9% and 66.5% in carriers of the TT allele and heterocygous
subjects, respectively. In subjects subjects homocygous for the CC allele this
increase was seen already earlier (OPP increase of 46.7% above baseline).
Conclusions: Our data indicate that healthy young carriers of the CC allele at
rs1061170 show abnormal choroidal blood flow regulation. This is interesting,
because elderly subjects with this genotype are at increased risk of having AMD.
Previously complement factor H polymorphisms were primarily linked to AMD via
inflammatory processes. The present study indicates that they may also be linked to
hypoxia and further studies are warranted to support this assumption.
Commercial Relationships: Reinhard Told, None; Doreen Schmidl,
None; Agnes Boltz, None; Stefan Palkovits, None; Semira Kaya, None; Gerhard
Garhofer, None; Leopold Schmetterer, None
Support: None
Interleukin 1-mediated Upregulation of Proinflammatory Genes are
Selectively Inhibited by a Platelet-activating Factor Antagonist in Human
Retinal Pigment Epithelial Cells
Pranab K. Mukherjee1, David E. Litner1, Julio Alvarez-Builla2, Nicolas G. Bazan1.
1
Neuroscience Cntr/Ophthalmology, LSU Health Sciences Center, New Orleans,
LA; 2University of Alcal, Madrid, Spain.
Purpose: Proinflammatory gene expression is induced in age-related macular
degeneration (AMD). The bioactive phospholipid platelet-activating factor (PAF) is
a potent inducer of pro-inflammatory gene expression. Therefore we have studied
here the ability of low molecular weight PAF-receptor antagonists to block
proinflamatory gene expression in RPE cells. The PAF receptor antagonists, LAU
(Louisiana Alcala Universities) compounds, are potent inhibitors of apoptosis and
mitigate inflammatory responses in different cell types. The purpose of this
research is to evaluate the effect of structurally-related LAU compounds on
interleukin-1 (IL-1)-mediated upregulation of NF-B and COX-2, in a human
retinal pigment epithelial cell line (ARPE-19).
Methods: ARPE-19 cells, grown overnight, were transfected with an NF-BLuciferas (LUC) construct containing four tandem repeats of NF-B enhancer
sequences (Promega) linked to luciferase reporter gene by Fugene-6, according to
the manufacturers protocol (Roche). A promoterless -galactosidase construct was

used to co-transfect as transfection control. Transfected cells were harvested, cell
extracts were made, and luciferase assays were performed using luciferin as a
substrate. In some experiments, an NF-B-LUC stable transfectant cell line (NFB-LUC-ST), generated in this laboratory, was also used. Western blot analysis of
COX-2 was performed using the COX-2 specific antibody (SantaCruz Biotech).
Results: Our results demonstrate that LAU compounds saliently inhibit IL-1induced NF-B-LUC activity in a concentration-dependent manner in ARPE-19
and NF-B-LUC stable transfectant (NF-B-LUC-ST) cell lines. Utmost inhibition
was achieved with 1M, and the least inhibition with 10nM. Inhibitory capacity
varied amongst the compounds assayed. Some were clearly efficacious (i.e. LAU09030), while others were not as effective (i.e. LAU-09001). Additionally, IL-1induced COX-2 activity was attenuated by LAU compounds in both cell lines. In
vitro LAU effects were complemented by in vivo cytoprotective activities in
ARPE-19 cells.
Conclusions: LAU compounds were able to inhibit substantially the IL-1-induced
induction of NF-B luciferase activity in ARPE-19 and NF-B-LUC-ST cell lines
and COX-2 induction. The inhibition of pro-inflammatory COX-2 and NF-B by
PAF receptor antagonists shows that the LAU compounds have therapeutic
potential for inflammatory/degenerative diseases that display enhanced induction of
proinflammtory signaling.
Commercial Relationships: Pranab K. Mukherjee, None; David E. Litner,
None; Julio Alvarez-Builla, None; Nicolas G. Bazan, None
Support: NIH NEI grant EY005121; Research to Prevent Blindness, Inc.
The Impact of Aging on Retinal Gene Expression in a Mouse Model of
Glaucoma
Molly M. Walsh1, Arjun Saha1, Mason Webb1, Paulo A. Ferreira2. 1Ophthalmology,
Duke University, Durham, NC; 2Ophthalmology, Duke University Medical Center,
Durham, NC.
Purpose: Increased intraocular pressure (IOP) is a hallmark risk factor for the
development of glaucoma . In addition, advanced age is yet another major risk
factor. Though several contributors including changes in vasculature, connective
tissue of the optic nerve head, oxidative stress and mitochondrial changes have
been implicated in the pathophysiology, the specific molecular mechanisms
underlying the degeneration of these neurons by increased IOP are not well
understood. To gain insight into the early molecular events induced by elevated
IOP and aging, we performed gene expression profiling to compare retinas in
young and old mice.
Methods: 10 week old and retired breeder C57Bl6 mice were injected with
hypertonic saline into their episcleral vein. Intraocular pressures were obtained
weekly with the TonoLab device. After intraocular pressures were elevated for 3
weeks, the mice were sacrificed. Retinas were removed and RNA was isolated.
Microanalysis was performed using the Affymetrix 1.0 ST
microchip.Immuonhistochemistry and western blots were used to confirm gene
expression changes at a protein level. Differences in gene expression between the
two groups were then analyzed.
Results: Significant changes (>2 fold change) were found in the expression of 255
genes (241 up, 14 down) that were unique to the younger mice. There was a unique
group of genes for the older population as well, including 94 upregulated and 12
down-regulate. In addition, there were 96 genes which were similarly affected
between both the young and old groups. In the olh hypertonic saline der mouse
population, genes related to tyrosine metabolism, glutathione metabolism, cell
adhesion-tight junction molecules, and mitochondrial functions were most notably
affected. Immunological responses including the classical, alternative, and lectininduced complement pathways were among those common to both groups.
Conclusions: This study has identified several pathways of interest involved in the
retinal pathophysiology of glaucoma, specifically with increased aging, and will
serve to identify potential targets which can be used to develop novel therapeutics
for the treatment of glaucoma.
Commercial Relationships: Molly M. Walsh, None; Arjun Saha, None; Mason
Webb, None; Paulo A. Ferreira, None
Support: K08
Nrf2 Signaling Inhibits Pathologic Choroidal Neovascularization
Ian F. Pitha1, Michael B. Sporn2, Rajendra S. Apte3. 1Ophthalmology, Barnes
Hosp/Washington Univ in St Louis, St Louis, MO; 2Pharmacology and Toxicology,
Dartmouth Medical School, Hanover, NH; 3Ophthalmology, Washington Univ - St
Louis, Saint Louis, MO.
Purpose: To test the hypotheses that (1) signaling of the transcription factor Nrf2
suppresses choroidal neovascularization (CNV) and that (2) pharmacologic
activation of Nrf2 inhibits CNV formation.
Methods: CNV was induced in 6 week-old wild-type (wt) and nrf2 knockout (nrf2/-) C57Bl/6J mice by rupture of Bruchs membrane using a krypton redcoherent

laser. On day 7 after treatment, CNV was visualized using fluorescein angiography
and CNV area was quantified using digital imaging software. Choroidal endothelial
cell growth from wt and nrf2-/- mice was quantified using a novel, in vitro
choroidal explant assay developed in our laboratory. The ability of wt and nrf2-/macrophages to regulate microvascular endothelial cell growth was assessed by an
in vitro coculture assay. To determine whether pharmacologic Nrf2 activation
inhibits CNV formation, wt and nrf2-/- mice were treated with vehicle control or
the potent Nrf2 activating agent CDDO-Im after rupture of Bruchs membrane.
Results: CNV area was significantly larger in nrf2-/- compared to wt control mice.
Choroidal endothelial cell proliferation was not increased in nrf2-/- mice, however,
macrophages isolated from nrf2-/- mice exhibited significantly reduced ability to
inhibit endothelial cell growth compared to macrophages isolated from wt mice.
The Nrf2 activating agent CDDO-Im inhibited CNV formation in a dosage
dependent manner. Mice treated with 3 micromole/kg mouse weight CDDO-Im by
had significantly reduced CNV area while mice treated with 0.3 micromole/kg did
not display significant reduction in CNV. Nrf2-/- mice were resistant to CDDO-Immediated inhibition of CNV formation.
Conclusions: Nrf2 signaling suppresses pathologic CNV and is necessary for
macrophage-induced inhibition of endothelial cell growth. Pharmacologic
induction of Nrf2 signaling inhibits CNV formation in an Nrf2-dependent manner.
Taken together these data suggest that activation of Nrf2 signaling has potential to
inhibit CNV in diseases such as exudative age-related macular degeneration.
Commercial Relationships: Ian F. Pitha, None; Michael B. Sporn, Reata
(F); Rajendra S. Apte, None
Support: Barnes Jewish Hospital Foundation
Wide Field Angiography in Diagnosis and Management of Familial Exudative
Vitreoretinopathy (FEVR)
Michael T. Trese1, Amir H. Kashani2, Antonio Capone, Jr.1A, Kimberly A.
Drenser1B. AOphthal/Beaumont, BWilliam Beaumont Hospital, 1Associated Retinal
Consultants, Royal Oak, MI; 2Ophthalmology, Associated Retinal Consultants,
William Beaumont Hospital, MI.
Purpose: To explore the role of wide-field imaging and angiography in the
diagnosis and management of FEVR
Methods: Retrospective review of patient records (July 2011 to December 2011)
with diagnosis of FEVR at a single, multi-physician, subspeciality, pediatric retina
practice. Only patients with clinical examination and wide-field imaging and
angiography results were included in this study.
Results: Fourteen patients (24 eyes) with clinical diagnosis of FEVR (stages
ranging from 1-5) underwent wide-field imaging and angiography (Optos Inc). The
patient population was 28% male. Average age was 23 (range 1-60 years old).
Average LogMAR visual acuity in 24 eyes was 0.31 (20/41 Snellen) and ranged
from 0 to no light perception (NLP). Two eyes with prosthesis and four others that
were NLP were excluded from the study. Here we report a number of
distinguishing peripheral vascular patterns not easily observed with standard
clinical examination or fundus photography. These include variably avascular areas
in the periphery (Figure 1A), small areas of bulbous-like vascular changes (Figure
1A), capillary staining and leakage (Figure 1A), and neovascularization (Figure
1B). Wide-field imaging also reveals exquisite details in patients with subretinal
exudation (Figure 1B), macular dragging (Figure 1B), exudative and tractional
retinal detachment (Figure 1B).
Conclusions: The most disturbing feature of FEVR is that it shows the potential for
unpredictable, lifelong reactivation from any stage. Reactivation is often heralded
by leakage and staining of capillaries that are not easily observed on clinical exam
or standard fundus photography. The use of serial wide-field angiography is very
helpful for diagnosis early disease (stage 1) and equally helpful for close
management of more advanced disease (stage 2) which may require image guided
laser
therapy.
Commercial Relationships: Michael T. Trese, None; Amir H. Kashani,

None; Antonio Capone, Jr., None; Kimberly A. Drenser, None
Support: None
AAV Mediated Systemic Low Molecular Weight Drug Delivery To The Retina
Matthew Campbell, Marian M. Humphries, Anna-Sophia Kiang, Anh T. Nguyen,

Paul F. Kenna, Peter Humphries. Genetics, Trinity College Dublin, Dublin,
Ireland.
Purpose: It has been estimated that up to 98% of systemically-deliverable low
molecular weight anti-neovascular and neuroprotective drugs, many of which
would have significant therapeutic potential in the treatment of degenerative retinal
conditions, are prevented from entering the retina via the peripheral circulation
because of the presence of the inner blood-retina barrier (iBRB). We have recently
reported an experimental platform in rodents for non-invasive systemic drug
delivery to the retina based on transient RNAi-mediated suppression of transcripts
encoding claudin-5, a component of the tight junctions of the inner retinal
vasculature. This process allows passive diffusion of clinically validated drugs
from the peripheral circulation into the retina while excluding larger potentially
harmful substances, and we have used it to radically improve vision in a range of
animal models.
Methods: An AAV-2/9 vector was generated with a doxycycline inducible gene
expressing claudin-5 shRNA. The system was tested via intra-vitreal and subretinal delivery routes and phenpotype assessments were made using electroretinography (ERG) and magnetic resonance imaging (MRI).
Extent of barrier modulation was assessed in animal models of wet AMD, and
Retinitis pigmentosa and systemic delivery of anti-neovascular agents and neuroprotective agents was assessed.
Results: We report herein on the use of barrier modulation in tandem with systemic
drug therapy to prevent retinal degeneration in animal models of Retinitis
pigmentoda (RP) and to suppress laser-induced-choroidal-neovascularisation
(CNV). These observations constitute the basis of a minimally invasive systemic
therapeutic modality for retinal diseases, including RP and AMD, where, in early
stage disease, the iBRB is intact and impervious to systemically administered
drugs.
Conclusions: AAV mediated modulation of the iBRB for the purposes of systemic
drug delivery to the retina of low molecular weight drugs has the potential to
radically improve the chronic treatment of the most common forms of retinal
degeneration. Levels of active compound needed to produce a therapeutic effect
can also be decreased significantly.
Commercial Relationships: Matthew Campbell, None; Marian M. Humphries,
None; Anna-Sophia Kiang, None; Anh T. Nguyen, None; Paul F. Kenna,
None; Peter Humphries, None
Support: Science Foundation Ireland (SFI), Health Research Board of Ireland
(HRB), Fighting Blindness Ireland
443 Drug Delivery II
Wednesday, May 9, 2012, 1:45 PM - 3:30 PM
Room 114 Paper Session
Nanoparticles in Porous Microspheres (NPinPMP) Sustain Delivery of Stable
Bevacizumab for 4 Months
Sarath K. Yandrapu1, Arun K. Upadhyay1, J Mark Petrash2, Uday B. Kompella1,2.
1
Nanomedicine and Drug Delivery Laboratory, Department of Pharmaceutical
Sciences, University of Colorado Anschutz Medical Campus, Denver, Aurora, CO;
2
Department of Ophthamology, University of Colorado Anschutz Medical Campus,
Denver, Aurora, CO.
Purpose: To develop NPinPMP using supercritical fluid technology for sustained
delivery of stable bevacizumab.
Methods: Using biodegradable poly(lactide-co-glycolide) family of polymers and
supercritical carbon dioxide pressure quench technology, nanoparticles containing
bevacizumab (NP) were loaded in porous microspheres (PMP). The prepared
NPinPMP formulation was characterized for mean particle size, morphology (SEM
and confocal microscopy), and in vitro release of bevacizumab in PBS pH 7.4 using
ELISA. The stability of released bevacizumab was evaluated using size exclusion
chromatography (SEC), circular dichorism (CD), and by SDS-PAGE. Further, in
vivo delivery of bevacizumab was evaluated following intravitreal administration
of Alexa Fluor conjugated bevacizumab in NPinPMP in a rat model. In vivo drug
delivery was monitored using Fluorotron MasterTM.
Results: NPinPMP were of a mean size of 9.6 m. The SEM and confocal images
indicated incorporation of bevacizumab NP in PMP. The in vitro drug release was
sustained for 4 months. Stability studies indicated the integrity of bevacizumab as a
monomer. NPinPMP sustained intravitreal bevacizumab delivery to a greater extent
than plain bevacizumab in the in vivo study.
Conclusions: Incorporation of bevacizumab NP in PMP allows sustained
intravitreal delivery of the protein drug for 4 months.
Commercial Relationships: Sarath K. Yandrapu, U.S. Provisional Pat. App.
61/561,256 (P); Arun K. Upadhyay, None; J Mark Petrash, None; Uday B.
Kompella, U.S. Provisional Pat. App. 61/561,256 (P)
Support: NIH grants R01 EY018940 and RC1 EY020361

The Potential of Nanolipossomes-Encapsulated Bevacizumab (Avalong) in
Prolong VEGF-Inhibition and Enhancing Choroidal Neovascularization Targeting Following Intravitreal Injection
Jose A. Cardillo1,2A, Alessandro J. Dare3,1, Acacio A. Lima Filho2, Anselmo G.
Oliveira4, Rubens Belfort, Jr.2A. 1Retina Department, Hospital de Olhos de
Araraquara, Araraquara, SP, Brazil; ARetina Department, 2Federal University of
So Paulo, So Paulo, SP, Brazil; 3Retina Department, Centro Brasileiro de
Especialidades Oftalmolgicas, CBEO, Araraquara, SP, Brazil; 4Faculdade de
Cincias Farmacuticas, Universidade Estadual Paulista, Araraquara, SP, Brazil.
Purpose: To determine the potential of new and optimized bevacizumab-liposomal
formulation to improve drug availability, bioactivity and vessels targeting after
intravitreal administration.
Methods: Bevacizumab was encapsulated into liposomes via the dehydrationrehydration method tested in an animal model. Left eyes of rabbits received
liposomal bevacizumab and the right eyes were injected with the conventional
soluble formulation. The free drug concentration in aqueous humor and vitreous
samples at Days 3, 7, 14, and 29 after the injection was determined by enzymelinked immune sorbent assay. The comparative inhibitory efficiency of
bevacizumab in both conventional and liposomal formulation was determined by
analyzing the gene expression of VEGF in human fibroblast cell line. Electronic
microscopy (EM) of the retina and electro retinography (ERG) was performed to
rule out and potential side effect.
Results: Mean concentration of free bevacizumab in the eyes receiving liposomal
bevacizumab compared with the eyes injected with soluble bevacizumab was 7
(24.5 versus 3.5 microg/mL) times higher at Days 29. Mean concentration of free
bevacizumab in the aqueous humor of both injected eyes was almost the same at
the different intervals. The In vitro inhibitory efficiency of the liposomalbezacizumab formulation was 6 times greater than the soluble Bevacizumab,
possible to its higher cell targeting and affinity. No significant EM or ERG
findings were observed.
Conclusions: Optimized pharmacokinetics and physicochemical properties
obtained with Bevacizumab in a liposomal formulation, potentially enhancing the
intravitreal half-life and its ability to interact with endothelial cells are important
factors to consider for the treatment of Choroidal Neovascularization and other
angiogenesis-dependent diseases of the eye.
Commercial Relationships: Jose A. Cardillo, None; Alessandro J. Dare,
None; Acacio A. Lima Filho, None; Anselmo G. Oliveira, None; Rubens
Belfort, Jr., None
Support: None
Biodegradable And Injectable In Situ Gelling Solution Controls The Release
Of Bevacizumab (avastin)
Yadong Wang1, Thomas R. Friberg2, Britta Rauck1, Carlos A. Medina-Mendez3,
Veeral S. Shah4. 1Bioengineering, University of Pittsburgh, Pittsburgh, PA;
2
Ophthalmology/UPMC Eye Center, Univ of Pittsburgh, Pittsburgh, PA;
3
Ophthalmology, Univ of Pittsburgh Medical Center, Pittsburgh, PA; 4University of
Pittsburgh Ophthal, Eye and Ear Institute, Pittsburgh, PA.
Purpose: Controlled release of therapeutics in the eye increases patient compliance
and should decrease side effects and treatment cost. The most common vehicles for
controlled release are micro- and nano-particles. However, these usually require the
presence of organic solvents that can denature protein drugs. Another class of
common vehicles is hydrogels, the most attractive of which are thermal gels that
are liquid at ambient temperature and gel in situ in the eye. However, current gels
are typically non-biodegradable. We designed a biodegradable polymer (PureSet)
specifically for ocular delivery. This report will discuss
the gelling, biocompatibility, and bevacizumab release properties of PureSet.
Methods: We determined the gelling response of the polymer with temperature,
confirmed the biodegradability in vitro, examined the force needed to inject
PureSet solution through 31G needles at ambient temperature, characterized the
release of bevacizumab in vitro, performed biocompatibility test in vitro using
retina pigmented epithelial cells and cornea endothelial cells, and tested in vivo
performance in rabbit eyes.
Results: PureSet solution (8-15%) gelling begins at around 27C and completes at
around 36C. The solution gels within seconds upon injection into artificial
vitreous fluid at 37C. The polymer degrades 22% within 45 days in vitro. The
injection force increases with solution concentration and injection speed. 8%
solution requires an easily manageable 6 N at 3 mm/s. Thus we were able to easily
inject the PureSet through a 31 gauge needle on a TB syringe, which is a typical
syringe-needle combination used for intravitreal injections. In vitro test following
ISO standard reveals no toxicity of the polymer extract and preliminary in vivo test
shows high biocompatibility. The polymer solution releases bevacizumab over 3
months in artificial vitreous fluid at 37C. 8% solutions injected via 31G needle
gelled into transparent spheres in rabbit eyes and preliminary examinations showed
no adverse host responses.

Conclusions: These data demonstrate the feasibility of in situ gelling of solutions
loaded with bevacizumab, the controlled release of bevacizumab, and the
biocompatibility of PureSet. A larger scale in vivo experiment is warranted to
examine this controlled release platform and to project possibility of clinical
translation for intraocular delivery of bevacizumab and other protein drugs.
Commercial Relationships: Yadong Wang, None; Thomas R. Friberg,
None; Britta Rauck, None; Carlos A. Medina-Mendez, None; Veeral S. Shah,
None
Support: UPMC Fox Eye Center Seed Grant
Microbubble Delivery of DiI to the Ocular Posterior Segment: Comparison of
Intravitreal and Suprachoroidal Injection Routes
Gabriela S. Seiler1A, Sydney E. Cartiff1B, Paul A. Dayton2, Jacklyn H. Salmon1B,
Brian C. Gilger1B. AMolecular Biomedical Sciences, BClinical Sciences, 1North
Carolina State University, Raleigh, NC; 2Biomedical Engineering, North Carolina
State University and The University of North Carolina, Chapel Hill, NC.
Purpose: To compare the distribution of dye to the ocular posterior segment via an
intravitreal (IV) or anterior suprachoroidal space (SCS) injection of free or
microbubble-contained 1,1-Dioctadecyl-3,3,3,3-tetramethylindocarbocyanine
iodide (DiI).
Methods: Fresh canine cadaver eyes received either an IV or SCS injection (250
uL) of free or microbubble-incorporated DiI. Eyes injected with microbubbles were
simultaneously imaged with ultrasound and either received 0, 1, or 3 pulses to
destroy the microbubbles and release the incorporated Dil once they reached the
ocular posterior segment. All eyes were frozen within 1 minute of injection using
2-methylbutane in liquid nitrogen, stored at -80C until sectioned at 5-10 um at 30C, and mounted on superfrost slides. Amount of dye in the retina as observed on
a fluorescent microscope was semi-quantified using a score of 0 (none) to 4 (retina
saturated with DiI) in representative areas of the superior, central, and inferior
retina by two masked observers. Mean cumulative scores (of the 3 anatomic areas)
were analyzed using Wilcoxon rank sum test and differences considered significant
at P<0.05.
Results: Ultrasound imaging illustrated that microbubble contrast was present in
the ocular posterior segment within 10 seconds of injection. Mean cumulative
scores of retinal dye were similar between IV (2.0 +/- 1.7) and SCS (1.8 +/- 0.88)
for free DiI, and slightly higher for 1 pulse microbubble delivery of DiI (IV: 2.9 +/1.4 ; SCS: 2.8 +/- 2.3). However, 3 pulse microbubble destruction resulted in
significantly higher delivery of DiI to SCS (P=0.043) (4.1 +/- 0.8) compared to IV
(2.7 +/- 1.1).
Conclusions: These results support the theory that microbubbles as a delivery
vehicle and site-targeted ultrasound may improve drug delivery to the ocular
posterior segment. Furthermore, these results suggest that the SCS may allow
enhanced targeted drug delivery to the ocular posterior segment and use of these
modalities together (i.e., microbubbles and SCS injections) may result in more
effective drug delivery to the retina.
Commercial Relationships: Gabriela S. Seiler, None; Sydney E. Cartiff,
None; Paul A. Dayton, None; Jacklyn H. Salmon, None; Brian C. Gilger, None
Support: None
The Impact Of Chemical Structure On The Use Of Tof-sims To Investigate
Drug Distribution
Jenifer Mains, Clive Wilson, Andrew Urquhart. SIPBS, University of Strathclyde,
Glasgow, United Kingdom.
Purpose: The use of of time-of-flight secondary ion mass spectrometry (ToFSIMS) to investigate the ocular drug distribution of two probe drug molecules.
Methods: Intravitreal injections of Amitriptyline hydrochloride and
Dexamethasone sodium phosphate were administrated to perfused and nonperfused ovine eyes. The vitreous humor, the lens, and the retina of the eyes were
then removed and divided into front, middle, and back sections. ToF-SIMS analysis
was performed on each cross- section of the vitreous humor using Bi3+cluster
source and used to map drug distribution.
Results: The utility of ToF-SIMS to map drug distribution is dependent on the
uniqueness of the chemical structure amongst natural components. Amitripyline
has a complex heterocyclic structure without covalently-bonded halogens and was
more difficult to fingerprint, therefore principal component analysis (PCA) was
employed in order to measure the distribution of the model drug. For
dexamethasone the method was more sensitive due to the fluorine moiety present in
the drug structure. In the positive ion spectra, four key drug fragment peaks were
identified and in the negative ion spectra, one key drug peak was imaged.
Differences in distribution were demonstrated between the non-living the living
eye. Figure 1 details the front, the middle and the back section of each tissue and
shows that dexamethasone diffused slowly through the vitreous in the non-perfused
eye (A) however, when perfused (B), distribution in the vitreous was faster and the

drug was shown to penetrate into the posterior retina and the lens.
Conclusions: The results illustrate the facility of ToF-SIMS in defining spatial
characteristics of drug distribution within ocular tissues. The specificity and
sensitivity is dependent on the availability of a drug molecule with non-biological
characteristics. Moreover, differences in drug movement through the vitreous
humor elicited by perfusion in the ovine eye were demonstrated, and illustrate the
importance of convective flow as a determinant of distribution.
Commercial Relationships: Jenifer Mains, None; Clive Wilson, None; Andrew

Urquhart, None
Support: AstraZeneca
Safety And Tolerability Of Single Escalating Doses Of Esba1008, A Singlechain Anti-vegf Antibody Fragment, In Patients With Exudative Age-related
Macular Degeneration
Georges Weissgerber1, Sushanta Mallick1, Esteve Travesa2, Colin Hughson1,
Thomas Valencia2, Hermann Schulze1, Kyounghwa Bae2, Silvia Boller1, Anne
Schmidt1, Dominik Escher1. 1ESBATech, an Alcon Biomedical Research Unit LLC,
Zurich-Schlieren, Switzerland; 2Alcon Research, Fort Worth, TX.
Purpose: Evaluation of the safety and tolerability of a single intravitreal (IVT)
injection of 0.5 mg, 3 mg or 4.5 mg of ESBA1008 in the dose escalation phase of
this ongoing prospective, Lucentis (0.5 mg) controlled, single-dose ascending,
double-masked, multi-center, randomized study.
Methods: Three cohorts of 7 patients of either sex 50 years of age have been
sequentially enrolled. In each cohort patients have been randomized to an IVT
administration in the study eye of ESBA1008 or Lucentis in a 5:2 ratio. The first
4 patients in each cohort were dosed sequentially. Escalation to the next cohort was
allowed only if the previous dose didn't meet the dose-escalation stopping criteria
(targeted and serious adverse events related to ESBA1008).
Study eyes were treatment-nave. Selection criteria included a new diagnosis of
exudative AMD or evidence of recent disease progression in the last 3 months, best
corrected visual acuity (BCVA) between 20/40 and 20/200, evidence of subretinal
fluid or retinal cystic changes with central subfield thickness of > 340 m, presence
of a primary subfoveal choroidal neovascularization (CNV) secondary to AMD, a
lesion area 50% of the total lesion area. Subretinal blood, if present must have
spared the fovea and must involve 50% of the lesion.
Results: Each of 3 escalating doses of ESBA1008 have been injected in 5 eyes of 5
patients. Doses were well tolerated as assessed by the absence of study medication
related targeted adverse events in the study eye within 7 (1) days of the IVT
injection (primary safety endpoint).
This favorable safety and tolerability evaluation was also supported by the other
ocular safety endpoints such as evaluation of study eye post-injection, BCVA,
ocular signs, posterior segment abnormalities, evaluation of retina
hemorrhage/detachment, evaluation of vitreal hemorrhage density and vitreous
cells, intraocular pressure and eyelid/pupil responsiveness.
Conclusions: ESBA1008, a potent inhibitor of VEGF, was safely administered to
15 patients at doses up to 4.5 mg/eye. The well-tolerated high doses, combined
with the smaller molecular size of scFvs compared to full-length IgG or Fab
fragments and the high affinity for VEGF, give ESBA1008 the potential to be
administered at intervals greater than the current monthly interval, thereby reducing
the treatment burden.
Commercial Relationships: Georges Weissgerber, ESBATech, An Alcon
Biomedical Research Unit LLC (E); Sushanta Mallick, ESBATech, An Alcon
Biomedical Research Unit LLC (E); Esteve Travesa, Alcon Research (E); Colin
Hughson, ESBATech, An Alcon Biomedical Research Unit LLC (E); Thomas
Valencia, Alcon Research (E); Hermann Schulze, ESBATech, An Alcon
Biomedical Research Unit LLC (E); Kyounghwa Bae, Alcon Research (E); Silvia
Boller, ESBATech, An Alcon Biomedical Research Unit LLC (E); Anne Schmidt,
ESBATech, An Alcon Biomedical Research Unit LLC (E); Dominik Escher,
ESBATech, An Alcon Biomedical Research Unit LLC (E)
Support: None

Update on QLT091001 in Subjects with Leber Congenital Amaurosis (LCA)
due to Lecithin:Retinol Acyltransferase (LRAT) or Retinal Pigment Epithelial
65 Protein (RPE65) mutations: Longer-term follow-up of subjects originally
treated with 7-day therapy
Robert K. Koenekoop1A, Eunice Esteban1B, Leah Wood1B, Ruifang Sui2, Juliana M.
Sallum3, Elias I. Traboulsi4, L I. van den Born5, Ava K. Bittner6, Gislin Dagnelie7,
David A. Saperstein8. AMcGill Ocular Genetics Laboratory, BPaediatric
Ophthalmology, 1McGill University Health Centre, Montreal, QC, Canada;
2
Ophthalmology, Peking Union Med College Hosp, Beijing, China;
3
Ophthalmology, UNIFESP, Sao Paulo, Brazil; 4Center for Genetic Eye Diseases,
Cole Eye Institute, Cleveland, OH; 5Medical Retina, Rotterdam Eye Hospital,
Rotterdam, The Netherlands; 6Ophthalmology, Johns Hopkins University,
Baltimore, MD; 7Ophthal-Lions Vision Cntr, Johns Hopkins Univ, Baltimore, MD;
8
Ophthalmology, Ophthalmology, Seattle, WA.
Purpose: To assess the safety and efficacy of an oral synthetic cis-retinoid
(QLT091001) in subjects with LCA due to mutations in the LRAT or RPE65 genes,
a severe form of childhood visual impairment.
Methods: This is ongoing follow-up of a Phase Ib open-label, proof-of-concept
clinical trial to evaluate the safety and efficacy of 7 days of oral QLT091001 in
subjects with LCA due to mutations in the RPE65 or LRAT genes. Primary visual
function tests are ETDRS best-corrected visual acuity (BCVA) and Goldmann
visual fields (GVFs). Complete ophthalmic and physical examinations,
electrocardiograms, and laboratory blood work are completed before and after
treatment at predetermined time points. Mutations were identified by Sanger
sequencing, confirmed by a CLIA certified lab.
Results: Fourteen subjects (ages 6-38 years) with LCA due to LRAT (N=7) or
RPE65 (N=7) mutations have been enrolled; 8 subjects maintained longer-term
improvements in one or both of GVF and BCVA after 7 days of treatment. In 12 of
17 eyes (6 of 9 subjects) with baseline GVFs up to 70 average diameter, clinically
meaningful improvements in retinal area of 27%-180% were seen, averaged over
time. Six subjects followed for 9-13 months showed sustained GVF improvements
ranging between 40%-50% on average over time. Notable VA improvements were
seen in 5 subjects, which, in 3 subjects were maintained up to 12 months beyond
the end of treatment. Changes in fMRI in a cohort of 3 subjects suggest potential
effects on the visual cortex. Some subjects reported meaningful improvements in
activities of daily living (ADLs). There were no serious adverse events. Transient
headache and photophobia were reported and reversible elevations in triglyceride
levels and reduction in HDL were recorded.
Conclusions: Longer-term follow-up of subjects originally treated with 7 days of
oral QLT091001 showed vision improvements in 8 of 14 LCA subjects.
QLT091001 was well tolerated and adverse events were transient and/or reversible.
Dormant photoreceptors may be able to respond to external manipulation.
Enrollment of RP subjects is ongoing and will be reported separately.
Commercial Relationships: Robert K. Koenekoop, FFB-C, CIHR, NIH,
Reseau, FRSQ (F), QLT Inc (C); Eunice Esteban, QLT In (C); Leah Wood, QLT
Inc (C); Ruifang Sui, None; Juliana M. Sallum, None; Elias I. Traboulsi,
None; L. I. van den Born, None; Ava K. Bittner, QLT Inc (C); Gislin Dagnelie,
QLT Inc (C); David A. Saperstein, QLT Inc (C)
Support: QLT Inc, Foundation Fighting Blindness Canada, CIHR, Reseau Vision,
FRSQ
478 Membrane Physiology and Signal Transduction
miR130a Increases Hyaluronan Synthesis In Orbital Fibroblasts By Inhibiting
The AMPK Signal Transduction Pathway
Naxin Guo1A, Collynn Woeller1B, Steven E. Feldon1A, Richard Phipps1A.
A
Ophthalmology, URMC, BEnviromental Medicine, 1University of Rochester,
Rochester, NY.
Purpose: Thyroid Eye Disease (TED) is characterized by orbital infiltration of
white blood cells and accumulation of the nonsulfated glycosaminoglycan,
hyaluronan (HA). HA contributes significant connective tissue expansion in TED
by virtue of its extraordinary hydrophilic nature. microRNAs (miRNAs) are
extensively involved in diverse biological processes. However, little is known
about the role of miRNAs in regulating the production of HA in orbital tissue. We
hypothesized that miRNAs would influence HA synthesis in orbital fibroblasts. In
this study,we report that miR130a increases both basal and TGF--stimulated HA
production in orbital fibroblasts. miR130as mechanism of action in regulating HA
production may be through inhibiting the adenosine monophosphate (AMP)-

activated protein kinase (AMPK) signal transduction pathway.
Methods: Primary orbital fibroblasts were isolated from TED patients undergoing
orbital decompression surgery. The cells were grown in RPMI 1640 media
containing 10% FBS. Non-specific control miR, miR130a, anti-miR130a or
AMPK-1 siRNA were introduced into orbital fibroblasts using lipofectamine. The
amount of HA in the cell culture supernatant was measured by ELISA. Relative
miR130a expression and HA Synthases (HAS) 1-3 mRNA expression were
measured by qPCR. AMPK-1 and AMPK-1 protein expression were detected
with western blot.
Results: We found miR-130a was highly expressed in TED orbital fat tissue
compared to fat from other tissues. Overexpression of miR-130a significantly
increased HA production both in basal and TGF-beta stimulated TED orbital
fibroblasts. When miR-130a was inhibited by an anti-sense RNA, the amount of
HA production decreased. Overexpression of miR-130a also significantly increased
HAS1 and 2 expression both in basal and TGF-beta stimulated TED fibroblasts.
Basal levels of HAS3 mRNA expression also increased, but the TGF-beta
stimulated levels of HAS3 mRNA did not change significantly. We also found that
one target of miR130a may be the AMPK subunit, AMPK-1. AMPK-1protein
levels were significantly decreased by overexpression of miR130a. Furthermore,
down regulation of AMPK-1 by siRNA significantly increased HAS 1 and 2
mRNA expression and HA production in orbital fibroblasts.
Conclusions: Our data indicate that miR130a is involved in HA synthesis and may
act via down-regulation of the AMPK signal transduction pathway. Reduction of
AMPK-1 leads to higher expression of HAS1 and HAS2 and significantly
enhances the production of HA. These studies provide new insight into the role of
miR130a and AMPK in the pathogenesis of TED.
Commercial Relationships: Naxin Guo, None; Collynn Woeller, None; Steven
E. Feldon, None; Richard Phipps, None
Support: EY017123, EY011708, Rochester Tissue/Eye bank and Research to
Prevent Blindness Foundation Unrestricted Grant.
Iduna Is A Neuroprotectin D1 (npd1) Target In Retinal Pigment Epithelial
Cell Survival Signaling
Veronica Balaszczuk1A, Pranab K. Mukherjee1B, Nicolas G. Bazan1B.
A
Neuroscience Center, BNeuroscience Center/Ophtalmology, 1LSUHSC, New
Orleans, LA.
Purpose: Iduna is a neuroprotective protein against glutamate NMDA receptormediated excitotoxicity both in vitro and in vivo through interfering with PAR
polymer-induced cell death. Mutation at the PAR polymer binding site abolishes
the PAR binding activity of Iduna and attenuates its protective actions. NPD1, a
docosahexanoic acid-derived mediator, induced cell survival through the
upregulation of Bcl2 class of survival proteins under oxidative-stress (OS) in RPE
cells. The goal of this study is to examine if NPD1 survival bioactivity engages
Iduna expression in RPE cells undergoing OS.
Methods: 72h grown ARPE-19 cells were serum starved overnight, oxidativestress was introduced, and then challenged with various concentrations (1nM-2M)
of NPD1 for 3, 4, 6, 8, 10, and 12h. Cell lysates were made, and Western blot
analysis was performed. Iduna protein was detected by either anti-Iduna antibody
(RNF146) or clone N201/35 containing anti-iduna (UC Davis/NIH Neiro Mab
Facility, CA). Immunocytochemical studies were performed on ARPE-19 cells
after 6h treatment with 50nM NPD1 under OS, and probed with Iduna-specific
antibody.
Results: Our results indicated that NPD1 and DHA at 50 and 100 nM
concentrations enhanced expression of Iduna in ARPE-19 cells undergoing OS.
However, OS or NPD1 alone did not have any effect on the Iduna expression.
Moreover, the NPD1-mediated expression of Iduna is time and concentrationdependent. The enhanced expression of Iduna by NPD1 started at least 4h after
initiation of OS. NPD1 treatment, maximized at 6h, continued until 10h, and
declined at 12h in ARPE-19 cells. Concentration kinetic of NPD1 (1 nM to 2 M)mediated expression of Iduna in OS ARPE-19 cells indicated that NPD1 as low as
10 nM was able to enhance Iduna expression and maximized at 50 nM, after which
it started to decline. Additionally, human primary retinal culture (hRPE) also
followed the same path. Immunocytochemical analysis of the Iduna expression in
ARPE-19 cells under oxidative stress by NPD1 corroborated the above
observations.
Conclusions: Here we identify that NPD1 exerted enhanced expression of Iduna in
RPE cells undergoing OS. To our knowledge, this is the first report that a bioactive
lipid molecule NPD1 mediates enhanced expression of Iduna in RPE cells. This
NPD1 mediated expression of Iduna may be a key regulator of cell survival in OS
mediated cell death.
Commercial Relationships: Veronica Balaszczuk, None; Pranab K.
Mukherjee, None; Nicolas G. Bazan, None
Support: NIH NEI grant EY005121, Research to Prevent Blindness, Inc

Dependence of Toll Like Receptor induced Responses on TRPV1 Activation in
Corneal Epithelial Cells
Hua Yang1, Zheng Wang2, Peter S. Reinach1. 1Biological Sciences, SUNY College
of Optometry, New York, NY; 2Ophthalmology, Mount Sinai School of Med, New
York, NY.
Purpose: Toll-like receptors (TLRs) activation in the corneal epithelium induces
innate immune responses, which are essential mediators of host defense during
pathogen infection. Transient receptor potential vanilloid 1 (TRPV1) channel
activation by severe corneal injury also induces many of the responses elicited by
TLR activation. This commonality between their effects prompted us to determine
in human corneal epithelial cells (HCEC) if TLR-induced responses occur as a
consequence of TRPV1 activation.
Methods: Western blot analysis and immunostaining probed for TLRs expression.
TRPV1 and TLR interaction was assessed using coimmunoprecipitation. Calcium
imaging evaluated Ca2+ transients in fura2-loaded HCEC. TLR2,3 and 4 agonists
used were lipopolysaccharide (LPS), lipoteichoic acid (LTA) and polyinosinic
polycytidylic acid [poly(I:C)], respectively. TRPV1 agonist/antagonist pair was
capsaicin (CAP) and capsazepine (CPZ). Gene silencing studies used TRIF siRNA
and the stably transfected shRNA MyD88 subline. ELISA determined
proinflammatory cytokine and chemoattractant release.
Results: TLR2,3 and 4 expression was identified in scrambled shRNA cells. CAP
(10 M) and LTA (5 g/ml)-induced more than 3-fold increases in IL-6 release that
were fully suppressed in TRPV1 siRNA transfected cells. Following exposure of
the scrambled shRNA subline to either LPS (10 ng/ml), CAP(10 M) or LTA (5
g/ml), p-IRAK4 formation was detected in the MyD88 immunoprecipitates.
However, in the MyD88 shRNA subline, none of these agonists induced p-IRAK4
formation were measurable. CAP and poly(I:C) (10 g/ml)-induced three and 4
fold increases in CCL5/ Rantes release, respectively, whereas in TRIF-siRNA
transfected cells these rises failed to occur. LPS-induced a 2.6-fold Ca2+ transient in
scrambled shRNA cells, which was fully blocked during exposure to CPZ. LPSinduced 3-fold increases in IL-6/IL-8 release, which was similar to rises elicited by
CAP. On the other hand, during exposure to CPZ, neither LPS nor CAP had any
effect on IL-6/IL-8 levels.
Conclusions: TLR2, and 4 mediate through TRPV1 activation stimulation of
MyD88 dependent signaling leading to increases in IL-6/IL-8 release. TLR3 is also
dependent on TRPV1 activation to induce increases in CCL5/Rantes release
through MyD88 independent signaling. These results suggest that TRPV1-linked
signaling provides a potential drug target for modulating responses induced by
TLR2,3 and 4 activation.
Commercial Relationships: Hua Yang, None; Zheng Wang, None; Peter S.
Reinach, None
Support: EY04795 and W81XWH090163
Comparison Of Pro-apoptotic Effects Of Avastin And Lucentis In Rat
Primary Neuronal Cells
Weiye Li1, Juan Wang2, Jianfeng Shen3, Jieping Zhang2, Lixia Lu2, Guo-Tong Xu2.
1
Ophthalmology, Drexel University College of Medicine, Philadelphia, PA; 2Tongji
Eye Institute, Tongji University School of Medicine, Shanghai, China; 3Lab of
Clinical Vision Sciences, Institute of Health Sciences (IHS), Shanghai, China.
Purpose: To detect and compare the pro-apoptotic effects of Avastin and Lucentis
by using a rat primary retinal neuronal cell culture.
Methods: Rat primary retinal neuronal (rPRN) cells were isolated from neuroretinas of Sprague-Dawley rats on postnatal day (P) 1 and maintained in
Neurobasal medium with B27 supplement. Cells were cultured for at least 7 days
before experiments. rPRN cells were identified based on their morphology and
expression of cell surface markers. The working concentrations are as follows:
Avastin (2.5 mg/ml, 10 folds of the clinical concentration in the vitreous) and
Lucentis (1 mg/ml, 10 folds of the clinical concentration in the vitreous). The rPRN
cells were treated with Avastin or Lucentis for 48hrs, respectively, while H2O2
(0.8uM and 0.16uM) treated as positive control, and no treated as negative control.
TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling), and
MMOP (Mitochondrial Membrane Potential) were applied as apoptotic parameters.
All Pairwise Multiple Comparison Procedures were used for statistic analysis. The
biochemical and molecular mechanisms of neuron apoptosis by anti-VEGF agents
were explored by using specific inhibitors of signaling pathways.
Results: A mixed rPRN cells were obtained and identified to show the possession
of characteristic morphology and specific cell markers of various retinal neurons.
And then they were used for following comparative study. After Avastin or
Lucentis treated for 48hrs, MMOP result, calculating the ratio of red to green
fluorescent intensity, showed no statistic significance between treated groups
(Avastin or Lucentis) and the negative control. But TUNEL assay showed Avastin
caused 34.66%10.75% cell apoptosis, while Lucentis caused 33.94%14.56% cell
apoptosis. The number of apoptotic cells significantly increased after either Avastin
or Lucentis treatment (compared with 12.83%9.88% cell apoptosis in the negative
control group), although no significance between the two treatment groups

(P=0.8925). The present data also indicate that both Erk and AKT pathways were
involved in the pro-apoptotic effects of Avastin and Lucentis.
Conclusions: In rPRN cells, super-clinical concentrations of Avastin (2.5mg/ml)
and Lucentis (1mg/ml) lead to neuron-apoptosis in vitro, although MMOP may not
be sensitive enough to show this change. These findings suggest that a long-term
and high-dose clinical application of anti-VEGF agents have potential side effect of
neuronal apoptosis. The comparative study showed there was no significant
difference regarding pro-apoptotic effects between Avastin and Lucentis, indicating
whether these two anti-VEGF agents can be interchangeable merits further study.
Commercial Relationships: Weiye Li, None; Juan Wang, None; Jianfeng Shen,
None; Jieping Zhang, None; Lixia Lu, None; Guo-Tong Xu, None
Support: Unrestricted Research Fund from the Clear Vision Foundation,
Philadelphia USA
In Vitro Hydrolysis of Latanoprost by Human Ocular Tissues
Fany L. Guerra1, Joseph Rager1, Sid Bhoopathy1, Ismael Hidalgo1, Karolien
Castermans2, Olivier Defert2, Sandro Boland2, Olivier Defert2. 1Absorption
Systems, Exton, PA; 2Amaken N.V., Diepenbeek, Belgium.
Purpose: Latanoprost is an isopropyl ester prostaglandin F2 analogue prodrug
indicated for the treatment of open-angle glaucoma. Latanoprost is hydrolyzed in
the eye to the biologically active metabolite, latanoprost acid. The objective of this
study was to assess the extent of latanoprost hydrolysis in various human ocular
tissues. Insight in the enzymatic activity of these tissues is useful for the
interpretation of human data and the design and testing of new drugs for
ophthalmology.
Methods: Whole human eyes were obtained from tissue banks within 24 hours
post-mortem. The eyes were dissected into aqueous humor, cornea, conjunctiva,
ciliary body, retina, choroid, and sclera. Each tissue was divided into two pieces,
blotted, and weighed. The two replicates from individual tissues were then
transferred to separate incubation tubes containing Glutathione-bicarbonated
Ringers (GBR) buffer, pH 7.4. Latanoprost was dosed at a concentration of 20
M, the tubes were incubated at 37C, and samples were collected at the onset of
the incubation and at pre-determined time points up to 4 hours. Disappearance of
latanoprost and formation of the active metabolite latanoprost acid were analyzed
using LC-MS/MS.
Results: Most of the dosed latanoprost disappeared within 2 hours of incubation
with the various human ocular tissues, except aqueous humor. The apparent
formation of latanoprost acid was slightly lower than the degradation of the parent
compound at each time point (possibly due to non-specific binding of latanoprost to
tissue), and accounted for approximately 40-90% of the dose at the end of the
incubation. Across tissues, the rate of formation of latanoprost acid correlated well
with the rate of degradation of the parent compound. When normalized for tissue
weight, the rate of hydrolysis of latanoprost was highest in choroid > ciliary body
cornea conjunctiva. These results are similar to data obtained with ocular tissues
from pigmented rabbits under similar experimental conditions.
Conclusions: The results demonstrate that latanoprost is extensively hydrolyzed in
a majority of human ocular tissues and that the tissues with the highest rate of
hydrolysis are similar between humans and pigmented rabbits. A good correlation
was also found between the disappearance of the parent compound and formation
of latanoprost acid.
Commercial Relationships: Fany L. Guerra, Absorption Systems (E); Joseph
Rager, Absorption Systems (E); Sid Bhoopathy, Absorption Systems (E); Ismael
Hidalgo, Absorption Systems (E); Karolien Castermans, Amaken N.V. (E);
Olivier Defert, Amaken N.V. (E); Sandro Boland, Amaken N.V. (E); Olivier
Defert, Amaken N.V. (E)
Support: None
signaling pathways that may be linked to NMDA induced cell survival or

excitotoxicity.
Results: Calcium imaging results have shown that s1r ligands, (+)-Nallylnormetazocine hydrochloride [(+)-SKF10047] and Pentazocine, in primary
RGCs inhibited the influx of calcium through NMDA receptors in a dose
dependent manner. Also, 100uM treatment of NMDA on primary RGCs caused a
greater than 2 fold induction of the transcription factor cAMP-responsive element
binding protein (pCREB) following 6 hrs of treatment.
Conclusions: S1r agonist pre-treatment decreased the levels of calcium influx
through NMDA receptors. Additionally, pCREB is elevated following NMDA
treatment. Since pCREB is considered an important signaling molecule implicated
in cell survival and synaptic plasticity, further studies are needed to investigate the
potential link that NMDA signaling plays in the survival of RGCs and if s1r
impacts NMDA induced pCREB expression.
Commercial Relationships: Brett H. Mueller, II, None; Yong H. Park,
None; Hai Ying, None; Thomas Yorio, None
Support: 1)Department of Defense: W81XH-10-2-0003 2)Training in the
Neurobiology of Aging: T32 AG020494
Effect of Fenugreek on Severe Evaporative Dysfunctional Tear Syndrome
Caterina Gagliano1A,2, Roberta Amato1A,2, Davide Scollo1A,2, Elina Ortisi1A,
Salvatore Caruso1B, Michele Reibaldi1A, Elena Lionetti1, Daniela Rocca1A,2, Giulia
Malaguarnera1A,1C, Teresio Avitabile1A. AOphthalmology, BGynecology,
C
Biomedical Science, 1Catania University, Catania, Italy; 2NEST (Neurovisual
Science Technology) srl, Catania, Italy.
Purpose: To value the role of postmenopausal insufficiently level of 17
Estradiol, Estrone, Testosterone in the pathogenesis of Dysfunction Tear Syndrome
(DTS), investigating the relationship between tear osmolarity and serum sex
hormones level in women with severe evaporative DTS, and value the efficacy and
safety of oral phytoestrogens (fenugreek) in the treatment of these patients.
Methods: Twenty-two postmenopausal women were enrolled in a double-blind,
randomized, placebo-controlled, two-period cross-over trial of oral preparation of
phytoestrogens (fenugreek seed extract 200 mg containing diosgenin 1.3%,
steroidal saponins 50% and alkaloids, as characterized by HPCL method and
gravimetric method). The participants were examined at day 0 and day 30 of each
period. The tests performed included laboratory blood analysis, tear osmolarity
measurement, Schirmer test, Tear Film Break Up Time (TF-BUT).
Results: At baseline the lowest circulating levels of 17 Estradiol, Estrone, and
Testosterone were correlated to the highest levels of tear film osmolarity in 22
postmenopausal women (rs = -0.59, -0.61, -0.58, respectively). After 30 days of
treatment we observed a significant (p<0.05) decrease of tear osmolarity and a
significant increase of Schirmers and TF-BUT values (p< 0.001) in the fenugreek
treated groups in both periods, in comparison with placebo.
Conclusions: This study showed that tear osmolarity is correlated negatively with
sex hormone levels, suggesting an important role of these hormones in the
pathogenesis of postmenopausal DTS which it is more common the involvement of
Meibomain Glands, and a daily treatment with oral phytoestrogens (fenugreek 200
mg) improved significantly signs and symptoms of severe evaporative DTS in
postmenopausal women, without any significant side
effect.

Inhibition of NMDA Induced Calcium Ion Influx in Retinal Ganglion Cells
through Sigma-1 Receptor Stimulation
Brett H. Mueller, II1, Yong H. Park2, Hai Ying1, Thomas Yorio1. 1Pharmacology &
Neuroscience, Univ of North Texas Hlth Sci Ctr, Fort Worth, TX; 2Pharmacology
& Neuroscience, UNT Health Science Center, Fort Worth, TX.
Purpose: Sigma-1 receptor (s1r) activation provides neuroprotective effects in
retinal ganglion cells (RGCs) in both in vivo and in vitro studies. The purpose of
this study is to determine the role s1rs play in controlling calcium dynamics
through activated NMDA receptors in primary RGCs. Additionally, this study aims
to identify cell survival/apoptotic pathways that are activated through the prolonged
stimulation of NMDA receptors, and assess the potential role that s1r plays in
regulating those pathways.
Methods: Purification and culture of RGCs were performed by sequential
immunopanning using Thy 1 antibody from P3-P7 Sprague-Dawley rats. Calcium
imaging was used to measure the intracellular changes in calcium when subjecting
these cells to 100uM of NMDA. Western blots were also performed to determine

time against ischemic injury significantly. Further studies are needed to find the
optimal composition of the used irrigation solution and to evaluate how long the
retinal ganglion cells tolerate ischemia under these conditions.
Commercial Relationships: Maximilian Schultheiss, None; Thoralf
Herrmann, None; Sven Schnichels, None; Karl U. Bartz-Schmidt,
None; Gnther Zeck, None; Martin S. Spitzer, None
Support: None
Commercial Relationships: Caterina Gagliano, None; Roberta Amato,

None; Davide Scollo, None; Elina Ortisi, None; Salvatore Caruso,
None; Michele Reibaldi, None; Elena Lionetti, None; Daniela Rocca,
None; Giulia Malaguarnera, None; Teresio Avitabile, None
Support: Grant N. 20078M2BFW/06 MIUR Italy
The ischemic tolerance time of the retina can be extended by PPV using
Dulbecco`s Modified Eagle Medium as irrigation solution
Maximilian Schultheiss1, Thoralf Herrmann2, Sven Schnichels1, Karl U. BartzSchmidt1, Gnther Zeck2, Martin S. Spitzer1. 1Department of Ophthalmology,
University of Tuebingen, Tuebingen, Germany; 2Department of Ophthalmology,
Natural and Medical Science Institute at the University of Tbingen, Reutlingen,
Germany.
Purpose: Up to date no therapy exists for the treatment of the central retinal artery
occlusion. In order to estimate and to possibly extend the time-window for any
future treatment, we investigate the survival-time of the ischemic retina.
Methods: Enucleated bovine eyes underwent Pars Plana Vitrectomy (PPV) 100
minutes after the animal's death. The eyes were filled with Dulbecco`s Modified
Eagle Medium and an oxygen bubble. Four hours after the start of PPV the retina
was analyzed from functional and morphological perspective. The functional cell
density and the spontaneous electrical activity of retinal ganglion cells was
measured with a 256 multielectrode array. The cellular morphology was
investigated by immunohistochemistry (Brn3a- and DAPI staining) on cryo
sections and the caspase 3/7 activity of the whole retina was evaluated.
Furthermore the Thy-1 mRNA expression was measured 24 hours after the
beginning of the PPV. The operated eyes were compared to eyes which underwent
sham operation and to eyes which suffered from 10-, 100- or 340 minutes of
ischemia.
Results: The eyes which underwent PPV showed a significantly better survival of
retinal ganglions cell compared to eyes which were left untreated for 340 minutes
or which received sham operation. Nevertheless, compared to the enucleated eyes
which were analyzed 10- and 100 minutes after the animal's death the PPV treated
eyes showed a slightly reduced number of functional retinal ganglion cells.
Conclusions: Performation of PPV and filling up the globe with Dulbecco`s
Modified Eagle Medium and an oxygen bubble can extend the retinal tolerance

Delta-Opioid Agonist Targets TNF- and NF-B During Retinal Ischemic
Injury
Shahid Husain, Yasir Abdul. Ophthalmology, Medical Univ of South Carolina,
Charleston, SC.
Purpose: To study the neuroprotective role of a -opioid agonist, SNC-121, and
determine potential targets in the optic nerve and retina for neuroprotection against
retinal ischemic injury.
Methods: Retinal ischemia was induced in Brown Norway rats by raising
intraocular pressure (IOP) above systolic blood pressure (155-160mmHg) for 45
minutes. Rats were treated with the -opioid-receptor agonist, SNC-121 (0.1-1
mg/kg; i.p.), 30 minutes post-ischemia, and daily up to 7 days. To quantitate postischemic functional recovery, electroretinograms (ERG) were performed seven
days following ischemic injury. Structural integrity of the retinal layers was
determined by hematoxylin-eosin staining. Selected rats were treated with a opioid-receptor antagonist, naltrindole (3 mg/kg, i.p.), 15 minutes prior to the SNC121 treatment. TNF-alpha and NF-B were measured by immunohistochemistry
and Western blot analyses.
Results: In eyes subjected to 45 minutes of ischemia, mean b-wave amplitudes
were significantly reduced when compared to the control eyes (control eyes 64675
vs. ischemic eyes 32159 volts; P<0.05), as determined by ERG measurement 7
days following retinal ischemia. In contrast, the b-wave amplitudes were
significantly greater in the animals treated with SNC-121 for 7 days (ischemic eyes
32159 vs. SNC-121 + ischemic eyes 50850 volts, P<0.05). SNC-121-mediated
retina neuroprotection was completely abolished when animals were treated with a
highly selective -opioid-receptor antagonist, naltrindole (3 mg/kg), 15 minutes
prior to SNC-121 treatment (SNC-121 + ischemic eyes 50850 vs. naltrindole +
SNC-121 + ischemic eyes 34857 volts; P<0.05). In addition, degeneration of the
inner retina resulted in 36% reduction in overall retina thickness, and this reduction
was almost fully recovered in SNC-121-treated animals. Pretreatment with
naltrindole reversed the structural retina protection induced by SNC-121. There
was a robust increase in TNF- and NF-B production in the optic nerve and inner
retina layers 4-hours post-ischemia. The production of both TNF- and NF-B was
attenuated in the presence of SNC-121.
Conclusions: These results provide evidence that post-ischemia activation of opioid-receptors provides retina neuroprotection. Current data also demonstrate that
production of TNF- and NF-B are early events in ischemic injury, which are
opposed by -opioid-receptor-activation. Overall, these results provide evidence
that the -opioidergic system exhibits the potential to protect the retina against
ischemic injury.
Commercial Relationships: Shahid Husain, None; Yasir Abdul, None
Neuroprotective Effects of Angiotensin II Type 1 Receptor (AT1-R) Blocker
via Modulating AT1-R Signaling and Decreased Extracellular Glutamate
Levels
Tomoyoshi Fujita, Kazuyuki Hirooka, Fumio Shiraga. Kagawa University Faculty
of Medicine, Ophthalmology, Miki, Japan.
Purpose: To investigate the mechanism of the neuroprotective effects of the AT1R blocker on neuronal death in retinal ischemia-reperfusion injury .
Methods: Retinal ischemia was induced by increasing intraocular pressure to 130
mmHg for 45 min. At 30 min prior to the ischemia, 1 mg/kg candesartan, which is
an AT1-R blocker (ARB), or saline was administered. Glutamate release from the
rat retina and intravitreal PO2 profiles were monitored during and after ischemia
using a microdialysis biosensor and oxygen-sensitive microelectrodes. ELISA was
used to measure changes in the expression of AT1-R. Expression of retinal mRNA
levels of NADPH oxidase membrane components, p47phox and p67phox were
measured by real-time polymerase chain reaction. Reactive oxygen species (ROS)
were measured using dihydroethidium. Data were analyzed using an independent
Students t-test or Dunnetts multiple comparison test.
Results: Administration of candesartan suppressed ischemia-induced increases in
the extracellular glutamate. Candesartan also attenuated the increase in intravitreal
PO2 during reperfusion. AT1-R expression peaked at 12 h after reperfusion (1.264
0.103 ng/ml; P < 0.001). Although there was an increase in the retinal mRNA
expression of p47phox and p64phox at 12 h after the reperfusion (193.0 35.3% of
control; P = 0.038, 329.6 94.6; P = 0.016), administration of candesartan

suppressed these expressions (75.6 18.0; P = 0.863, 84.0 19.2; P = 0.998). The
production of ROS was detected at 12 h after reperfusion (60.20 1.99% of
control; P < 0.001). After candesartan administration, a significant suppression of
this upregulation was observed (18.14 0.93; P < 0.001)
Conclusions: NADPH oxidase-mediated ROS production increased at 12 h after
reperfusion. Candesartan may protect neurons by decreasing extracellular
glutamate immediately after reperfusion and by attenuating oxidative stress via a
modulation of the AT1-R signaling that occurs during ischemic insult.
Commercial Relationships: Tomoyoshi Fujita, None; Kazuyuki Hirooka,
None; Fumio Shiraga, None
Support: None
Activation of Stress Pathways by Ischaemia in Human Organotypic Retinal
Cultures
Andrew Osborne1, David C. Broadway2, Julie Sanderson1. 1School of Pharmacy,
University Of East Anglia, Norwich, United Kingdom; 2School of Biological
Science, University of East Anglia, UK, Norwich, United Kingdom.
Purpose: To investigate activation of p38 and JNK in Human Organotypic Retinal
Cultures (HORCs) exposed to simulated ischaemia and the role played by these
stress-activated pathways in ischaemia-mediated retinal ganglion cell death.
Methods: HORCs were dissected from donor human globes within 24 hours post
mortem and cultured in serum-free DMEM/HamF12 medium. Ischaemia was
simulated by oxygen/glucose deprivation (OGD) in which HORCs were cultured
for 3 hour in glucose-free DMEM in a modular incubator gassed with
95%N2/5%CO2 in the presence or absence of p38 (SB203580) or JNK (SP600125)
inhibitors. Following exposure to OGD the HORCs were returned to control
medium containing inhibitors for a further 21 hours. Lactose dehydrogenase (LDH)
was measured in culture medium to indicate levels of cell death and numbers of
RGCs assessed by THY-1 mRNA expression and NeuN immunohistochemistry.
Phosphorylated p38 and JNK levels were detected by Western blot and
immunohistochemistry.
Results: Simulated ischaemia (OGD) resulted in a decrease in RGC markers
(~45% reduction in THY-1 mRNA; n=6; p<0.05; ~40% reduction in NeuNlabelling; n=9; p<0.05) and an increase in release of LDH (~45%; n=6; p<0.05).
Both p38 and JNK activation increased following 3 hours OGD (2.8-fold & 2.5fold respectively at t=60min; n=3; p<0.05). Activated JNK was co-localised with
NeuN in the ganglion cell layer of HORCs whilst phosphorylated p38 was
primarily localised to the inner nuclear layer. Neither SP600125 (90nM) nor
SB203580 (10M) significantly inhibited OGD-mediated loss RGCs (THY-1
mRNA; n=3) nor limited LDH release from HORCs (n=5). However, inhibition of
the p38 pathway under control conditions significantly increased LDH release by
30% (n=5; p<0.05) and induced a loss of THY-1 mRNA (n=3; p<0.05).
Conclusions: Simulated ischaemia increased activation of the p38 and JNK stress
pathways in the human retina. Inhibition of p38 or JNK did not prevent loss of
RGCs suggesting that activation of these stress pathways is not part of the
neurodegenerative process in this model. To the contrary, p38 inhibition induced
loss of RGC markers and increased cell death, suggesting activation of the p38
pathway may mediate survival mechanisms in retinal ganglion cells.
Commercial Relationships: Andrew Osborne, None; David C. Broadway,
None; Julie Sanderson, None
Support: The Edith Murphy Foundation, The Humane Research Trust, The
Norwich Glaucoma Research Fund
Retinal Neuroprotection Against Ischemia/Reperfusion Damage Induced By
Global And Local Hypothermia
Ezequiel M. Salido, Damian Dorfman, Monica Chianelli, Ruth E. Rosenstein.
Human Biochemistry, University of Buenos Aires (UBA), CABA, Argentina.
Purpose: Retinal ischemia could provoke blindness. There is no effective treatment
against retinal ischemic damage. We investigated whether a brief global or local
hypothermia could induce ischemic tolerance in the rat retina.
Methods: Ischemia was induced in male Wistar rats by increasing intraocular
pressure to 140 mm Hg for 50 minutes. One day before ischemia, animals
underwent a 20-minute period of hypothermia by lowering the whole body
temperature to 32C or by cooling only one eye to 32C for 30 min. Two weeks
after ischemia, animals were subjected to electroretinography (ERG) and
histological analysis. Moreover, glutamate uptake was assessed using 3Hglutamate, and glutamine synthetase activity was assessed by a spectrophotometric
assay.
Results: Retinal ischemia induced a significant decrease in oscillatory potentials,
and scotopic ERG a- and b-wave amplitude, which was significantly prevented by
global or monocular hypothermia. Retinal ischemia induced a significant decrease
in glutamate uptake and glutamine synthetase activity, whereas monocular
hypothermia prevented the effect of ischemia on glutamate recycling. The
intravitreal injection of supraphysiological concentrations of glutamate mimicked

electroretinographic and histological alterations provoked by ischemia, which were
significantly abrogated by hypothermic preconditioning.
Conclusions: These results indicate that hypothermia significantly protected retinal
function and histology against ischemia/reperfusion injury. Hypothermic
preconditioning could provide a relatively low-risk approach for treating retinal
ischemic pathologies.
Commercial Relationships: Ezequiel M. Salido, None; Damian Dorfman,
None; Monica Chianelli, None; Ruth E. Rosenstein, None
Support: CONICET / UBA / ANPCyT
Molecular Mechanisms of Blood-Retinal Barrier Breakdown in IschemiaReperfusion Injury
Arivalagan Muthusamy, Cheng-Mao Lin, Heather Lindner, Sumathi Shanmugam,
Steven F. Abcouwer, David A. Antonetti. Department of Ophthalmology & Visual
Sciences, University of Michigan Kellogg Eye Center, Ann Arbor, MI.
Purpose: Formation and maintenance of the blood-retinal barrier (BRB) requires
assembly of multiple tight junction (TJ) proteins including the MARVEL family
(occludin, tricellulin, and marvel D3 proteins) and the claudin family. Recently, we
demonstrated that ischemia-reperfusion (IR) injury induces retinal vascular
permeability that occurs within 4 h after reperfusion and persists for at least 48 h.
Previous studies demonstrated that VEGF causes vascular permeability through
rapid phosphorylation of the TJ protein occludin on serine 490 (Ser-490), which
leads to its ubiquitination and translocation away from TJ complexes. Therefore,
the present study was designed to test the hypotheses that retinal IR injury causes
vascular permeability through rapid alterations in tight junction protein
expression, modification and complex formation.
Methods: Rats were subjected to ischemia for 45 minutes followed by reperfusion
up to 4 h. Blood-retinal barrier breakdown after IR was confirmed by leakage of
intravenously injected Evans blue dye. Changes in retinal TJ protein content and
occludin phosphorylation were assessed by Western blotting. Occludin
ubiquitination was determined by immunoprecipitation (IP) followed by Western
blotting. To observe the effect of IR on TJ organization, the localization of TJ
proteins was examined by immunohistochemistry (IHC) of retinal flat mounts.
Results: Retinal vascular permeability was significantly increased 4, 24 and 48 h
after reperfusion. IR caused rapid occludin Ser-490 phosphorylation occurring
between 15 and 60 min after reperfusion, while poly-ubiquitination of occludin was
evident after 15 min. IHC indicated that IR diminished TJ protein content at
endothelial cell junctions; however, there were no changes in total protein contents
of occludin, claudins (1, 5, 10, and 16) and Zonula occluden-1 (ZO-1).
Conclusions: These data are consistent with retinal IR injury causing vascular
permeability through rapid phosphorylation and ubiquitination of occludin leading
to its translocation and subsequent disassembly of the tight junction complexes.
This model suggests that occludin phosphorylation may represent a valid target for
therapeutic prevention of BRB breakdown and subsequent edema.
Commercial Relationships: Arivalagan Muthusamy, None; Cheng-Mao Lin,
None; Heather Lindner, None; Sumathi Shanmugam, None; Steven F.
Abcouwer, None; David A. Antonetti, None
Support: JDRF
Expression of Nox Family Proteins In Normal and Ischemic RGCs
Galina Dvoriantchikova1A, Jeff Grant1B, Andrea Rachelle C. Santos1A, Eleut P.
Hernandez1A, Valery I. Shestopalov1A,1C, Dmitry V. Ivanov1A,2. ABascom Palmer
Eye Institute, Department of Ophthalmology, BDepartment of Physiology and
Biophysics, CDepartment of Molecular Cell and Developmental Biology,
1
University of Miami Miller School of Medicine, Miami, FL; 2Vavilov Institute of
General Genetics RAS, Moscow, Russian Federation.
Purpose: Increasing evidence suggests that NADPH oxidase complexes are an
important source of cellular ROS and oxidative stress-mediated damage after
ischemia/reperfusion (IR) injury in neurons. This study was designed to investigate
superoxide production and the expression of the Nox family of NADPH oxidase
proteins in normal and ischemic RGCs.
Methods: We evaluated the in vivo and in vitro expression levels of members of
the Nox family and related subunits in normal RGCs and after ischemia by
quantitative RT-PCR. IR injury was induced by unilateral elevation of intraocular
pressure via direct corneal canulation. RGCs isolated by immunopanning from
retinas were exposed to oxygen and glucose deprivation (OGD). Corresponding
changes in protein abundances in RGCs were analyzed by immunohistochemistry
and immunocytochemistry. The level of ROS generated was assayed by
dihydroethidium (DHE). The Nox inhibitors VAS2870, AEBSF and ML090 were
then tested to determine if it altered the production of ROS within the RGCs.
Results: We report that RGCs produced ROS after OGD. The Nox inhibitors
VAS2870, AEBSF and ML090 decreased the burst stimulated by the OGD. We

found that RGCs express catalytic Nox1, Nox2, Nox4, Duox1, as well as regulatory
Ncf1/p47phox, Ncf2/p67phox, Cyba/p22phox, Noxo1, Noxa1 subunits and Rac1,
Rac2 and Rac3 under normal conditions and after ischemia. However, RGCs
express only low levels of catalytic Nox2, Nox4 and Duox1, and regulatory
Ncf1/p47, Ncf2/p67 subunits, but exhibit significantly higher level of catalytic
subunit Nox1 and subunits required for optimal activity of Nox1, including
Cyba/p22phox, Noxo1, and Rac1. The level of catalytic Nox3 and Duox2, and
regulatory Ncf4/p40phox subunits were statistically insignificant in RGCs.
Importantly, OGD treatment increased the expression level of Nox1, while the
levels of Nox2 and Nox4 were reduced after OGD
Conclusions: Our study showed that catalytic subunits Nox1, Nox 2, Nox4, Duox1
and related regulatory subunits are present in normal and ischemic RGCs. The
expression of proteins of the Nox family in RGCs suggests that NADPH-oxidase
related production of ROS can play a role in normal RGC function, but excessive
Nox-derived ROS production after IR can mediate RGC death. Importantly, high
levels of catalytic subunit Nox1 and subunits required for optimal activity of Nox1
in RGCs suggest that this enzyme could be a major source of ROS in ischemic
RGCs produced by NADPH oxidases.
Commercial Relationships: Galina Dvoriantchikova, None; Jeff Grant,
None; Andrea Rachelle C. Santos, None; Eleut P. Hernandez, None; Valery I.
Shestopalov, None; Dmitry V. Ivanov, None
Support: AHA Scientist Development Award 0735014B (DI), NIH grant
EY020613 (DI), NEI Core Center Grant P30 EY014801 to BPEI
Deletion of Arginase 2 Prevents Retinal Ganglion Cell Loss and Blocks
Formation of Acellular Capillaries after Ischemia/Reperfusion Injury
Zhimin Xu1A, Harumasa Yokota2, Subhadra P. Narayanan1A, Robert W. Caldwell1B,
Ruth B. Caldwell1A,3. AVascular Biology Center, BDepartment of Pharmacology and
Toxicology, 1Georgia Health Sciences University, Augusta, GA; 2Ophthalmology,
Asahikawa Medical University, Asahikawa, Japan; 3VA Medical Center, Augusta,
GA.
Purpose: Excessive activity of the urea cycle enzyme arginase has been implicated
in a variety of cardiovascular diseases. Our lab is investigating whether this enzyme
could serve as a therapeutic target in ischemic retinopathy. Our previous studies
have shown that deletion of arginase 2 (Arg2) significantly reduces retinal neuronal
and vascular injury in a model of retinopathy of prematurity. In this study, we have
evaluated the role of Arg2 in neuronal and capillary degeneration resulting from
retinal ischemia/reperfusion (I/R) injury.
Methods: Wild type (C57BL/6) or Arg2 (-/-) mice were prepared in a model of I/R
injury. I/R injury was produced by raising the intraocular pressure to 110 mmHg
for 40 minutes followed by reperfusion for various times. The contralateral eye was
used as sham control. NeuN staining and confocal imaging of whole mounted
retinas was used to quantify neuronal cells in the ganglion cell layer (GCL) at 1
week after I/R. For this, 10 confocal images were randomly taken around the optic
nerve at the ganglion cell layer in each retina. Then the ratio of the number of
NeuN positive cells relative to that of the sham control eye was calculated.
Formation of acellular capillaries was assessed by using trypsin digest procedures
to isolate the retinal vasculature at 4 weeks after I/R.
Results: I/R in the wild type mice resulted in a 60% reduction in the number of
NeuN positive GCL neurons compared to the sham eyes (p<0.05). This neuronal
cell loss was largely prevented in the Arg2-deficient mice which had a 40%
increase in the number of NeuN positive GCL neurons compared to the WT I/R
retinas (p<0.05). The formation of acellular capillaries was also significantly
decreased in the Arg2-deficient mice. Whereas the number of acellular capillaries
in the WT I/R retinas was increased by 8 fold above sham controls (p<0.001), the
acellular capillaries in the Arg2-/- I/R retinas were increased by only 2 fold above
the sham controls (p< 0.001).
Conclusions: Deletion of arginase 2 prevents the loss of GCL neurons and reduces
the formation of acellular capillaries induced by I/R injury. These data suggest that
inactivation of arginase 2 may offer a new strategy for preventing neuronal cell
death as well as retinal vasculature damage in ischemic retinopathy.
Commercial Relationships: Zhimin Xu, None; Harumasa Yokota,
None; Subhadra P. Narayanan, None; Robert W. Caldwell, None; Ruth B.
Caldwell, None
Support: NEI-R01-EY04618; NEI-R01-EY11766; VA Merit Review
Kingdom; 3Department of Sports Medicine, Medical Clinic, University Hospital

Heidelberg, Heidelberg, Germany.
Purpose: This study aimed to quantify structural and functional changes of the
macula during acute exposure to high altitude and to assess their structure/function
relationship.
Methods: Spectral domain optical coherence tomography, fundus controlled
microperimetry and best-corrected visual acuity (BCVA) were used to quantify
changes of central retinal structure and function in 14 healthy subjects between
baseline recordings (341 m) and during acute exposure to high altitude (4559 m).
Data from high-resolution macular volume scans and central microperimetry were
analyzed for all 9 subfields of the ETDRS grid.
Results: Detailed longitudinal analysis revealed increased total retinal thickness
(TRT) in all four peripheral ETDRS grid subfields during acute altitude exposure
(TRTperiphery = 2.802.16 m; mean95%CI). This change is inverted towards
the central four subfields (TRTcentral = -1.892.05 m) with significant reduction
of TRT in the foveola (TRTfoveola = -6.621.92 m) at altitude. BCVA revealed
no significant difference compared to baseline (-0.040.10 logMAR).
Microperimetry showed stable mean retinal sensitivity in all but the foveolar
subfield (MSfoveolar = -1.381.15 dB). At baseline recordings before and >2
weeks after high altitude exposure, all subjects reached prior levels with no sign of
persisting structural or functional sequels.
Conclusions: Significant changes of TRT occur in the central retina of healthy
subjects during acute exposure to high altitude. The gradient from central reduction
to peripheral increase of TRT reflects the relatively higher contribution of nerve
fibers and vessels to the TRT in peripheral ETDRS subfields. The observed
structural changes did not have an overall impact on key functional outcome
measures such as BCVA and retinal mean sensitivity. For the first time a
quantitative approach has been used to assess these changes during acute, nonacclimatized high altitude exposure.
Commercial Relationships: M Dominik Fischer, None; Gabriel Willmann,
None; Andreas Schatz, None; Kai Schommer, None; Ahmad Zhour,
None; Eberhart Zrenner, None; Karl U. Bartz-Schmidt, None; Florian
Gekeler, None
Support: fortuene Junior F1222708, Ewald und Karin Hochbaum Stiftung
Spatio-Temporal Characteristics Of Oxygen Diffusion In The Vitreous Humor
Karthik Murali1, Ramiro Ribeiro2, James Weiland1, Mark Humayun1. 1Biomedical
Engineering, University of Southern California, Los Angeles, CA; 2Doheny Eye
Institute, Los Angeles, CA.
Purpose: To investigate the spatio-temporal characteristics of oxygen diffusion in
the vitreous humor of the eye with the aim of creating an in-vitro model for ocular
oxygen diffusion.
Methods: A point source of oxygen was created in 3 different media: saline,
enucleated pig eyes vitreous, and live rabbits vitreous. 2 platinum/Iridium (80/20)
electrodes with exposed surface area of 0.3mm2 each were used to perform
electrolysis under a monophasic current pulse train. The pulse amplitude was
1.5mA, pulse duration was 200 s, and the pulse period was 10 ms. Under these
conditions, 0.078 nmol of O2/min was generated. An oxygen probe was placed
0.5mm away from the oxygen producing electrode (anode) and oxygen tension was
recorded over time in the aforementioned media. In the enucleated pig eyes and in
the rabbit eyes, the electrodes were placed close to the optic disk.
Results: The data collected suggests that oxygen diffusion in saline is faster than
oxygen diffusion in the other two media. The oxygen tension in saline increased
from 30mmHg to more than 200 mmHg in less than a minute when the oxygen
source is 0.5mm away from the oxygen measuring probe. Since the oxygen probe
cannot measure partial pressures above 200mmHg, there are no data points after 1
minute. In both in-vitro and in-vivo vitreous humor, the oxygen tension rises from
5mmHg to about 55mmHg over 30 minutes, when the oxygen source is 0.5mm
away from the oxygen measuring probe.
Conclusions: The data collected suggests that in vitro tests of oxygen diffusion
cannot be modeled on saline since the diffusion dynamics of oxygen in saline is
very different from that of vitreous humor. Enucleated pig eyes seem to be a good

Structural and Functional Changes in the Macula During Acute Exposure to
High Altitude
M Dominik Fischer1,2, Gabriel Willmann1, Andreas Schatz1, Kai Schommer3,
Ahmad Zhour1, Eberhart Zrenner1, Karl U. Bartz-Schmidt1, Florian Gekeler1.
1
Centre for Ophthalmology, University of Tuebingen, Tuebingen, Germany;
2
Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, United

model for in-vivo experiments.
Commercial Relationships: Karthik Murali, None; Ramiro Ribeiro,

None; James Weiland, None; Mark Humayun, None
Support: None
cAMP Promotes Fluid Transport Across Porcine Ciliary Epithelium By
Enhancing Gap Junctional Permeability
King Wah Angela Cheng, King Kit Li, Chi Ho To, Chi Wai Do. School of
Optometry, Hong Kong Polytechnic University, Hong Kong, Hong Kong.
Purpose: To determine the effect of cAMP on fluid transport across porcine ciliary
epithelium.
Methods: Electrical parameters including transepithelial potential difference (PD),
short-circuit current (Isc) and transmural resistance (Rt) were continuously
monitored in modified Ussing-Zhrahn-type chamber. Lucifier Yellow dye transfer
technique was employed to study the gap junctional permeability. Whole-cell patch
clamp recording was used to study Cl- channel activity.
Results: The addition of 1-100 M 8-bromo-cAMP to the aqueous surface
consistently stimulated Isc by 60-80% across porcine ciliary epithelium. This effect
was completely abolished by either bathing Cl- substitution or pretreatment with
3.5 mM heptanol (a gap junction blocker), indicating that NPE-Cl- channels and/or
gap junctions between PE and NPE are the possible cellular site(s) of cAMPinduced responses. Addition of 10 M 8-bromo-cAMP significantly increased the
rate of Lucifier Yellow dye transfer from PE to NPE cell in isolated porcine PENPE cell couplets (n=24; p<0.05). However, cAMP had no effect on whole-cell Clcurrent in both isolated native PE (n=4) and NPE (n=7) cells.
Conclusions: Our results suggest that cAMP stimulates Isc primarily by increasing
the gap junctional permeability between PE and NPE cells. In the absence of gap
junctions linking the two cell layers, the effects of cAMP on Cl- efflux are,
however, subtle in both cell types.
Commercial Relationships: King Wah Angela Cheng, None; King Kit Li,
None; Chi Ho To, None; Chi Wai Do, None
Support: Niche Areas J-BB76; ICRG G-YG14 and A-SA27
Evaluation of the Modulation of Organic Cation Transporter (OCT) in the
Tear Disposition of its Substrates in Rabbits
Thirumurthy Velpandian1, Jayabalan Nirmal1, Anju Sirohiwal1, Sundararajan B.
Singh2, Thavaraj Vasantha3, Raj V. Azad4A, Supriyo Ghose4B. 1Ocular
Pharmacology & Pharmacy, Dr RP Centre for Ophthalmic Sciences, All India
Institute of Medical Sciences, New Delhi, India; 2Biophysics, All India Institute of
Medical Sciences, New Delhi, New Delhi, India; 3Indian Council of Medical
Research, New Delhi, India; Avitreoretinal diseases & Trauma, BPediatric
Ophthalmology & Oculoplasty, 4Dr.RPCentre for Ophthalmic Sciences, All India
Institute of Medical Sciences, New Delhi, India.
Purpose: Endogenous cationic compounds are known to exist in the tear fluid.
Therefore, the positioning and modulation of Organic Cation Transporters (OCT)
in cornea, conjunctiva and lacrimal gland gain interest in understanding the
precorneal disposition of xenobiotics for their pharmacological and toxicological
significance. This study was conducted to evaluate the role of OCT in the fate of
topical and intravenous substrates in their precorneal disposition using their
blockers as pharmacological tools. Gene expression studies were also conducted to
identify the presence of the subtype of OCT in tear glands.
Methods: Albino rabbits of either sex weighing 1.5-2.0 kg were used for the study.
Tear kinetics of OCT substrates (tetraethylammonium (TEA), metformin) after
topical and intravenous administration were assessed in the presence and absence
of blockers (quinidine & atropine) applied topically. Tear samples were collected
using Schirmers strips at various time intervals and subjected for the simultaneous
quantification of substrates and blockers using LC-MS/MS. Invivo Gamma
Scintigraphy was also used to image the fate of 99Tc labelled TEA with or without
OCT transporter blockade in rabbits. Rabbit lacrimal tissue samples were subjected
for the evaluation for the expression of OCT1, OCT2 & OCTN2 genes using RTPCR.
Results: Topical pretreatment (30 min prior to substrate) of OCT blockers
significantly decreased the pre-corneal clearance of OCT substrates thereby
increased their tear levels after topical administration. Surprisingly, intravenous
administration of TEA reached significant concentration in the tear after single
intravenous administration at the Tmax of 60 min and was significantly inhibited
by the blockers. Gene expression studies revealed the presence of OCTN2 in
lacrimal glands whereas OCT 1 & 2 were undetected.
Conclusions: OCTs are functionally active in the precorneal disposition of cationic
substrates. OCTs present in corneal epithelium and conjunctiva are positioned from
apical to basolateral and basolateral to apical in tear glands. For the first time, this
study revealed that intravenously administered xenobiotics (OCT substrates) are
capable of reaching precorneal area through tear secretion and can have
physiological and pharmacological relevance.
Commercial Relationships: Thirumurthy Velpandian, None; Jayabalan
Nirmal, None; Anju Sirohiwal, None; Sundararajan B. Singh, None; Thavaraj
Vasantha, None; Raj V. Azad, None; Supriyo Ghose, None
Support: AIIMS Intramural grant F.6-1/2010/Acad
Potential Role of Anoctamin-6 in Cell Volume Regulation of Human
Trabecular Meshwork Cells
Juni Banerjee1A, Ang Li1A,2, Chi Ting Leung1A, Kim Peterson-Yantorno1A, W. Daniel
Stamer3, Mortimer M. Civan1A,1B. APhysiology, BMedicine, 1Univ of Pennsylvania
Perelman Sch of Medicine, Philadelphia, PA; 2Anatomy, University of Hong Kong
Li Ka Shing Faculty of Medicine, Hong Kong SAR, China; 3Ophthalmology, Duke
University, Durham, NC.
Purpose: Cell volume of trabecular meshwork (TM) cells has been posited to
regulate aqueous humor outflow resistance. Regulation of TM cell volume depends
on activity of swelling-activated Cl- channels (ICl,swell) whose identity is
unknown in these cells. Most tissues express anoctamins Ano1 or Ano2, which are
Ca2+-activated Cl- channels (CaCCs). Anoctamins are reported to be components
of epithelial ICl,swell, but agreement about their functions and location is
incomplete. We are testing whether anoctamins may participate in TM cell volume
regulation.
Methods: Transformed normal human TM5 and glaucomatous GM3 TM cells and
HEK293 cells were studied. Gene expression was measured by reversetranscription PCR (RT-PCR) and real-time PCR, protein product by western blots,
and membrane currents by whole-cell ruptured-patch recording.
Results: TM5 cells highly expressed Ano6, but Ano1-2 were not detected (N=3).
Ano4 and Ano7-10 were also expressed. HEK293 cells expressed both Ano1 and
Ano6, (N=2) whereas GM3 cells expressed Ano2 and Ano6 but not Ano9 (N=2).
The Ca2+ ionophore ionomycin (5 micromolar) triggered CaCC currents in TM5
cells, with activating and inactivating currents at depolarizing and hyperpolarizing
potentials, respectively. At +100 mV, 5 micromolar ionomycin increased currents
from 13 4 to 60 12 pA/pF. The CaCC blocker tannic acid (100 micromolar)
inhibited these CaCC currents by 78 4% (N=3). Knockdown (75%) of Ano6
markedly reduced ionomycin-activated currents to 12 and 19 pA/pF in duplicate
experiments. ICl,swell after 20% hypotonicity reached 188 40 pA/pF from 8 3
pA/pF. The CaCC inhibitors CaCCinh-A01 (50 micromolar) and tannic acid (100
micromolar) inhibited ICl,swell by 45 5% and 96 1%, respectively (N=4), but
the selective inhibitor of Ano1 and Ano2, T16Ainh-A01 (20 micromolar), was
ineffective (N=3).
Conclusions: Transformed normal TM cells did not express the best characterized
channels of CaCCs, Ano1-2, but highly expressed Ano6, and this expression
differed from that of transformed glaucomatous TM cells. The early data suggest
that Ano6 may play a role in CaCCs, and that anoctamins may modulate ICl,swell
and thereby TM cell volume regulation.
Commercial Relationships: Juni Banerjee, None; Ang Li, None; Chi Ting
Leung, None; Kim Peterson-Yantorno, None; W. Daniel Stamer,
None; Mortimer M. Civan, None
Support: NIH Grants EY13624 (M.M.C.) and Core Grant EY 01583
Lack of Bicarbonate Transport by SLC4A11 in Bovine Corneal Endothelial
Cells
Supriya S. Jalimarada1, Eranga Vithana2, Joseph Bonanno1. 1Sch of Optometry,
Indiana University, Bloomington, IN; 2Singapore Eye Research Institute,
Department of Ophthalmology, Yong Loo Lin School of Medicine, National
University of Singapore, Singapore, Singapore.

Purpose: Bicarbonate transport is an integral part of the fluid transport/pump
mechanism in corneal endothelium (CE). SLC4A11 is a member of the SLC4
superfamily of bicarbonate transporters and has been characterized as a putative
sodium borate transporter. Using primary cultures of bovine corneal endothelial
cells (BCEC), we ask if SLC4A11 functions as a Na-dependent bicarbonate
transporter.
Methods: SLC4A11 expression in BCEC was knocked down using an SLC4A11
siRNA mixture. Knockdown was analyzed by western blot. Solute transport was
assessed by measuring intracellular pH (pHi) and intracellular sodium
concentration [Nai], using fluorescent dyes. Cells grown on coverslips were
transfected with either scrambled-sequence or siSLC4A11 or treated with
transfection reagent only and subjected to analysis 72 hrs post-transfection. Na and
HCO3 transport was determined by examining pHi and [Nai] responses to
CO2/HCO3 pulses in control and transfected cells. Additionally, effect of
knockdown on Na/H+ exchange was assessed using Na free pulses on pHi.
Results: siRNA transfection of BCEC resulted in ~70% knockdown of SLC4A11.
Perfusion with CO2/HCO3 rich ringer induced a brief acidification (0.07 pH units)
followed by alkalinization (0.2 pH units) in control cells. No significant difference
in rates or amplitude of acidification and alkalinization was observed for the
siSLC4A11 transfected cells, indicating lack of bicarbonate transport by SLC4A11.
Addition of CO2/HCO3 induced a sharp increase in [Na]i. The initial rate of Na
influx was not significantly different in siRNA treated cells. However, the
amplitude of the [Na]i increase was 30% less (p=0.04) and the steady-state [Nai]
was 46% lower (p= 0.005) in siSLC4A11 cells compared to control indicating
decreased [Na]i. Na free pulses showed a 34% and 38% decrease in acidification in
the absence and presence of EIPA (an inhibitor of Na+/H+ exchanger),
respectively, compared to control.
Conclusions: Lack of changes in pHi to CO2/HCO3 in siSLC4A11 transfected
cells indicates absence of any significant bicarbonate transport by SLC4A11.
However, a decrease in sodium influx and lower steady state in SLC4A11
knockdown to CO2/HCO3, along with reduced Na free acidification & EIPA
sensitivity suggests Na+/H+ exchanger like activity
Commercial Relationships: Supriya S. Jalimarada, None; Eranga Vithana,
None; Joseph Bonanno, None
Characterization of Transient Receptor Potential Channel TRPM1 Purified
From Insect Cells
Melina A. Agosto, Zhixian Zhang, Feng He, Theodore G. Wensel. Biochemistry and
Molecular Biology, Baylor College of Medicine, Houston, TX.
Purpose: In retinal ON-bipolar cells, post-synaptic signal transduction is mediated
by a G protein-coupled receptor cascade, which in turn controls the open state of a
cation-selective channel. The transient receptor potential channel TRPM1 has
recently been implicated in this pathway. TRPM1 is a 180 kDa protein which likely
forms a multimeric channel complex. However, virtually nothing is known about
the structure of the TRPM1 channel or its close relatives. To enable biochemical
and structural studies, baculovirus-expressed TRPM1 was purified from insect
cells.
Methods: TRPM1 with a C-terminal 1D4 epitope tag was affinity purified from
Sf9 cells. Cell membranes were detergent solubilized and incubated with sepharose
conjugated to the monoclonal antibody 1D4. After washing, TRPM1 was eluted
from the resin with the nonapeptide corresponding to the 1D4 epitope. The purified
protein was reconstituted into proteoliposomes by mixing the protein with
solubilized lipids, then removing the detergent with Bio-Beads polystyrene
adsorbent. For structural studies, the purified protein was mixed with amphipol,
followed by detergent removal with Bio-Beads. Protein-amphipol complexes were
examined by electron microscopy and single particle reconstruction.
Results: Both blue native PAGE and size exclusion chromatography indicate that
the purified protein is a homogeneous oligomeric complex of approximately 500600 kDa. Successful proteoliposome formation was confirmed by flotation of the
protein in a sucrose gradient, and pull-down experiments with 1D4 antibody
indicated symmetric orientation of the protein in the liposome membranes.
Preliminary electron microscopy suggests that TRPM1 forms a complex with a
small transmembrane domain and a larger basket-like cytoplasmic domain.
Conclusions: A single-step affinity purification scheme has been developed for
TRPM1, with a yield of ~1-2 mg/L spinner culture. Future studies will focus on
biochemical characterization of the purified protein and demonstration of channel
function in proteoliposomes, as well as structure determination by electron
microscopy.
Commercial Relationships: Melina A. Agosto, None; Zhixian Zhang,
None; Feng He, None; Theodore G. Wensel, None
Support: NIH Grants F32EY200672, T32EY007102, R01EY07981, R01EY11900
Diversity of Expression of Voltage-Gated Sodium Channels in the Rat Retina

George R. Ehring1, Jia C. Li1, Howard B. Rind2, Simon R. Levinson3. 1Bioilogical
Sciences, Allergan Pharmaceuticals, Irvine, CA; 2Integrated BioSolutions
Consulting, Laguna Niguel, CA; 3Physiology & Biophysics, University of
Purpose: Many of the cell types in the retina generate sodium currents. The
sodium channel isoforms underlying these currents have not been completely
characterized. In this study, we applied qPCR and immunolocalization to determine
the expression patterns of sodium channel isoforms in rat retina.
Methods: In Sprague Dawley rats, we measured retinal sodium channel gene
expression at four ages p7, p15, p30 and at 3-4 months using qPCR. Amplicons
from the qPCR reaction product were annealed to pGEM-T vectors, amplified and
sequenced for confirmation of the target sodium channel sequences. Using isoformspecific antibodies, we examined channel protein localization with confocal
microscopy.
Results: The mRNA for NaV1.1, NaV1.2, and NaV1.6 was strongly expressed
throughout development. Expression of NaV1.3 mRNA decreased 3-fold from p7 to
p15, but then remained stable to the age of four months. We demonstrated a low
level of expression of NaV1.8 and NaV1.9 mRNA and greater expression of NaV1.7,
three genes normally considered to be expressed only in the PNS. Using confocal
microscopy, we found substantial immunoreactivity of the Nav1.7, 1.8, and 1.9
isoforms in the retina. Nav1.7 and 1.9 appear to be localized to photoreceptors and
to synaptic structures in the outer plexiform layer, while Nav1.8 is expressed in a
subset of retinal ganglion cells and their processes.
Conclusions: A diverse set of sodium channel isoforms are expressed in the
mammalian retina with different pattern of cellular localization for each suggesting
unique contributions to signal processing and integration in the complex neuronal
network of the retina.
Commercial Relationships: George R. Ehring, Allergan, Inc. (E); Jia C. Li,
Allergan, Inc. (E); Howard B. Rind, Allergan, Inc. (C); Simon R. Levinson,
Allergan, Inc. (C)
Support: None
The Association Of Altered Aquaporin 9 Expression With Apoptosis Of
Retinal Ganglion Cells
Akiko Miki1, Akiyasu Kanamori1, Maiko Naka2, Akira Negi1, Makoto Nakamura1.
1
Kobe university of medicine, osaka, Japan; 2Mitsubishikobe Hospital, Kobe,
Japan.
Purpose: Aquaporins are a family of integral membrane proteins that allow water
to cross the plasma membrane. They are critically involved in the maintenance of
ionic and osmotic balance in the central nervous system (CNS). Aquaporin
9(AQP9) belongs to an aquaglyceroporin, is permeable to non-charged solutes such
as lactate, an energy substrate in the CNS, and is reportedly expressed in retinal
ganglion cells (RGCs). Previously we reported that elevated intraocular pressure
reduced the AQP9 expression in rat RGCs. However, the causative relationship
between the AQP9 loss and RGC death remains unknown. The purpose for this
study is to investigate the association of altered AQP9 expression with apoptotic
death of RGCs.
Methods: RGC5 cells were deprived of serum and the optic nerve was transected
to induce apoptosis of RGCs in rats. The retinal and RGC5 AQP9 expression at
various time points was quantified by real time RT-PCR and western blotting. Cell
survival was assessed by Live/Dead assay. The effect of AQP9 knockdown using
short interference RNA on the RGC5 apoptosis was evaluated by activated caspase3 immunohistochemistry.
Results: Both serum deprivation and optic nerve transection reduced protein
expression of AQP9 (p= 0.0067 and p= 0.01,respectively).The AQP9 gene
expression was also reduced in the latter (p=0.0012). The AQP9 knockdown
accelerated the total number of death (p= 0.011) and apoptosis (p<0.001) of serumdeprived RGC5 cells.
Conclusions: AQP9 plays a critical role in RGC survival. The reduced expression
of AQP9 induces apoptotic death of RGCs.
Commercial Relationships: Akiko Miki, None; Akiyasu Kanamori,
None; Maiko Naka, None; Akira Negi, None; Makoto Nakamura, None
Support: None
A Study Of The Intracellular Electrical Properties And Transport
Mechanisms Of Porcine Pigmented And Non-pigmented Ciliary Epithelial
Cells
Jingru Xiao1, King Kit Li1, Chi Wai Do1, Chi Ho To1,2. 1School Of Optometry,
Hong Kong Polytechnic Univ, Hong Kong, Hong Kong; 2Department of
Ophthalmology, Sun Yat-sen University, Guangzhou, China.
Purpose: To investigate the mechanism of ion transport across ciliary epithelium
of porcine eye, the electrical membrane properties of primary cultures of pigmented

ciliary epithelial cells (PE) and non-pigmented ciliary epithelial cells (NPE) were
characterized.
Methods: The membrane potential of primary culture of PE and NPE cells from
porcine eyes was simultaneously measured by an anionic potential-sensitive
fluorescent dye, bis(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC4(3)]. The
effects of bathing ion substitution and transporter inhibitors were investigated.
Results: The membrane potential of single NPE and PE cells were 690.87mV(n=184) and -632.08mV(n=105) respectively. All of the single NPE
cells, PE cells and Coupling NPE-PE cells showed similar electrical responses
towards different bathing solutions and drugs with similar magnitude. Reducing
extracellular sodium concentration hyperpolarized the intracellular potential, while
chloride replacement caused significant depolarization. Addition of Ba2+
immediately reduced membrane potential. Ouabain also induced depolarization but
in a slower manner. A chloride channel blocker, diphenylamine-2-carboxylate
(DPC) markedly hyperpolarized intracellular potential, while there showed no
effect after the blockage of Na-K-2Cl cotransport by bumetanide.
Conclusions: Both the PE and NPE cells from porcine eye possess K+ channel,
Na+/K+-ATPase and DPC-sensitive chloride channel. The membrane potential is
strongly dependent on the extracellular chloride concentration. The experimental
paradigm allows the study of NPE and PE responses simultaneously and may
reveal differential transport characteristics of these epithelial cells in orchestrating
the aqueous humor production.
Commercial Relationships: Jingru Xiao, None; King Kit Li, None; Chi Wai
Do, None; Chi Ho To, None
Support: glaucoma niche J-BB76, GRF B-Q04F, 1-BB8W, A-SA27. G-YG14
TRPC1 and 3 Channels Mediate Store-operated Calcium Entry in Mouse
Retinal Ganglion Cells
Tunde Molnar, Eerik Elias, Amber M. Frye, Wei Xing, Ning Tian, David Krizaj.
Ophthalmology and Visual Sciences, Moran Eye Center, University of Utah, Salt
Lake City, UT.
Purpose: To characterize the properties of store-operated calcium entry (SOCE) in
mouse retinal ganglion cells (RGCs). We also assessed whether SOCE in mouse
RGCs is mediated by TRPC1 and/or TRPC3 channels which were shown to
function as store-operated channels in non-excitable cells.
Methods: Fura-2 calcium imaging was performed in dissociated mouse RGCs
prepared from wild type, transgenic Thy1:CFP and Trpc1/Trpc3 (TRPC1/3-/-)
double knockout retinas. Whole-cell patch-clamp recordings were made from
RGCs in whole mount retinas prepared from wild type mice. Store-operated signals
were evoked with prolonged depletion of intracellular Ca2+ stores within the
endoplasmic reticulum (ER). Immunohistochemistry and RT-PCR was performed
using primary antibodies and primers for mouse SOC channel candidates.
Results: Cell-type specific RT-PCR showed expression of Trpc1-7, Stim1-2,
Orai1-3 transcripts in wild type mouse RGCs. Resting [Ca2+]i levels in dissociated
wild type RGCs were 53 7 nM. Glutamate and high K+ -containing saline
elevated [Ca2+]i to ~1 M, indicating maintained excitability of dissociated RGCs.
Depletion of ER stores in Ca2+-free saline supplemented with cyclopiazonic acid
(CPA) induced SOCE (187 7 nM) which was manifested as [Ca2+]i overshoots
following the return to control Ca2+ -containing saline in the presence of L-type Ca
channel blockers. SOCE was reduced in mice lacking TRPC1 and 3 isoforms and
was further diminished by SOC channel blockers SKF96365 and Gd3+ (98 12
nM). Depletion of intracellular stores with CPA induced an inward current of ~5-10
pA in Ca2+-free saline and in the presence of TTX & NMDA, AMPA/kainate and
voltage-gated Ca2+ channel blockers. The store depletion-evoked current was
antagonized by SKF96365 and Gd3+.
Conclusions: We found that store depletion elicits cation influx and [Ca2+]i
increases in mouse retinal ganglion cells. The suppression of depletion-evoked
Ca2+ signals in double KO mice indicating that TRPC1 and/or TRPC3 channels
contribute to RGC SOCE. Our data suggests SOCE is prominently expressed in
mouse RGCs, highlighting a role for ER stores in RGC excitability and retinal
output.
Commercial Relationships: Tunde Molnar, None; Eerik Elias, None; Amber
M. Frye, None; Wei Xing, None; Ning Tian, None; David Krizaj, None
Support: Knights Templar Eye Fundation 2011-12, NIH EY13870, Foundation
Fighting Blindness, Int retina Research Fundation, Research to Prevent Blindness
Effects of Cardiotonic Steroids on Trabecular Meshwork Cells: Search for
Mediator of Ouabain-Enhanced Outflow Facility
Mortimer M. Civan1A,1B, Juni Banerjee1A, Kim Peterson-Yantorno1A, Chi Ting
Leung1A, Ang Li1A,2. APhysiology, BMedicine, 1Univ of Pennsylvania Perelman Sch
of Medicine, Philadelphia, PA; 2Anatomy, University of Hong Kong Li Ka Shing
Faculty of Medicine, Hong Kong SAR, China.
Purpose: Ouabain administration (at least 30 nM, for 4 hrs or more) has been
reported to reduce aqueous humor outflow resistance. We previously found that
ATP release and ectoenzymatic conversion to adenosine may link cytoskeletal
remodeling and outflow resistance modulation. We now tested whether altered
ATP release might also be a mediator of ouabain's effect on outflow resistance.
Methods: ATP release from human TM5 trabecular meshwork cells was measured
by the luciferin-luciferase reaction, matrix metalloproteinases (MMPs) by
zymography, cell Na+ concentration by SBFI fluorometry, gene expression by realtime PCR, cell volume by electronic cell sorting, cell viability by LDH and MTT
assays, and the actin cytoskeleton by confocal microscopy of phalloidin-stained
cells.
Results: Contrary to expectation, ouabain at concentrations 10 nM or higher
inhibited swelling-triggered ATP release after 4 hrs or longer. Inhibition was
enhanced by increasing ouabain concentration and exposure time. Similar effects
were produced by the reversible cardiac aglycone strophanthidin. Ouabain (4 hrs,
30 nM and 100 nM) did not alter gene expression of the ATP-release pathways, and
cell viability was unchanged by exposure to ouabain (30 nM to 1 M).
Preincubation with 30 nM ouabain for 4 hrs did not detectably change Na+ level,
the regulatory volume decrease (RVD) or the actin cytoskeleton of TM5 cells, but
did inhibit hypotonicity-elicited ATP release. Moreover, even when N-methyl-Dglucose replaced Na+ in the extracellular fluid, ouabain still inhibited swellinginitiated ATP release at 100 nM. In the absence of ouabain, extracellular ATP
stimulated MMP secretion, which was largely blocked by inhibiting conversion of
ATP to adenosine, as expected. In contrast, ouabain reduced ATP release, but did
not alter secretion of MMP-2 and MMP-9 from cells pretreated for 4 hrs or less.
Conclusions: The results suggest that: (1) ouabain can trigger enhancement of
outflow facility independent of its transport and actin-restructuring effects exerted
at higher concentration and longer duration; (2) ouabain exerts parallel independent
effects on ATP release and outflow facility; and (3) these effects likely reflect
ouabain-induced changes in the scaffolding and/or signaling functions of Na+, K+activated ATPase.
Commercial Relationships: Mortimer M. Civan, None; Juni Banerjee,
None; Kim Peterson-Yantorno, None; Chi Ting Leung, None; Ang Li, None
Support: NIH Grants EY13624 (M.M.C.), and Core Grant EY 01583.
Spatial Expression, Protein Trafficking and Post-Translational Modification
of AQP5 in Mammalian Cornea and Lens
Kulandaiappan Varadaraj1,2, Murali Varadaraj3, George J. Baldo3, Anil G.
Menon4, Sindhu S. Kumari1. 1Physiology and Biophysics, State University of New
York, Stony Brook, NY; 2SUNY Eye Institute, New York, NY; 3Department of
Science, InSTAR Program, Ward Melville High School, East Setauket, NY;
4
Department of Molecular Genetics, Biochemistry and Microbiology, University of
Cincinnati College of Medicine, Cincinnati, OH.
Purpose: The present investigation studies the spatial expression, protein
trafficking and post-translational modifications of AQP5 in the transparent tissues
of eye, namely cornea and lens.
Methods: Spatial expression and protein trafficking were studied by
immunostaining and western blotting analyses in the wild type (WT), and AQP5
knockout (AQP5-/-) mice. Western blotting was also performed to test for
expression of AQP5 in rabbit. Protein trafficking and post-translational
modifications were investigated using immunostaining and immunoblot analyses.
Protein Kinase A (PKA) - induced phosphorylation effect on mouse AQP5
expression and trafficking was investigated in vitro using stably expressing MDCK
cells and ex vivo by culturing mouse corneal cells.
Results: Immunostaining of WT mouse cornea and lens sections showed
expression of AQP5 in epithelial cells, and keratocytes of cornea, as well as in
epithelial and fiber cells of lens. Immunoblotting of proteins from cornea, isolated
lens epithelial cells, cortical fiber cells and nuclear fiber cells of both rabbit and
mouse all showed the expression of AQP5. As expected, both immunostaining and
immunoblotting of AQP5-/- mice cornea and lens did not show anti-AQP5 antibody
binding. Both rabbit and mouse corneas and lenses expressed non-phosphorylated
and phosphorylated forms of AQP5 protein. Expression of non-phosphorylated
form of AQP5 was about ten-fold higher than the phosphorylated form. In vitro
studies using mouse AQP5 expressed in MDCK cells also showed both nonphosphorylated and phosphorylated protein bands. These results suggest AQP5
may play a significant role in maintaining the transparency and homeostasis of the
avascular cornea and lens. PKA agonist cAMP (100 M), reduced AQP5 plasma
membrane localization and promoted AQP5 internalization; in contrast, PKA
antagonist H-89 (20 M) retained AQP5 protein in the plasma membranes of the
MDCK cells (transfected to express AQP5), corneal and lens epithelial cells. When
the cells were pretreated with H-89 before exposure to cAMP there was not much
internalization of AQP5 protein compared to the PKA activation, suggesting H-89
blocks the action of cAMP.
Conclusions: To our knowledge, this is the first report on the spatial expression of
AQP5 protein in the corneal keratocytes, and lens epithelial and fiber cells. This
study documents the role of PKA in the localization of AQP5 in the plasma

membrane. Our data suggest AQP5 may be regulated by the PKA signaling
pathway in the cornea to maintain water homeostasis to prevent dry eye syndrome,
and in the lens for microcirculation to supply nutrients and to remove metabolic byproducts to keep the lens transparent.
Commercial Relationships: Kulandaiappan Varadaraj, None; Murali
Varadaraj, None; George J. Baldo, None; Anil G. Menon, None; Sindhu S.
Kumari, None
Ocular GluR1 And GluR2 Expression Following Retinal Injury
Yong H. Park1,2, Hidehiro Oku3, Masahiro Fukuhara4, Takuji Kurimoto4,
Tsunehiko Ikeda3, Thomas Yorio1,2, Adnan Dibas1,2. 1Pharmacology &
Neuroscience, UNT Health Science Center, Fort Worth, TX; 2North Texas Eye
Research Institute, Fort Worth, TX; 3Department of Ophthalmology, Osaka
Medical College, Osaka, Japan; 4Department of Ophthalmology, Hyogo College of
Medicine, Hyogo, Japan.
Purpose: Changes in the expression of AMPA receptors subunits (GluR1-4) have
been reported in a number of diseases. However, such changes and mechanisms
remain to be evaluated for retinal injury after optic nerve crush. This study was
designed to analyze changes in the expression of GluR1 and GluR2 following optic
nerve crush.
Methods: The optic nerve of the right eye of Wistar rats was crushed. Retinal
ganglion cells (RGCs) were retrogradely labeled by applying fluorogold onto the
left superior colliculus one week prior to crushing. Retinal injuries were induced by
optic nerve crush in rat eyes. Real-time PCR (qPCR) was used for measuring
changes in GluR1, GluR2, and -actin messages. Changes in glial fibrillary acidic
protein (GFAP) and GluRs expression were also followed in total retinal extracts
using western blotting.
Results: The mean number (SD) of RGCs labeled retrogradely from superior
colliculus was 2089.6 85.2/mm2 in rats without any treatment, which decreased
to 1090.8 77.6 (52.2%) and 496.6 87.3/mm2 (23.7%) on day 7 and 14,
respectively. GluR1 and GluR2 mRNA levels were decreased at 7 and 14 days.
While GFAP (a marker of astrogliosis) increased at 2, 7 and 14 days, GluR2
decreased significantly at 7 and 14 days (by 30%) as determined by western
blotting. Interestingly, glutamine synthetase initially increased at 2 days, but
decreased at 7 and 14 days post optic nerve crush as determined by western
blotting.
Conclusions: The reduced expression of GluR1 and GluR2 suggest dysfunctional
ion coupling in retina following optic nerve crush and likely impaired retinal
function. The increased GFAP suggests astrogliosis while the changes in glutamine
synthetase indicate abnormalities in Muller cells function. Further studies are
needed to identify the site of GluR1 and GluR2 changes and to characterize the
mechanism involved in down regulation of the glutamate receptors.
Commercial Relationships: Yong H. Park, None; Hidehiro Oku,
None; Masahiro Fukuhara, None; Takuji Kurimoto, None; Tsunehiko Ikeda,
None; Thomas Yorio, None; Adnan Dibas, None
Support: None
Transient Receptor Potential Vallinoid 1 Expression and Localization In
Rabbit and Human Eye
Carmen M. Martinez-Garcia, Sr.1, Covadonga Paneda, Sr.2, Patricia GallegoMuoz1, Ana I. Jimenez, Sr.2, Tamara Martinez2, Roberto Cantalapiedra1. 1Cell
Biology, University Of Valladolid, Valladolid, Spain; 2I+D Deparment, Sylentis,
Madrid, Spain.
Purpose: Transient receptor potential (TRP) is a superfamily of cation channels
permeable to calcium. It is activated by a wide range of stimuli such as heat
(>43C), low pH (<6.5), hypoxia, hypertonicity, etc. TRPV1 receptor expression in
the visual system has been localized in the epithelium and endothelium of the
cornea, in the ophthalmic branch of the trigeminal nerve and in the developing
retina. Our aim was to demonstrate the expression of this receptor in the whole eye.
Methods: Thirteen New Zealand White adult rabbits of approximately 2 Kg were
used in this study and five human eyes obtained from the pathology department.
Antibodies anti-human TRPV1 rabbit polyclonal antibody, anti-rabbit TRPV1
sheep polyclonal antibody and vimentine monoclonal antibody were used.
Total RNA was isolated from the following ocular structures: lacrimal gland,
ciliary body, retina and lens. Real time PCR was performed using StepOnePlus
detection system. The following gene-specific probes and primers available as
Taqman assays were used: Oc03397394_m1 (TRPV1) and 824823 B3 (HPRT1)
Results: TRPV1 protein was found in the corneal epithelium and endothelium and
in the basal layer of the conjunctiva. TRPV1 protein was also found in the epithelia
of the ciliary body, in the pars plana and plicata and epithelium of lens. In the
retina, the retinal pigmented epithelium and Mller cells in the inner layer of the
structure were found positive for this protein. At the mRNA level, TRPV1 was
found in the lacrimal gland, retina, ciliary body, lens and cornea. The highest
expression was found in the lens, whereas the lowest levels were found in the
retina.
Conclusions: The specific cell localization of TRPV1 in the epithelia and Mller
cell colocalizes with places very actively involved in exchange of ions and water
flow in the eye, thus a role of TRPV1 as osmosensor channel could be expected in
this particular cells.
Commercial Relationships: Carmen M. Martinez-Garcia, Sr., None;
Covadonga Paneda, Sr., Sylentis (E); Patricia Gallego-Muoz, None; Ana I.
Jimenez, Sr., Sylentis (I); Tamara Martinez, Sylentis (E); Roberto
Cantalapiedra, None
Support: CENIT CEYET
Paracellular Permeability In Corneal Endothelial Monolayers Of Varying
Density
Jorawer S. Singh1A, Joyce E. Young1B,2, Sangita P. Patel3,2. ASchool of Medicine,
B
Ophthalmology, 1University at Buffalo, Buffalo, NY; 2Research Service,
VAWNYHS, Buffalo, NY; 3Ophthalmology, University at Buffalo, SUNY Eye
Institute, Buffalo, NY.
Purpose: The paracellular pathway is a primary route for water movement across
the corneal endothelial monolayer. Although fluid influx is known to increase at
low monolayer densities, underlying changes in the paracellular pathway are not
well defined. This study investigates alterations in transendothelial electrical
resistance (TER) and diffusional paracellular permeability (Papp) in corneal
endothelial monolayers of varying endothelial cell density (ECD) to test the
hypothesis that monolayers of lower ECD have lower TER and greater Papp.
Methods: Bovine eyes were obtained from local abattoirs. Passage 1 bovine
corneal endothelial cells were seeded onto 0.33 cm2 permeable polycarbonate
transwell inserts in two different media at varying dilutions to obtain monolayers of
different ECD (DMEM + 10% FBS, 1:4, for ECD 1500-2999 cells/mm2; MEM +
0.5% FBS, 1:16, for ECD 1000-1499 cells/mm2). TER was monitored using an
epithelial voltohm meter in an Endohm chamber (World Precision Instruments,
Sarasota, FL) and resistance measurements for blank inserts were subtracted.
Cultures were determined to be confluent once TER plateaued. Confluent cultures
were normalized for 48 hrs in MEM + 0.5% FBS. Prior to the permeability assay,
cultures were equilibrated in physiologic Ringers solution for 30 minutes. Papp to
75 g/ml sodium fluorescein was measured by apical sample fluorophotometer
counts over 120 minutes following addition to the basolateral chamber. Papp was
calculated in cm/s as apical counts per time interval divided by basolateral counts
per volume times the area of the transwell aperture. Following the assay, transwell
membranes were removed, cells were fixed, nuclei were stained with DAPI, and
photographed with a fluorescence microscope to count ECD.
Results: Papp and TER data was grouped by ECD into 3 categories (category [mean
SD, n]): low, 1000-1499 cells/mm2 (1195 163, n = 12); mid, 1500-1999
cells/mm2 (1721 132, n = 5); and high, 2000-2999 cells/mm2 (2250 282, n =
11). Although groups had significantly different ECDs (One-Way ANOVA,
p<0.0001), no significant differences were observed in TER measurements or Papp
at 120 minutes. TER plateau values in ohms were: low ECD, 33.5 8.4; mid ECD,
31.8 13.9; and high ECD, 31.3 10.9. Papp measurements were (in cm/s): low
ECD, 9.07 2.64 10-5; mid ECD, 8.47 1.67 10-5; and high ECD, 9.54 3.68
10-5.
Conclusions: Within the range of 1000-2999 cells/mm2, the endothelial monolayer
demonstrates no significant change in TER or Papp. Despite the large range of ECD,
monolayer barrier integrity persists. In humans, corneal edema is not seen until
ECD falls below 1000 cells/mm2. This study supports that observation and
emphasizes the critical role of maintaining junctional integrity.
Commercial Relationships: Jorawer S. Singh, None; Joyce E. Young,
None; Sangita P. Patel, None
Support: Funded in part by Ralph Hochstetter Medical Research Fund in honor of
Dr. Henry C. and Bertha H. Buswell (SPP). Unrestricted Dept Challenge Grant
from Research to Prevent Blindness, NY, NY.
Acetazolamide Increases cAMP in Cultured Porcine Nonpigmented Ciliary
Epithelium and Elicits Subcellular Translocation of H+-ATPase
Amritlal Mandal, Mohammad Shahidullah, Nicholas A. Delamere. Physiology,
College of Medicine, Univ of Arizona, Tucson, AZ.
Purpose: The carbonic anhydrase inhibitor acetazolamide (ACTZ) reduces
aqueous humor (AH) secretion and intraocular pressure in species that do or do not
concentrate bicarbonate in the AH. Earlier studies showed ACTZ increased the
production of cAMP by rat renal cortical slices in vitro (Rodriguez HC et al. J.
Clin. Invest. 53:122-130, 1974). Because cAMP signaling in the ciliary body is
known to affect AH secretion, studies were carried out to examine the possibility
that ACTZ elicits a cAMP response in non pigmented ciliary epithelium (NPE).

Methods: Porcine NPE was established in primary culture according to our
published method. Using an approach based on centrifugation (Lin PH et al.
Biochemistry. 26 :731-736, 1987), a plasma membrane-rich fraction was isolated
from the NPE and used for western blot analysis of soluble adenylate cyclase (sAC)
and H+-ATPase (V-ATPase) abundance. cAMP was measured by RIA using a
commercial kit (Perkin Elmer).
Results: When cultured NPE cells were exposed to ACTZ (500M) for 10 min the
abundance of sAC protein detected in the plasma membrane-rich fraction was
doubled, suggesting subcellular sAC translocation. Cells exposed to ACTZ+IBMX
for 1 -10 min displayed a rapid increase in cAMP which peaked at 2 min and
remained significantly elevated for 10 min. ACTZ treatment for 10 min was found
to cause a significant increase in V-ATPase B1 subunit protein in the plasma
membrane-rich fraction, pointing to subcellular translocation of H+-ATPase. A
similar increase in V-ATPase B1 subunit protein was observed in the plasma
membrane-rich fraction obtained from cells exposed for 10 min to 8-Br-cAMP, a
cell permeable cAMP analog.
Conclusions: The findings suggest ACTZ increases cAMP in a response that may,
in part, involve activation of sAC. Subcellular translocation of H+-ATPase that
occurs in cells exposed to ACTZ appears to be a cAMP-dependent response. This
raises the possibility that some functional effects of carbonic anhydrase inhibitors
on the NPE may be related to cAMP signaling.
Commercial Relationships: Amritlal Mandal, None; Mohammad Shahidullah,
None; Nicholas A. Delamere, None
Transepithelial Iron Transport and Polarized Iron Reuptake in Retinal
Pigmented Epithelial Cells
Mary C. McGahan, Steven Nagar, Malgorzata Goralska, Lloyd N. Fleisher, Jill
Harned. Dept of Molec Biomed Sci, North Carolina State University, Raleigh, NC.
Purpose: Our earlier studies have shown that iron levels in the intraocular fluids
and the crystalline lens are carefully controlled by a functioning blood ocular
barrier (BOB) system. However, the mechanisms by which iron is taken up into the
eye and how intraocular levels of iron are regulated are not known. The purpose of
this study was to determine how iron is transported across the retinal pigmented
epithelium (RPE), which forms an important part of the BOB's.
Methods: Canine RPE were grown in transwell culture plates where the apical and
basolateral surfaces are separate. Formation of tight junctions was determined by
transepithelial resistance and polarization of cells determined by directional
secretion of glutamate. Tight junctional, polarized RPE were incubated with 59Fe
labeled transferrin on either the apical or basolateral sides and samples of media
collected from the opposite side at 1-24h were counted. At 24h, RPE were washed
with fresh media and efflux of iron determined on both sides at 6 and 24h.
Results: At 24h transepithelial iron movement was greater in the apical to
basolateral direction (5.6% of total iron loaded appeared in the basolateral
compartment) vs. basolateral to apical (1.6% of the total iron loaded appeared in
the apical compartment). Efflux measurements at 6h demonstrated a higher amount
of iron secreted into the basolateral (18.7% of total cell lysate iron) vs. apical
compartment (5.5%) of cells loaded with iron on the apical side. Interestingly, at
24h there is a loss of greater than 95% of the counts present at 6h in the basolateral
compartment of cells loaded with iron on the apical side, while there is a 5-fold
increase in iron in the apical compartment of these same cells at 24h.
Conclusions: There is polarized movement of iron across the RPE monolayer, with
greater movement of iron in the apical to basolateral direction. Most interesting are
the results of the efflux experiments where RPE loaded with iron from the apical
side initially (6h) had more iron enter the basolateral than apical compartment.
However, at 24h there is almost no measurable iron present in the basolateral
compartment, while iron continues to accumulate in apical compartment. This
result indicates that there is an increase in transferrin receptor on the basolateral
surface after iron loading which is responsible for the removal of iron from the
surrounding medium. Iron movement into and out of cells is a complex process
involving a number of different proteins. It is apparent from these results that there
is a dynamic polarized movement and localization of these components that is
altered by iron.
Commercial Relationships: Mary C. McGahan, None; Steven Nagar,
None; Malgorzata Goralska, None; Lloyd N. Fleisher, None; Jill Harned, None
Support: NIH EY-04900-29
479 Retina: Physiology, Pharmacology and Toxicology
Investigation Of New Dyes For Chromovitrectomy: Preclinical

Biocompatibility Of Trisodium, Orangell And Methil Violet
Rodrigo A. Souza-Lima, Sr.1, Emmerson Badaro1, Eduardo B. Rodrigues2, Milton
Moraes-Filho1, Mauricio Maia3, Fernando M. Penha4, Carsten H. Meyer1, Michel
E. Farah1. 1Ophthalmology, Federal University of Sao Paulo, Sao Paulo, Brazil;
2
Ophthalmology, Vision Institute, Florianopolis, Brazil; 3Ophthalmology, Paulista
School of Medicine, Assis, Brazil; 4Ophthalmology, Bascom Palmer Eye Institute,
Miami, FL.
Purpose: To investigate the retinal toxicity by Electroretinography (ERG) and
fundoscopy after intravitreal injection of the biological stains in two
concentrations: Trisodium (0.50 g/L and 1.00 g/L), Orangell (0.25 g/L and 1.00
g/L) and Methil Violet (1.00 g/L and 0.50 g/L).
Methods: Nineteen New Zealand Albinos rabbits were assigned in six groups (n =
3 in each group except for the group 5 n=4). The animals in group 1 received
Trisodium (8-Hydroxypyrene-1,3,6-Trisulfonic Acid Trisodium) in the dose of 0.50
g/L and group 2 received 1.00 g/L. Group 3 received Orangell in the dose of 0.25
g/L and group 4 received 1.00 g/L. Group 5 received Methil Violet in the dose of
1.00 g/L and group 6 received 0.50 g/L. A volume of 0,1 mL of dye was injected in
the right eyes, whereas the left eyes received the same volume of balanced salt
solution (BSS) as control. ERG recordings were performed at baseline and 7 days
after intravitreal injection. Ephios handheld system (Ephis AB, Rejmyre,
Sweden), ERG-jet and skin electrodes were used. Scotopic and fotopic curves were
measured (Fig 1). Amplitude of waves were obtained by transferred data to
software Mjolner v1.3:0.5. The responses at 1 week after injection were compared
with baseline levels. A decrease in the post-injection amplitude of more than 66%
was considered remarkable.
Results: At clinical examination by indirect ophthalmoscopy 7 days after dye
injection, all eyes were negative for cataract, hemorrhage, retinal detachment, and
intraocular opacities. Amplitude analysis of minimal scotopic b-wave (ROD) ,
maximum (MAX) scotopic a- and b-wave, photopic single flash cone b-wave
(SFC) and photopic 30 Hz flicker b-wave (FLI) showed no significant reduction in
either dye injected or control eyes. Descriptive comparison between dyes showed
that main amplitude decreasing, was in the Methil Violet groups (5 and 6) in both
scotopic an photopic recordings (Table 1). One rabbit in group 5 (Methil Violet
1.00g/L) was found dead 7 days after dye injection. No other adverse effects were
observed in our study.
Conclusions: Trisodium, Orangell and Methil Violet can be applied in future
studies in order to prove the capacity to stain preretinal membranes and vitreous
without toxicity. The three dyes did not induce significant ERG amplitude
reduction in this preliminary experimental research. Further histopathological
analysis may corroborate ERG amplitude analysis. Trisodium, Orangell and Methil
Violet may be promising vital dyes for ocular surgery.
Commercial Relationships: Rodrigo A. Souza-Lima, Sr., None; Emmerson
Badaro, None; Eduardo B. Rodrigues, None; Milton Moraes-Filho,
None; Mauricio Maia, None; Fernando M. Penha, None; Carsten H. Meyer,
None; Michel E. Farah, None
Support: None
The Effects of EPA and EPA/DHA Combination in Cultured Human RPE
Cells Under Oxidative Stress
Andrea R. Carvalho1, Anna Salas Torras1, Miguel Zapata2, Laura Distefano2,
Emilio Segovia2, Jose Garcia-Arumi2. 1Ophthalmology, Vall d'Hebron Research
Institute, Barcelona, Spain; 2Ophthalmology, Vall d'Hebron Hospital, Barcelona,
Spain.
Purpose: Previous studies demonstrated that omega-3 long-chain polyunsaturated
fatty acids within the eye have an anti-angiogenic, neuroprotective and antiinflammatory effects. As the first and most important cell population damaged in
AMD is the retinal pigment epithelium, the aim of this study was evaluate the
effect of eicosapentaenoic acid (EPA) and a combination of EPA and
docosahexaenoic acid (EPA/DHA) in ARPE-19 cultured cells under oxidative
stress.
Methods: To better mimic aged-RPE, cultured ARPE-19 cells were exposed to
10M of H2O2 for a week to induce adaptive response. Adapted cells were than
exposed to 300M of H2O2 with or without EPA (10, 50 and 100M) and
EPA/DHA (150, 75 and 37M final concentration) in 0.03% dimethyl sulphoxide
(DMSO) serum free media. It was evaluated cellular metabolism by alamar blue
assay, apoptosis using apoptag staining, nitric oxide formation (Griess assay), and
VEGF release in media by ELISA. Data represent the average of two or three
experiments SD. Statistical significance was determined by Students two-tailed
t-test. A p value less than 0.05 were considered significant.
Results: Viability results were expressed as an optical density percentage in
relation to the controls. Cellular viability curves were drawn up on 96-well plates
by showing about 20.000 cells per well. EPA and EPA/DHA treated cells showed
15% 3% and 27,4% 1% respectively more metabolic activity in a chemical
reduction of alamar blue than H2O2 treated cells; there were no statistical
difference between the different concentrations tested.

Immunofluorescence to detect apoptotic cells showed no difference in treated and
control groups regarding number of stained cells. NO2- in media was bellow limit
of detection (125pmol) in all samples tested.
VEGF levels in culture media after 3h of treatment were 41.7pg/mL 7.7;
30.2pg/mL 3.8; 16.9pg/mL 5.7; 62.9pg/mL 1.9; and 131.5pg/mL 3.9 for
EPA 10M, EPA 50M, EPA 100M, control and H2O2 300M treated cells
respectively. Statistical values between EPA treated cells with controls and H2O2
treated cells were significant in all concentrations. In EPA/DHA treated cells
VEGF levels were 53.27pg/mL 5.4; 45.57pg/mL 0.9; 39.8pg/mL 2.7;
64.2pg/mL 9.2; and 131.5pg/mL 3.9 for 37, 75, 150M final concentration of
EPA/DHA, control and H2O2 300M treated cells respectively. Only
concentrations of 75M (p= 0.029) and 150M (p= 0.04) showed statistical
significance in relation of H2O2 treated cells.
Conclusions: EPA and EPA/DHA improved metabolism of cells under oxidative
stress. EPA and EPA/DHA (higher concentrations) decreased VEGF release from
oxidative stressed cells. EPA and EPA/DHA can have a valuable anti-angiogenic
effect in AMD treatment.
Commercial Relationships: Andrea R. Carvalho, None; Anna Salas Torras,
None; Miguel Zapata, None; Laura Distefano, None; Emilio Segovia,
None; Jose Garcia-Arumi, None
Support: None
Treating X-linked Juvenile Retinoschisis with Carbonic Anhydrase Inhibitors
Jonathan Y. Chi1, Ian M. MacDonald2. 1UBC MD Undegraduate Program,
Vancouver, BC, Canada; 2Ophthalmology, Royal Alexandra Hospital, Edmonton,
AB, Canada.
Purpose: X-linked juvenile retinoschisis (XLRS) is an X-linked bilateral
vitreoretinal dystrophy, characterized by areas of macular schisis or splitting of the
neural layers of the retina resulting in cystic cavities. The condition affects from
1:5000 to 1:25000. Typically, visual acuity of affected males ranges from 20/60 to
20/120. XLRS results from defective or absent retinoschisin, a cell-adhesion and
cell-cell interaction protein secreted by retinal neurons. Currently, there are no
highly effective medical or surgical treatments for the condition. The purpose of
this case report is to document the use of a carbonic anyhydrase inhibitor as a
medical treatment modality and describe its efficacy in restoring retinal anatomy
and function.
Methods: Dorzolamide (a carbonic anyhydrase inhibitor) 2% eye drops twice daily
were prescribed to a patient with XLRS. Spectral-domain optical coherence
tomography (OCT) (Spectralis) and multifocal electroretinogram (mfERG) were
used to assess retinal anatomy pre- and post-treatment. mfERG responses from
both eyes were recorded according to ISCEV standards.
Results: We report the case of a 31 year old male with a family history of
retinoschisis. Fundus examination showed foveal stellate cystic changes OU and an
infero-temporal region of retinoschisis OD. Pinhole corrected vision to 20/80 OU.
OCT revealed schisis cavities and cystoid changes in the retina in both eyes.
Dorzolamide drops were initiated in both eyes. Two months post-treatment, the
OCT of the both eyes showed central flattening in the retina. Six months posttreatment pinhole had improved to 20/60-2 OD and 20/70-1 OS. The patient noted
subjectively brighter vision and a wider field after dorzolamide treatment.
mfERG amplitudes and latencies also improved over five months of treatment. On
average, mfERG amplitudes increased 248.6%, 5.8%, and 53.6% in rings 1, 2, and
3, respectively. On average, mfERG latencies became faster by 24.8%, 1.4%, and
3.5%, respectively.
Conclusions: A measurable degree of flattening of the central retinoschisis was
seen on OCT after two months of dorzolamide treatment. Vision improved from
20/80 OU pinhole to 20/60-2 OD and 20/70-1 OS pinhole. mfERG responses also
improved after five months of treatment. There have been several previous reports
of dorzolamide flattening schisis cavities in XLRS. This report is the first to
describe an improvement in the mfERG after the use of dorzolamide in XLRS.
Commercial Relationships: Jonathan Y. Chi, None; Ian M. MacDonald, None
Support: None
Cntf Induces A Transient Restoration Of Cone Function And Day Vision In
Cngb3-achromatopsia
Andras M. Komaromy1,2, Simone Iwabe1, Jessica S. Rowlan1, Alison D. Duncan1,
Shilpa Rao3, Annie Oh1,4, Rong Wen5, Gustavo D. Aguirre1. 1Clinical Studies, Univ
of Pennsylvania, Sch of Vet Med, Philadelphia, PA; 2Coll Vet Med, Michigan State
Univ, East Lansing, MI; 3Microarray Facility-Bioinformatics Group, Univ of
Pennsylvania, Philadelphia, PA; 4Coll Vet Med, Western University of Health
Sciences, Pomona, CA; 5Bascom Palmer Eye Inst, University of Miami, Miami,
FL.
Purpose: Intravitreal bolus injection of CNTF leads to deconstruction and
regeneration of photoreceptor outer segments in normal dogs, and improves the
success rate of viral CNGB3 gene replacement therapy in older dogs (> 1 year) with
CNGB3-achromatopsia. The present work evaluates the effect of intravitreal CNTF
alone on retinal gene expression and function in dogs with CNGB3-achromatopsia.
Methods: Five CNGB3-mutant dogs (age: 10 months) and 5 normal control dogs
(age: 8 months) were injected unilaterally with 12 g of intravitreal CNTF; the
contralateral eyes served as controls and were injected with PBS. ERGs were
recorded at baseline and at 1 week post injection, when retinas were collected for
RNA extraction. Retinal gene expression was evaluated by qRT-PCR and Agilent
Canine Oligo Microarray, containing 42,034 60-mer oligonucleotide probes. Three
additional mutant dogs (ages 10, 13 and 45 months) were treated with intravitreal
CNTF and followed by ERG, visual behavior testing, and gene and protein
expression evaluated by qRT-PCR and IHC at 1, 7, and 94 weeks post treatment.
Results: CNTF treatment severely reduced ERG amplitudes at 1 week in both
normal and CNGB3-mutant dogs. However, cone function and day vision were
transiently restored in the mutant dogs. At 5-weeks following treatment, scotopic
retinal function normalized but cone function and day vision deteriorated back to
baseline. In both normal and mutant retinas, these functional observations were
associated with marked, but transient shortening of photoreceptor outer segments
and decrease in photoreceptor-specific gene expression at 1 week. The most upregulated genes at 1 week post CNTF included FGG, C3, and AQP3; examples of
the most down-regulated genes were ARR3 and GUCA1A.
Conclusions: The CNTF-induced changes in photoreceptor morphology and retinal
gene expression are similar between normal and CNGB3-mutant retinas at 1 week.
However, mutant retinas transiently regain cone function reminiscent of the 3-4
week old developing CNGB3-mutant retina. Since functional beta subunits of the
cone CNG channels are lacking, CNTF might have facilitated the alpha subunits to
form functional homotetrameric channels in the cone outer segments, resulting in
restored cone function.
Commercial Relationships: Andras M. Komaromy, None; Simone Iwabe,
None; Jessica S. Rowlan, None; Alison D. Duncan, None; Shilpa Rao,
None; Annie Oh, None; Rong Wen, Neurotech USA (C); Gustavo D. Aguirre,
None
Support: Supported by NIH Grants EY006855, 17549, 18586, 19304, K12EY015398, P30-EY001583, P30EY14801, RPB, FFB, Hope for Vision.
Cytotoxicity Of Ketorolac Tromethamine 0.4% Ophthalmic Solution In
ARPE-19 and R28 Cells In Vitro
Ian H. Chan1, Maria F. Estrago-Franco2, Rhina M. Piche Lopez3, Gail M. Seigel4,
Cristina M. Kenney5, Baruch D. Kuppermann6. 1Ophthalmology, Gavin Herbert
Eye Institute UC Irvine Medical Center, Irvine, CA; 2Ophthalmology, Clinica Dres
Estrago, Corrientes, Argentina; 3Ophthalmology, University of California, Irvine,
Fountain Valley, CA; 4Center for Hearing and Deafness, University of Buffalo,
Buffalo, NY; 5Ophthalmology, Univ of California-Irvine, Irvine, CA; 6Gavin
Herbert Eye Inst Dept Ophthal, University of California Irvine, Irvine, CA.
Purpose: To study effects of Ketorolac Tromethamine (KT) ophthalmic solution
0.4% (Acular LS, Allergan, Irvine, CA) in human ARPE-19 and rat neurosensory
(R28) cells in vitro.
Methods: ARPE-19 & R28 cells were treated for 24 hours with 400g/ml(4X),
200g/ml(2X),100g/ml(X), 50g/ml(1/2X) and 25g/ml(1/4X) of KT. Cell
viability (CV) was measured using the trypan blue dye-exclusion assay.
Mitochondrial membrane potential (m) was measured using JC-1 assay kit.
Mitochondrial dehydrogenase (MD) activity was determined using WST-1
colorimetric assay. Activity of caspase 3-7 was measured by fluorescence caspase
kit.
Results: The mean CV of ARPE-19 and R28 cells cells was reduced after 24 hour
exposure to 4X dose of KT, (38.311.2%,[p<0.001] versus 96.21.5% for the
untreated cells for ARPE-19 ) while for the R28 cells it was 64.412.5,[p<0.001]
versus 89.45.1% for the untreated cells. KT reduced the m of ARPE-19 cells at
the 4X dose, (0.690.04, [p<0.001] versus 1.10.1 for untreated controls). There
was no significant change in m of R28 at any of doses. The MD activity was
reduced in the ARPE-19 cells at the 4X dose only, (0.0460.002, [p<0.01] versus
0.650.1 for untreated controls) while the MD activities of R28 cells were reduced
in the 4X, 2X and X doses of KT, (0.0460.02,[p<0.001], 0.070.02,[p<0.001] and
0.430.05,[p<0.001] respectively versus 0.60.1 for the untreated controls). There
was no significant increase in caspase 3-7 activity in either cell line at any dose of
KT.
Conclusions: Ketorolac tromethamine decreases the cell viability of ARPE-19 and
R28 cells at highest tested doses. As seen with the JC-1 and MD assays, KT
appears to have variable toxicity to the mitochondria .
Commercial Relationships: Ian H. Chan, None; Maria F. Estrago-Franco,
None; Rhina M. Piche Lopez, None; Gail M. Seigel, None; Cristina M. Kenney,
None; Baruch D. Kuppermann, Allergan (C)
Support: Discovery Eye Foundation, Polly & Michael Smith Foundation,
Beckman Initiative for Macular Research, Lincy Foundation, Iris & B. Gerald
Cantor Foundation, Research to Prevent Blindness

Ocular Distribution of the Visual Cycle Modulator (VCM) 14C-ACU-4429 in
Beagle Dogs
Terry Podoll1, Suliman Al-Fayoumi1, Elizabeth Prescott2, Gang Sun2, John W.
Chandler1, Ryo Kubota1. 1Acucela Inc., Seattle, WA; 2Covance Laboratories Inc.,
Madison, WI.
Purpose: To evaluate the ocular distribution and characterize the pharmacokinetic
profile of ACU-4429, a novel VCM, and its metabolites in beagle dogs after single
and repeated oral doses.
Methods: Oral 14C-ACU-4429 0.3 mg/kg (40 Ci/kg/day) was administered as a
single dose or repeated doses (once daily for 7 days) to a total of 36 male beagle
dogs. Mass balance was assessed through 168 hours after a single dose or through
336 hours after the last repeated dose. Blood was collected at multiple timepoints
up to 192 hours following the final dose; blood and plasma were analyzed for
radioactivity and metabolic profiling. Ocular tissues were collected at multiple
timepoints up to 168 hours after the final dose and analyzed for radioactivity (right
eyes) or metabolic profiling (left eyes).
Results: On average, 95% of the administered dose of radioactivity was recovered,
including 46% in urine and 44% in feces. Radio-HPLC analyses detected 29 peaks
of radioactivity in urine, 18 peaks in feces, and 38 peaks in plasma, indicating
ACU-4429 was extensively metabolized after oral administration in dogs; overall,
the parent compound accounted for <2% of radioactivity in excreta. In plasma, the
fraction of parent compound relative to total radioactivity was <1%. The metabolic
profile in plasma was qualitatively similar to that observed in completed rat and
human radiolabel ADME studies. Among ocular tissues, the highest levels of
radioactivity were present in melanin-containing tissues (iris-ciliary body, choroid,
and RPE). Association of ACU-4429 with melanin was thought to be reversible, as
radioactivity levels in these tissues declined over time. In RPE, Cmax of total
radioactivity occurred at 12 hours post-dose, compared to 4 hours in plasma. In
RPE, parent compound accounted for >90% of total radioactivity at all timepoints,
and Cmax was 234-fold higher than in plasma (457 vs 1.95 ngeq/g) after a single
dose of 14C-ACU-4429.
Conclusions: In beagle dogs, orally administered 14C-ACU-4429 was
preferentially distributed to melanin-containing ocular tissues, including RPE, the
proposed site of action for ACU-4429. ACU-4429 exposure was markedly higher
in RPE relative to plasma, supporting specific targeting of RPE by orally
administered ACU-4429.
Commercial Relationships: Terry Podoll, Acucela Inc. (E); Suliman AlFayoumi, Acucela Inc. (E); Elizabeth Prescott, Acucela Inc. (F); Gang Sun,
Acucela Inc. (F); John W. Chandler, Acucela Inc. (E); Ryo Kubota, Acucela Inc.
(E)
Support: None
Proteoglycans Gene Expression Analysis In Developing Rat Retina Explants
Sumitted Bevacizumab
Paloma G. Krempel1A, Monique Matsuda1A, Alfred Sholl-Franco2A, Andr Luis F.
Portes3, Mnica V. Marquezini1B, Thiago Puntar2B, Nadia Campos O. Miguel2B,
Mario L. Monteiro1A. ALaboratory of Ophthalmology, BLaboratory of Experimental
Air Pollution, 1University of Sao Paulo, Sao Paulo, Brazil; ANeurobiology
Program, BCell Biology and Development Program, 2Federal University of Rio de
Janeiro, Rio de Janeiro, Brazil; 3Retina and Vitreous Service, Bonsucesso General
Hospital, Rio de Janeiro, Brazil.
Purpose: Bevacizumab (BVZ), an anti-vascular endothelial growth factor (VEGF)
agent has been used to treat several vasoproliferative disorders in adult eyes, and
more recently, in developing eyes of neonates with Retinopathy of Prematurity.
Extracellular matrix and cellular membrane proteoglycans (PG) are essentials for
retinal cell development as part of control mechanisms of neuron differentiation,
neurite and axon outgrowth, neural cell adhesion and apoptosis. Chondroitin
(CSPG) and Heparan Sulfated Proteoglycans (HSPG) are the most abundant PG in
the developing nervous central system as retina. Neurocan, a chondroitin sulfated
PG, is an extracellular matrix molecule that can inhibit neurite and axon outgrowth.
On the other hand, syndencan-3, a cellular membrane HSPG has the opposite
function: stimulate axonal and neurite growth by binding with basis Fibroblast
Growth Factor. The purpose of this research is to investigate the effects of BVZ on
extracellular matrix and/or cellular membrane PGs during rat retina development.
Methods: Thirty 2-days-old Lister Hooded rats were euthanized and had their
retinas dissected and cut in 1mm explants. Retinal tissue was divided in two
groups: control and treated (experimental group) with 0.5 mg/mL of BVZ for 48
hours. After incubation, retinas were analyzed with Real Time RT-PCR for mRNA
evaluation for: neurocan and syndecan-3, using SYBR Green. The mRNA
expression analysis was done with relative comparison method (Ct) and data
were analyzed by Rotor Gene ScreenClust HRM (Qiagen) software. The amount of
neurocan and syndecan-3 mRNA was calculated relative to the amount of
ribosomal protein ARBP as reference gene. Comparisons between the groups were
performed using the Students unpaired, considering a p value less than 0.05 as
statistically significant.
Results: According to mRNA expression analysis, similar patterns were observed
for the controls and experimental group for neurocan mRNA content. However, a
significant decrease in syndecan-3 mRNA level, about 6.5 times lower, was
observed in the BVZ-treated group compared to controls.
Conclusions: Our results demonstrated that BVZ could alter PG gene expression of
syndecan-3, therefore could affect retinal cells differentiation during development,
since syndecan-3 is important in neurite and axon outgrowth for adequate
formation of neural networks in retina. However, more studies are necessary to
investigate BVZ action, and suggest caution of its use, particularly on developing
retina, as currently used in children with retinopathy of prematurity.
Commercial Relationships: Paloma G. Krempel, None; Monique Matsuda,
None; Alfred Sholl-Franco, None; Andr Luis F. Portes, None; Mnica V.
Marquezini, None; Thiago Puntar, None; Nadia Campos O. Miguel,
None; Mario L. Monteiro, None
Support: FAPESP 2011/12271-3
Visual and Anatomic Findings after Discontinuation of Hydroxychloroquine
in Patients Diagnosed with Toxicity
Brandon Wong1, Lee M. Jampol2, Marie Brenner2, Alice T. Lyon2, Alfredo Sadun1,
Amani A. Fawzi1. 1Ophthalmology, Doheny Eye Institute, Keck School of
Medicine, USC, Los Angeles, CA; 2Ophthalmology, Northwestern University,
Chicago, IL.
Purpose: Hydroxychloroquine (HCQ) toxicity can be associated with poor visual
prognosis if discontinued after the appearance of visible retinal changes. The
purpose of this study is to report visual and anatomic findings in patients who
discontinued HCQ due to toxicity.
Methods: Retrospective review of patients who stopped HCQ due to toxicity
identified 14 patients of whom 7 patients at Doheny and 4 at Northwestern had
available follow up examinations using Humphrey visual field (HVF), fundus
autofluorescence (FAF), and/or spectral domain optical coherence tomography
(SD-OCT).
Results: Mean patient age was 48.5 years (range 22-80), treatment duration 7.70
5.96 years, cumulative dose 914.2 870.6 grams, duration of follow up after
toxicity diagnosis 11.5 months (range 1-36). At baseline, average HVF 10-2 defect
depth was 6.71 10.47 dB, HVF 30-2 mean defect (MD, -6.93 30.33 dB) and
pattern standard deviation (PSD, -17.80 4.63 dB). Final follow up HVF 10-2
defect depth was 6.12 9.88 dB (compared to baseline, p = 0.14), HVF 30-2 MD
was -6.33 24.92 dB (p = 0.5), and HVF 30-2 PSD was 17.50 4.27 dB (p =
0.45). Of 11 patients with follow up, 8 were symptomatic. Of these, 5 patients had
serial SD-OCT after stopping HCQ. Comparing baseline to 6 months follow-up
showed thickening of the RPE and areas of re-establishment of the external limiting
membrane and the inner/outer segment junction. (Figure) Two asymptomatic
patients were diagnosed based on FAF abnormalities (normal SD-OCT and HVF
10-2) and one was diagnosed based on SD-OCT. None showed changes in FAF,
SD-OCT, or HVF 10-2 after 1, 15, and 14 months of follow up, respectively.
Conclusions: Patients with clinically visible lesions in HCQ toxicity showed
borderline statistically significant visual improvement following drug cessation.
They also showed evidence of outer retinal remodeling early after discontinuing
HCQ on SD-OCT. Longer follow-up is needed for further functional
improvements, if any. Patients without clinically visible lesions showed no
improvements or worsening on FAF or SD-OCT. Our findings emphasize the
importance of physician vigilance and early detection of HCQ toxicity using newer
modality imaging approaches and long-term follow up after cessation.

Commercial Relationships: Brandon Wong, None; Lee M. Jampol,
None; Marie Brenner, None; Alice T. Lyon, None; Alfredo Sadun,
None; Amani A. Fawzi, None
Support: None
Development of Repletion of Intracellular Glutathione, Intact, for AMD and
Diabetic Retinopathy, Orally, with Advanced-Glutathione
Harry B. Demopoulos. Cell.Redox Corp, Elmsford, NY.
Purpose: The need for glutathione (GSH) in Ophthalmology is well known. Its
use, however, was delayed because exogenous, ordinary glutathione does not cross
cell membranes, and in addition, the unstable thiol undergoes oxidative
desulferation, leaving injurious Ophthalmic Acid. We have succeeded in
developing an Advanced-Glutathione that avoids both issues.
Methods: AMD and Diabetic Retinopathy display uncontrolled free radical
reactions, initiated (1)in AMD by blue light excitation of deposits in RPE, tobacco
smoke, and dysfunction of the Complement System, and (2) in Diabetes by
hyperglycemia. These destroy intracellular glutathione, causing declines: in Redox
Potential, and Antioxidant Defenses of RPE, Neuro-Retina, Endothelial Cells, and
Vascular Smooth Muscle. The direct free radical damage includes: peroxidation of
membrane lipids and proteins; apoptosis of RPE cells with loss of dependent rods;
decreased production of Pigment Epithelium Derived Factor (PEDF); free radical
promotion of pathologic angio-genesis and of Matrix Metalloproteinases. We chose
as a logical model of long-term, progressive, human losses of intracellular
glutathione, non-symptomatic, HIV-positive individuals. Progressive, Human
losses are documented in the Peripheral Blood Mononuclear Cells (PBMC's) which
correlate with declining Clinical status. We used 3 doses:: 1, 2 and 3 gm/d, orally,
in an 8 week period, involving timed phlebotomies to secure the points for Area
Under the Curve (AUC) analyses. A total of 10,000 GSH assays were conducted.
Results: Pharmacokinetics of this drug showed a significant dose-response in
rapidly repleting intact glutathione in PBMC's, doubling over baseline; p value
0.0005, based on AUC's. There were no safety issues.
Conclusions: The drug is planned for Phase 2,3 Clinical Trials in AMD and
Diabetic Retinopathy, with proposed End Points that include, among others, the
Choroidal Neo Vascularization of AMD, and Proliferative Diabetic Retinopathy.
Free Radical Pathology, Oxidative Stress and Glutathione insufficiency promote
pathologic angiogenesis.
Commercial Relationships: Harry B. Demopoulos, Cell. Redox Corporation
(E)
Support: None
Ocular Safety of Dasatinib in a Rabbit Model
Alfred K. Yu1A, Shawn A. Morales2, Krisztina I. Forward3, Lynn K. Gordon4, David
G. Telander1B. AOphthalmology & Vision Science, BOphthalmology, 1University of
California, Davis, Sacramento, CA; 2Ophthalmology, University of California, Los
Angeles, Pasadena, CA; 3Ophthalmology, Univ of California, Davis Sch of Med,
Davis, CA; 4Jules Stein Eye Inst, Univ of California-Los Angeles, Los Angeles,
CA.
Purpose: Dasatinib is a pharmacological inhibitor of the Src family of kinases. Src
is the key kinase regulating focal adhesion kinase (FAK) activation. FAK has an
important role in the integrin signaling cascade involved in cell adhesion,
migration, and invasion. Expression of some of these integrins has been found to be
involved in retinal disease. The purpose of this study was to determine if Dasatinib
is toxic in the rabbit eye after intraocular injection. Toxicity of Dasatinib was
assessed for its potential therapeutic use in retinal disease.
Methods: Eight New Zealand Albino rabbits were placed in four treatment groups
(2 animals each) receiving dosages of 300nM, 450nM, 600nM, and 900nM of
Dasatinib. Rabbits were anesthetized using ketamine hydrochloride (50 mg/kg),
xylazine (5 mg/kg), and acepromazine (0.5 mg/kg) administered subcutaneously 15
minutes before the procedure. One drop each of 1% tropicamide and 2.5%
phenylephrine hydrochloride was applied topically to achieve pupillary dilation.
One drop of proparacaine 0.5% was instilled in each eye before treatment to act as
an anesthetic. Rabbits received a dose of Dasatinib intraocularly every week for 6
weeks. Rabbits underwent an indirect ophthalmoscopy examine before each
injection. Electroretinogram (ERG) was performed at week 1 and week 4. Rabbits
were dark adapted for 1 hour prior to ERG. After rabbits were euthanized eyes
were processed and embedded in paraffin for histological examination.
Results: Upon subjective appraisal of the ERG waveforms, the functional integrity
of the retina remained fully intact. When comparing baseline b-wave amplitudes
and b-wave amplitudes at 4 weeks there were no significant changes to the b-wave
amplitude or the b-wave latency time. This is apparent in both scotopic and
photopic ERG responses.
Conclusions: ERG assessment of Dasatinib toxicity revealed that Dasatinib does
not alter retinal function. Cone and rod function remained stable. Dasatinib can be a
useful therapeutic agent in treating retinal diseases such as proliferative

vitreoretinopathy and proliferative diabetic retinopathy.
Commercial Relationships: Alfred K. Yu, None; Shawn A. Morales,
None; Krisztina I. Forward, None; Lynn K. Gordon, None; David G. Telander,
None
Support: NIH Grant EY019909 (DGT, LKG)
Effects Of Light Exposure, pH, Osmolarity And Solvent On The Retinal
Toxicity Of Vital Dyes In Chromovitrectomy
Elaine F. Costa1, Nilana Barros1, Fernando M. Penha1, Eduardo Dib1, Eduardo B.
Rodrigues1, Octaviano Magalhes, Jr.1, Milton N. Moraes-Filho1, Carsten Meyer2,
Mauricio Maia1, Michel E. Farah1. 1Ophthalmology, Federal Univ of Sao Paulo,
Sao Paulo, Brazil; 2Ophthalmology, University of Bonn, Bonn, Germany.
Purpose: To investigate the influence of pH, osmolarity, solvent and dye-light
interaction in currently use and novel dyes in order to identify the deleterious
effects of dye-related retinal toxicity.
Methods: Cultured cells derived from human retinal pigment epithelium (RPE),
ARPE-19 were exposed to seven different BSS solutions with and without trypan
blue (TB) 0.5 mg/ml with adjusted values of pH (4; 5; 6; 7; 7.5; 8 and 9), with
osmolarity ranging from 317-345 mOsm, and to other seven glucose solutions with
and without TB 0.5 mg/ml (2.5; 5.0; 10; 20; 30; 40; 50%), with osmolarity ranging
from 142 to 2530 mOsm with pH of 7.0-7.6, for 10 minutes. Mannitol solutions
were used as an osmotic control agent. ARPE -19 cells were also incubated with
0.05 mg/ml of six dyes diluted in BSS - TB, brilliant blue (BriB), bromophenol
blue (BroB), fast green (FG), light green (LG) and indigo carmine (IC) - in the
presence of two different light sources (high brightness xenon and mercury vapor)
for 10 minutes to evaluate dye-light interaction. Cell viability was asessed using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay. The
ANOVA statistical test was used to compare the cell viability results.
Results: Solutions with a non-physiological pH, below 7 and above 7.5, proved to
be remarkable toxic to RPE cells even without TB. All solutions containing glucose
were significantly more deleterious than the BSS control (p<0.001) even in an isoosmolar range. Mannitol solutions did not lead to any harm effect in all solutions
tested. Among tested dyes, only LG and FG were toxic to RPE cells (p<0.001).
Light exposure did not increase retinal toxicity either with the xenon light or with
the mercury vapor lamp. Evaluating dye light-interaction,TB was statically
different from BriB when exposed to mercury light; also, IC had a decrease in cell
viability compared to BriB with xenon light.
Conclusions: Several factors may contribute to increase the toxicity effect of the
dye in the retina. Non-physiologic values of pH and the use of glucose as a solvent
appear to be more deleterious to the RPE cells than the dye itself. Light exposure
with the stained retina should not be a concern when xenon or mercury vapor is
used as light source and specially when applied in a short period of time.
Commercial Relationships: Elaine F. Costa, None; Nilana Barros,
None; Fernando M. Penha, None; Eduardo Dib, None; Eduardo B. Rodrigues,
None; Octaviano Magalhes, Jr., None; Milton N. Moraes-Filho, None; Carsten
Meyer, None; Mauricio Maia, None; Michel E. Farah, None
Support: None
Evaluation Of The Surgical Behaviour And Toxicity Of Colored
Perfluorocarbon Liquids In Pig Eyes
Miguel A. Zapata1, Fabio Trinidade1, Andrea Carvalho1, Anna Salas1, Laura
Distefano1, Jose Garcia-Arumi2. 1Ophthalmology, Hospital Vall Hebron,
Barcelona, Spain; 2Ophthalmology, Hospital Vall dHebron, Barcelona, Spain.
Purpose: The perfluorocarbon liquids (PFCLs) are widely used in vitreoretinal
surgery due to their physicochemical properties. Although theyre useful
complications have been reported mainly related to their intraocular retention,
which can be attributed to the transparent nature of thesen compounds. The use of
colored PFCLs would help in a more complete and safe removal of these
substances.
Methods: Twenty-eight eyes of 14 hybrid pigs (15Kg) were used in this
experiment. Intraoperative behaviour and toxicity of 2 colored PFCLs (blue and
yellow) were evaluated by performing 23G pars plana vitrectomy. In the control
group transparent perfluoro-n-octane (PFnO) was used. PFCLs were left in the
vitreous cavity for one week according to different surgical protocols: a) 1 - 1.5 ml
in a balanced salt solution (BSS) filled eye; b) 0.2 ml with air-fluid exchange at the
end; c) 0.1 ml subretinaly. Colour fundus photography, fluorescein angiography
and electroretinography (ERG) were performed in all eyes before surgery and
euthanasia. One week after surgery eyes were enucleated and fixed in 4%
formaldehyde solution, for paraffin inclusion. Serial cuts of 3 m were stained in
standard hematoxiline/eosine to evaluate retinal integrity, number of retinal
ganglionar cells, including life and dead cells, nerve fiber layer changes, inner
retina vascular pattern and correlation of retina and NFL thickness. To evaluate

apoptosis deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) was also
performed.
Results: During the exchanges and under BSS, colored PFCLs were superior to the
transparent PFnO. On retinal surface visualization the transparent PFCL was better
but both colored PFCLs studied didnt affect significantly its manipulation. Blue
colored PFCL was better seen in the subretinal space than the yellow and the
transparent PFnO. Colored PFCLs showed no histological changes when compared
to the transparent PFnO. There were no remarkable changes in the ERGs of the
compared groups. TUNEL assay was negative in all samples.
Conclusions: Colored PFCLs facilitate the view during air exchanges and
generally provide a better visualization and blue colored PFCL was also better
visualized in the subretinal space, which can help to minimize its intraocular
retention.
Commercial Relationships: Miguel A. Zapata, None; Fabio Trinidade,
None; Andrea Carvalho, None; Anna Salas, None; Laura Distefano, None; Jose
Garcia-Arumi, None
Support: None
Intravitreal Injection of Integrin Peptide ALG-1001 for the Induction of
Posterior Vitreous Detachment in Rabbit Eyes
Hugo E. Sepulveda-Vazquez1A, Jose Luis Guerrero-Naranjo1A, Fernando
Schoonewolff1A, Hugo Quiroz-Mercado2, Hampar L. Karageozian3, John Y. Park4A,
Vicken Karageozian3A, Sonia Corredor1B, Abelardo Rodriguez-Reyes1B, Marc
Kirshbaum4. ARetina, BPathology, 1Assoc Para Evitar la Ceguera en Mexico, IAP,
Mexico, Mexico; 2Ophthalmology, Ophthalmology, Denver Health Medical Center,
Denver Health Medical Center, CO; AAllegro, 3S K Pharmaceuticals, Inc, San Juan
Capistrano, CA; AR & D, 4Allegro Pharmaceuticals, Inc, Santa Ana, CA.
Purpose: To evaluate the safety and assess the effect of intravitreal injections of
ALG-1001 on the vitreoretinal junction for a facilitating effect on posterior vitreous
detachment (PVD) in rabbit eyes.
Methods: Prospective, preclinical trial. A first group of five New Zealand white
rabbits received 100 L intravitreal injections of 2.5 mg of ALG-1001 Integrin
Peptide in their right eyes. A second group of four New Zealand white rabbits
receive 100 L intravitreal injections of 2.5 mg of ALG-1001 Integrin Peptide plus
0.02% Disodium EDTA solution in their right eyes. A third group of four New
Zealand white rabbits received 100 L intravitreal injections of balanced salt
solution (BSS) in their right eyes (control group). No rabbit received intravitreal
injections in their left eye. Slit lamp examination, dilated fundus exam,
electroretinogram (ERG) and ultrasonography (USG) were performed before and
one day after the injections in all eyes. Adverse events were evaluated. Vitrectomy
was performed in all eyes one day after the injection to evaluate the presence of
posterior vitreous detachment and vitreous liquefaction in all treated eyes. All
rabbits were euthanized after vitrectomy, eyes enucleated and light and scanning
electron microscopy performed.
Results: In the first group PVD occurred in all eyes injected (100%), 3 of them
were PVD grade 3, one grade 2 and one grade 1; vitreous liquefaction was found
during vitrectomy in 4 of 5 eyes (80%). In the second group with combined drugs
we found PVD in four injected eyes (100%), 2 eyes with grade 3 PVD and 2 eyes
with grade 1; vitreous liquefaction was found in 2 out of 4 eyes (50%). In the third
group injected with BSS we found PVD in only 2 eyes (50%), 1 eye with stage 2
PVD and one with stage 1, 2 eyes presented no PVD. We observed vitreous
liquefaction in only 1 of 4 eyes in this control group (25%). PVD was not observed
during the USG in the left eye of any rabbit.
Conclusions: Intravitreal injection of ALG-1001 integrin peptide seems to be a
safe procedure for producing total PVDs, with no adverse effects. Integrin peptides
could be useful as an adjunct for vitrectomy. Studies with larger sample sizes are
needed to confirm these findings.
Commercial Relationships: Hugo E. Sepulveda-Vazquez, None; Jose Luis
Guerrero-Naranjo, None; Fernando Schoonewolff, None; Hugo QuirozMercado, Consultor (C); Hampar L. Karageozian, Employee Allegro
Ophthalmics (E); John Y. Park, Employee Allegro Ophthalmics (E); Vicken
Karageozian, Employee Allegro Ophthalmics (E); Sonia Corredor,
None; Abelardo Rodriguez-Reyes, None; Marc Kirshbaum, Employee Allegro
Ophthalmics (E)
Support: None
Non-Invasive Monitoring of Changes in the Rat Retina Induced by Nicotine
Toxicity and Diabetes Mellitus
Nima Tirgan1A, Adam Boretsky2, Praveena Gupta1B, Bernard F. Godley1C, Ronald
G. Tilton1D, Massoud Motamedi1E. ADepartment of Ophthalmology, BOphtha &
Visual Sci, COphthal & Visual Sciences, D3Department of Internal Medicine,
Division of Endocrinology, EOphthalmology, 1Univ of Texas Medical Branch,
Galveston, TX; 2Ctr for Biomed Engineering, Univ of Texas Medical Branch,
Houston, TX.
Purpose: To non-invasively investigate the effects of nicotine and early stage
diabetes on retinal structure using spectral domain optical coherence tomography in
an established rodent model.
Methods: Sprague-Dawley rats (n=45) were examined using a combination of
confocal scanning laser ophthalmoscopy and spectral domain optical coherence
tomography (Spectralis, Heidelberg Engineering) to determine changes in retinal
structure in response to nicotine exposure (initiating at 0.3mg/kg to a final dose of
2.1 mg/kg), diabetes (induced with 60 mg/kg body weight streptozotocin) and the
combined effects of nicotine exposure and diabetes. Retinal thickness in superior,
inferior, nasal and temporal quadrants were determined based on SD-OCT volume
scans (20x20) centered on the optic disc. Segmentation of discrete retinal layers
was performed on a subset of SD-OCT cross-sections to further examine changes
in individual layers from each treatment group.
Results: The control group did not experience any significant change throughout
the study. The nicotine treatment group experienced an average decrease in total
retinal thickness (TRT) of 9.4 m with the majority of the loss localized within the
outer nuclear layer. Although the results were not statistically significant, the
diabetic group exhibited a trend toward decreasing TRT. However, segmentation
did reveal significant thinning within the ONL of the diabetic group. The
combination of nicotine and diabetes revealed a significant increase of 8.9 m in
the TRT.
Conclusions: We demonstrated significant changes in retinal morphology in
response to nicotine exposure, diabetes and with the combined effects of nicotine
and diabetes. These findings may have implications in determining treatment
strategies for diabetic smokers.
Commercial Relationships: Nima Tirgan, None; Adam Boretsky,
None; Praveena Gupta, None; Bernard F. Godley, None; Ronald G. Tilton,
None; Massoud Motamedi, None
Support: National Institute of Environmental Health Sciences, Research to Prevent
Blindness (RPB)
AL-78898A Inhibits Complement Deposition in a Primate Light Damage
Model
Robert J. Collier, Sherry Smith, Hayden Hoang, Elizabeth Martin, Yu Wang, Li
Zhu, Richard Ornberg, Carmelo Romano. Retina Discovery Research, Novartis
Institutes for Biomedical Research - FTW, Fort Worth, TX.
Purpose: Increased risk for development of AMD has been associated with
modifications in complement related genes (C2, C3, CFB and CFH). The purpose
of this study was to evaluate the role of complement activation in light-exposed
retinas and determine if complement inhibition can prevent complement deposition
in the retina.
Methods: Two days prior to light exposure, cynomologous monkeys received an
intravitreal injection (100 L) of AL-78898A (0.15 - 2.1 mgs), a C3 inhibitor.
NHPs were exposed to light for 30 min. Forty-eight hours after light exposure
tissues were collected for light microscopy and immunohistochemistry (C1q, C3,
Factor B, Factor H and membrane attack complex (MAC)). MicroVue EIA kits
(Quidel) for C3a-desArg and sC5b-9 were used to quantify activated complement
in retina and choroid/RPE samples.
Results: Two days after light exposure, RPE damage and diffuse pyknotic
photoreceptor nuclei were observed. C1q expression was not observed while C3,
FB, FH and MAC expression was present over the choriocapillaris, RPE, inner and
outer segments and ONL in light exposed retinal regions. Retinal C3a and sC5b-9
levels and RPE/ choroid sC5b-9 levels were significantly higher in vehicle dosed
eyes compared to normal eyes. C3a levels were not significantly elevated in light
exposed RPE/ choroid samples. Treatment with AL-78898A significantly reduced
the light-induced elevation of retinal C3a (0.150 - 2.1 mg) and sC5b-9 (0.35, 0.525
and 2.1 mg) and RPE/ choroid sC5b-9 (0.525 - 2.1 mg) complement levels
measured in this model.
Conclusions: Visible-light exposure results in activation of the alternative
complement pathway (positive C3 expression, negative C1q expression). Treatment
with AL-78898A significantly reduced the light-induced elevation of complement
levels measured in this model and demonstrates the potential utility of a C3
inhibitor for treatment of AMD.
Commercial Relationships: Robert J. Collier, Novartis Institutes for
Biomedical Research (E); Sherry Smith, Novartis Institutes for Biomedical
Research (E); Hayden Hoang, Novartis Institutes for Biomedical Research (E);
Elizabeth Martin, Novartis Institutes for Biomedical Research (E); Yu Wang,
Novartis Institutes for Biomedical Research (E); Li Zhu, Novartis Institutes for
Biomedical Research (E); Richard Ornberg, Novartis Institutes for Biomedical
Research (E); Carmelo Romano, Novartis Institutes for Biomedical Research (E)
Support: None

Preclinical Safety Of Intravitreal Docosahexaenoic Acid In A Rabbit Model
Rosa Dolz-Marco1, Roberto Gallego-Pinazo1, M Dolores Pinazo-Duran2, Sheila
Pons-Vazquez2, Manuel Diaz-Llopis1. 1University and Polytechnic Hospital La Fe,
Valencia, Spain; 2"Santiago Grisolia" Ophthalmic Research Unit. "Dr Peset"
Hospital, Valencia, Spain.
Purpose: To evaluate the retinal toxicity of intravitreal administration of the
purified fatty acid docosahexaenoic (DHA) in rabbit eyes.
Methods: Sixteen New Zealand albino rabbits were selected. Six concentrations of
DHA (Brudy Laboratories, Barcelona, Spain) were prepared: 10, 5 and 2.5 g/1
L; and 50, 25, and 5 g/50 L. A volume of 0.05 ml of each concentration was
injected intravitreally in the right eye of 2 rabbits. As a control, 0.05 ml of saline
solution was injected into in the right eye of 4 animals. Retinal safety of intravitreal
DHA was assesed by electroretinography (ERG) -changes between before and 1
week after the injection-; and by histologic examination of the retinal samples
obtained after sacrificing the rabbits.
Results: We evidenced a severe intraocular inflammation in eyes treated with 10
g/1 L, 5 g/1 L and 25 g/1 L DHA concentrations. The ERG studies amplitude and time of A and B waves- did not show significant difference (p<
0,01) between control and DHA-injected eyes. The histologic examination did not
evidence any retinal abnormality in the rabbits injected with different
concentrations of DHA.
Conclusions: DHA may be a safe intravitreal drug in the rabbit model up to 50
g/50 L. Further studies are warranted to evaluate the safety and efficacy in
human.
Commercial Relationships: Rosa Dolz-Marco, None; Roberto Gallego-Pinazo,
None; M Dolores Pinazo-Duran, None; Sheila Pons-Vazquez, None; Manuel
Diaz-Llopis, None
Support: None
In Vitro and In Vivo Cytotoxic Effects of Withaferin A on Retinal Epithelial
Pigmented Cells
Taimur Safder, Erica Person, Mark Cohen, Ridhwi Mukerji, Abbas Samadi, Patrick
Grogan. University of Kansas Medical Center, Kansas City, KS.
Purpose: Withaferin A (WA) is a naturaceudical product derived from Withania
somnifera that has demonstrated anti-cancer activity against several cancer types
including leukemia, colon, pancreas, thyroid, head and neck, breast, glioblastoma,
and cutaneous and uveal melanoma. Control Retinal Pigment Epithelial (RPE) cells
were exposed to WA and revealed an IC50 = 1.8M in a comparable range to the
IC50s of many other cancer lines [Leukemia SUPB15 = 0.13 M, Thyroid DRO811 = 0.65 M, KAT4B = 0.9 M, Breast MCF-7 = 0.58 M, MDA-MB231 = 0.54
M, Melanoma SKMEL28 = 0.62 M, UM 92.1= 2.50 M, OMM 2.3 = 1.0 M,
and Fibroblast MRC-5 = 3.95 M as systemic control]. Further in vivo and vitro
experiments were conducted to observe any cytotoxic effects of Withaferin A on
RPE Cells in vitro and in vivo.
Methods: In addition to MTS assay, cell proliferation and viability assay were
performed through trypan blue exclusion assay to confirm RPE suppression. To
determine if apoptosis was the primary mechanism, annexin V/PI staining and
Western blot for caspase 3 and PARP were completed. An in vivo experiment
compared retinal histology of mice treated with 8mg/kg/day to control.
Experimental protocol was in compliance with the ARVO statement for the use of
animals in ophthalmic and vision research and institutional animal care and use
committee requirements.
Results: Results of trypan blue exclusion confirmed our preliminary findings of
dose dependent toxicity of RPE cells to Withaferin A (p =0.006 at 72hrs). Annexin
V/PI and Western blot confirmed the mechanism of apoptosis when RPE cells were
directly exposed to WA. However, histological evidence from the in vivo
experiment suggests no damage was done to the RPE or retina at therapeutic IP
dosing of WA.
Conclusions: WA is a promising naturiceudical agent for the treatment of multiple
cancer types. While it has a cytotoxic effect on RPE cells in vitro, the same
principle does not apply in vivo when applied to a murine model. The retinal-blood
barrier could prevent the drug from reaching the retina and RPE and explain the
difference between the in vivo and in vitro results. As WA transitions to human
trials, close attention to the affects in the retina are indicated.
Commercial Relationships: Taimur Safder, None; Erica Person, None; Mark
Cohen, None; Ridhwi Mukerji, None; Abbas Samadi, None; Patrick Grogan,
None
Support: The Kansas Lions Sight Foundation
Wenhu Huang, Mark Zorbas. Pfizer Global R&D, San Diego, CA.
Purpose: PF-04523655, a synthetic and chemically-modified 19 base-pair siRNA
designed to temporarily inhibit expression of the RTP801 gene, is currently in
Phase II trials for the treatment of wet AMD and DME. Preclinical studies have
shown a dose-related suppression of RTP801 expression in rat CNV and DME
models. A GLP chronic toxicity study was conducted to evaluate the safety profile
of PF-04523655 following intravitreal administration in the monkey to support
Phase II repeat dose clinical studies.
Methods: Male and female cynomolgus monkeys (Macaca fascicularis) received
monthly bilateral intravitreal injections of vehicle control (phosphate buffered
saline; PBS), or 0.3, 1, or 3 mg/eye of PF-04523655 in a dose volume of 50 L for
13 or 49 weeks (4 or 13 doses given at monthly intervals). The main study groups
were sacrificed 4 days after the last dose. Recovery animals sacrificed 4 weeks
following the last dose were included in the vehicle control and high-dose (3
mg/eye) groups. Assessment of toxicity was based on mortality, clinical signs,
electrocardiography, ophthalmic examinations, ocular photography, intraocular
pressure measurements, electroretinography, clinical and anatomic pathology, and
immunogenicity/ antigenicity evaluations.
Results: PF-04523655 was well tolerated based on clinical signs. Intravitreal
administration of both the vehicle control (PBS) or PF-04523655 test material
typically resulted in mild and reversible mid anterior segment and vitreal
inflammatory response, the severity of which was comparable between the control
group and PF-04523655 treatment groups, whereas the incidence of these findings
was greater in eyes receiving 3 mg/eye (high-dose group). Posterior lens capsular
haze was noted in three males given 3 mg/eye started on week 16 and was transient
in two animals and progressed to an incipient posterior capsular cataract in one
animal. As for the neural retina, there was no test article related finding in
histopathology, electroretinogram, or ophthalmic examinations. In addition, no
changes were noted on body weight or electrocardiographic parameters that were
attributable to test article treatment.
Conclusions: The test article, PF-04523655, when administered monthly via
bilateral intravitreal injection to cynomolgus monkeys, was well tolerated
following 49 weeks of treatment. Procedure-related mild and transient ocular
inflammation was noticed in all treatment groups. The cataract in one high dose
animal was considered adverse. There was no clinical, histomorphologic or
electroretinographic evidence of a test article-related effect on the retina.
Commercial Relationships: Wenhu Huang, None; Mark Zorbas, None
Support: None
Electroretinogram Is Not Altered by KAT II Inhibitor in Rats
Chang-Ning Liu, Chris J. Somps, Chris Houle. Drug Safety R&D, Pfizer Inc,
Groton, CT.
Purpose: Selective inhibitors of kynurenine aminotransferase type II (KAT II) are
being developed to treat cognitive impairment associated with schizophrenia.
Reduced neurotransmission at the NMDA subtype of the glutamate receptor has
been identified as a primary neurochemical deficit in schizophrenia. Since
kynurenic acid (KYNA) is a negative NMDA receptor modulator in brain synapses,
decreasing synaptic KYNA concentration by inhibiting its production in astrocytes
should enhance NMDA signaling. However, KYNA and KAT II have also been
detected in the inner retina of rodent, rabbit and human eyes. In addition, glycine,
another positive NMDA modulator, has been found to be associated with visual and
electroretinogram (ERG) disturbances. Thus, we conducted the current in vivo
study in order to determine if a selective KAT II inhibitor causes changes in ERG
responses in rats.
Methods: Male Sprague-Dawley (SD) rats (8/group) were administered PF04859989 subcutaneously at 100 mg/kg (previously shown to decrease KYNA
concentration in the prefrontal cortex) or vehicle two hours prior to ERG
acquisition. Scotopic and photopic luminance responses, photopic adaptometry and
flicker responses were measured following 1 hour of dark adaptation. Plasma and
vitreous samples were obtained for determination of PF-04859989 concentrations
at the end of the ERG session.
Results: No significant differences were found between vehicle and PF-04859989
dosed SD rats in ERG parameters tested. PF-04859989 levels in vitreous humor
were 0.93 fold the levels in plasma. The amplitude of the scotopic ERG oscillatory
potentials was not correlated to the exposure of PF-04859989 in the vitreous humor
(r = -0.17, P >0.05).
Conclusions: A single subcutaneous dose of 100 mg/kg PF-04859989, a KAT II
inhibitor, did not result in changes in ERG in adult SD rats.
Commercial Relationships: Chang-Ning Liu, Pfizer Inc (E); Chris J. Somps,
Pfizer Inc (E); Chris Houle, Pfizer Inc (E)
Support: None

A 49-Week Chronic Study of PF-04523655 Given by Intravitreal Injection to
Cynomolgus Monkeys with a 13-Week Interim Evaluation

Insights Into Microvasculature Topography And Its Coupling With Neuronal
Demands In The Human Retina
Chandra Bala1,2, Geoffrey Chan1,2, Paula Yu1,2, William Morgan1, Ian McAllister1,
Dao-Yi Yu1,2. 1Physiology & Pharmacology, Centre for Ophthalmology and Visual
Science, The University of Western Australia, Australia; 2Physiology and
Pharmacology, Australian Research Council Centre of Excellence in Vision
Science, The University of Western Australia, Australia.
Purpose: Retinal vascular diseases are a major cause of blindness worldwide.
Retinal vulnerability to injury stems from the disparity between high neuronal
energy demands and the limited blood supply available to retinal structures. It
remains unclear how intra-retinal capillary organization is coupled to the
heterogeneous configuration of neuronal metabolic activity between different
retinal layers. This study provides important quantitative information concerning
the topographical characteristics of retinal microvasculature, in relation to neurons,
using our recently developed perfusion-fixation-staining technique in human
cadaveric eyes.
Methods: The macula-papillary region, 2mm nasal to the fovea, was studied in 14
undiseased human eyes (mean age 40 6.1 years). Novel micropipette technology
was used to cannulate the central retinal artery and label the retinal microcirculation
using a Phalloidin perfusate. Co-localization of capillary networks within retinal
layers was studied using anti-gamma-Synuclein, anti-Goalpha and antiParvalbumin antibodies; labeling for retinal ganglion cells (RGCs) and axons, ON
bipolar cells and horizontal cells, respectively. Confocal microscopy based
techniques were used to document topographic relationships between
microvascular distribution and neuronal populations. Reproducible methodology
was also employed to quantify capillary diameter, capillary density (expressed as
percentage of tissue volume) and capillary loop area within retinal layers.
Results: Laminar capillary networks were identified in the nerve fibre layer (NFL),
the deep portion of the inner nuclear layer (INL) and outer plexiform layer.
Complex three-dimensional networks were identified in the RGC layer and the
superficial portion of the INL. Capillaries in the NFL were predominantly oriented
parallel to the trajectory of RGC axons while consistent capillary-neuronal spatial
relationships were not identified within remaining layers. Capillary density and
loop area measurements were significantly different between retinal layers (both P
< 0.05), however, regions of greatest capillary density and loop area did not
correlate with previously demonstrated regions of high metabolic demand (density
of 17.95% in INL vs 22.32% in RGC layer). There was no difference in capillary
diameter between different layers (P > 0.05).
Conclusions: There is significant disparity in capillary topography between retinal
layers. Greatest capillary density does not correlate exactly with previously
described regions of maximal neuronal energy consumption. These findings may
have relevance to understanding novel patho-physiological mechanisms involved in
retinal vascular diseases.
Commercial Relationships: Chandra Bala, None; Geoffrey Chan, None; Paula
Yu, None; William Morgan, None; Ian McAllister, None; Dao-Yi Yu, None
Support: National Health and Medical Research Council of Australia, Australian
Research Council Centre of Excellence in Vision Science and Ophthalmic Research
Institute of Australia
Effects of Ethambutol on the ERG a-Wave Amplitude From the Isolated
Superfused Vertebrate Retina
Siarhei Siapich1A, Michaela Hartleb1A, Toni Schneider2A,2B, Gabriele Thumann1A,1B,
Peter Walter1A. ADepartment of Ophthalmology, BIZKF Biomat, 1RWTH Aachen
University, Aachen, Germany; AInstitute of Neurophysiology, BCenter of
Molecular Medicine (CMMC), 2University of Cologne, Cologne, Germany.
Purpose: Long-term therapy with the anti-tuberculosis drug ethambutol is known
to have neurotoxic effects and cause opticus neuropathy. Our objective was to
study the acute toxic effects of ethambutol on the a-wave response of
electroretinogram (ERG) of isolated superfused bovine retinas.
Methods: Isolated bovine retinas were perfused with phosphate buffered saline
(PBS) containing 1mM L-aspartate to block further synaptic transmission in order
to record the effects of ethambutol on photoreceptors. After light stimulation
electric field potentials were recorded as a transretinal potential using Ag/AgClelectrodes. Three different light intensities were tested (100 mlux, 1 lux, 10 lux).
After reaching a stable ERG amplitude, ethambutol (0.98mM, 3.1 mM und 9.8
mM) was added to the perfusing solution und was perfused for 90 min. Thereafter,
ethambutol was washed out during 90 min with PBS containing 1mM L-aspartate.
Changes in a-wave amplitude before, during and after ethambutol-application were
calculated and plotted.
Results: 0.98 mM ethambutol did not show any significant effect on the a-wave
amplitude at any light-intensity; 3.1 mM ethambutol reduced the a-wave amplitude
by 2-folds independent of light-intensity. The inhibition was found to be only
partially reversible by washing with PBS containing 1mM L-aspartate, however at
lower light-intensities recovery was better (1.4 to 1.65-folds). 9,8 mM ethambutol
reduced the a-wave amplitude up to 4-fold, increasing slightly at higher lightintensities. Partial recovery was observed at 100 mlux and 1 lux by washing with
PBS containing 1mM L-aspartate; however recovery at 10 lux was minimal.
Conclusions: Ethambutol shows dose dependent inhibitory effect on
photoreceptors, decreasing the a-wave amplitude with increasing concentration.
The inhibition is only partial reversible within the 90-minute washout. Considering
the cumulative effect of ethambutol during the long-term therapy, it is essential that
the exact dosage is calculated to avoid concentrations that lead to irreversible
neuronal damage.
Commercial Relationships: Siarhei Siapich, None; Michaela Hartleb,
None; Toni Schneider, None; Gabriele Thumann, None; Peter Walter, None
Support: None
Spontaneous And Light Evoked Retinal Ganglion Cell Activity In Early
Developmental Course Of GNAT2-DTA Transgenic Mouse
Chao Sun1A, Deborah v. List1A, Barbara Chapman1A,1B. ANeurobiology, Physiology
and Behavior, BCenter for Neuroscience, 1University of California, Davis, Davis,
CA.
Purpose: GNAT2-DTA mouse expresses diphtheria toxin under a cone-specific
human cone transducin alpha-subunit (GNAT2) promoter (Fong et al., 2005). In
this mouse line, both cone and rod photoreceptors fail to ever develop at all in the
ventral retina. In this study, we try to understand whether loss of photoreceptors
may have effects on retinal ganglion cell (RGC) function during early
development.
Methods: We used 60-channel multi-electrode array (MEA) recordings to quantify
patterns of spontaneous and light evoked retinal ganglion cell activity in GNAT2DTA mice and wild type mice at different postnatal ages
Results: 1. At postnatal day 6, the spontaneous waves of correlated RGC activity in
GNAT-DTA mice were similar to those in wild-type mice. 2. In GNAT-DTA
strain, spontaneous firing rates increased by P12. 3. At P12, full-field light flashes
evoked reliable responses in many RGCs in GNAT-DTA mice strains, with equal
preservation of on and off responses.
Conclusions: Although cone and rod photoreceptors in the ventral retina of
GNAT-DTA mice were never develop, the activity of many RGCs of GNAT-DTA
mice remained normal.
Commercial Relationships: Chao Sun, None; Deborah V. List, None; Barbara
Chapman, None
Neuroregenerative Properties Of Transcription Factor Brn3b In An Elevated
IOP Rat Model Of Glaucoma
Dorota L. Stankowska, Alena Z. Minton, Shaoqing He, Raghu R. Krishnamoorthy.
Cell Biology and Anatomy, NTERI, Univ of North Texas Hlth Sci Ctr, Fort Worth,
TX.
Purpose: Glaucoma is an optic neuropathy, commonly associated with an increase
in intraocular pressure (IOP) leading to neurodegenerative changes in the optic
nerve head and apoptosis of retinal ganglion cells. Insult to axons at the lamina
cribrosa is an early event that precedes other neurodegenerative changes seen in
various animal models of glaucoma. Hence, strategies to promote regeneration of
the optic nerve have gained prominence in recent years. Brn3b is a class 4 POU
domain transcription factor which has been shown to play key role in regulating
retinal ganglion cell axon outgrowth and pathfinding during development in
projection neurons. The purpose of this study was to determine if overexpression of
Brn3b could promote an axonal regenerative response in the optic nerve in an
elevated IOP rat model of glaucoma.
Methods: Adult male retired breeder Brown Norway (BN) rats were intravitrealy
injected with adeno-associated virus encoding either Brn3b (AAV-Brn3b) or the
empty vector (AAV-MCS). Experimental glaucoma was induced in rats by IOP
elevation using the Morrisons method (injection of hypertonic saline through
episcleral veins). Transgene expression of Brn3b was assessed by
immunohistochemistry using an anti-flag antibody. Two weeks following the
injection with the viral vectors BN rats were sacrificed. Retinas sections through
the optic nerve head were obtained and stained for Brn3b as well as
neuroregenerative markers including growth associated protein 43 (GAP-43), actin
binding LIM protein 1 (AbLIM) and acetylated tubulin alpha 1 (ac-Tuba-1) by
immunofluorescence technique.
Results: An increased immunostaining for Brn3b was observed two weeks postintravitreal injection of AAV-Brn3b. An upregulation of the axon-specific GAP-43
protein within the retina as well as in the posterior region of optic nerve was
observed after AAV-Brn3b injection. Overexpression of Brn3b promoted the
upregulation of two other neuroregeneration markers namely, ac-Tuba-1 and
AbLIM.
Conclusions: The AAV-mediated expression of Brn3b produced an increase in

expression of markers of nerve regeneration in vivo in the Morrisons elevated
intraocular pressure (IOP) model of glaucoma in rats. AAV-Brn3b may be a
suitable agent for promoting regeneration following axonal injury during glaucoma.
Commercial Relationships: Dorota L. Stankowska, None; Alena Z. Minton,
None; Shaoqing He, None; Raghu R. Krishnamoorthy, None
Support: W81XWH-10-2-003
Effects of Hydrogen Peroxide (H2O2) On Retinal Function In Vitro
David Cia1A, Nathalie Jacquemot1A, Jacques Cluzel1A, Adrien Rossary1B, Michel
Doly1A. AEA 2667 Biophysique des Handicaps Sensoriels, BEA 4233 Nutrition
Cancrognse et Thrapie anti-tumorale CLARA CRNH Auvergne, 1Clermont
Universit, Clermont Ferrand, France.
Purpose: The study was to investigate the effects of hydrogen peroxide (H2O2) on
retinal function from isolated rat retina and evaluate the H2O2-induced oxidative
stress.
Methods: Retinas were isolated from rat eyes and placed in a chamber
continuously perfused with a nutrient solution. The ERG was recorded every 3
minutes. Once the ERG was at a steady state, H2O2 was added at various
concentrations (0.1-1mM) to the perfusion medium for 3 hours. The amplitudes and
implicit times of the b-, a- and PIII-waves vs. time were plotted to obtain the
survival curves. The malondialdehyde (MDA) content in perfusion medium was
evaluated by HPLC as a biomarker for oxidative stress.
Results: Application of H2O2 on isolated retinas produced a rapid increase in the bwave amplitude followed by a significant decrease, as compared to the unexposed
retinas. The amplitudes of the a- and PIII-wave decreased significantly. The
implicit time of the a-wave remained unchanged; the implicit time of the b-wave
showed a moderate increase and then returned to its normal value; whereas the
implicit time of the PIII-wave increased significantly and then returned to its
normal value. The H2O2-associated changes of the ERG amplitudes and implicit
times were dose dependent. The content of MDA in perfusion medium increased in
a dose dependent manner.
Conclusions: Exposure of retina to H2O2 impairs retinal function as shown by the
changes of the ERG amplitudes and implicit times, and induces oxidative stress as
reflected by the increase of MDA.
Commercial Relationships: David Cia, None; Nathalie Jacquemot,
None; Jacques Cluzel, None; Adrien Rossary, None; Michel Doly, None
Support: None
Involvement Of Calpain In The Degeneration And Dysfunction Of Rat Retinal
Nerve Fiber Layers Due To Acute Ocular Hypertension
Rie Suzuki1, Takayuki Oka1, Yoshiyuki Tamada1, Thomas R. Shearer2, Mitsuyoshi
Azuma1,2. 1Senju Pharmaceutical Co Ltd, Kobe, Japan; 2Integrative BiosciencesDentistry, Oregon Health Sciences University, Portland, OR.
Purpose: Increased intraocular pressure (IOP) in human glaucoma leads primarily
to damage of the retinal ganglion cell layer (GCL). Electrophysiological studies
also suggest impairment of the inner plexiform layer and inner nuclear layer
(IPL+INL). But damage to the photoreceptors (PR) is controversial. Our recent
studies with acute ocular hypertensive rats confirmed damage predominantly in the
GCL and IPL+INL, and only partial damage to the PR. Calpain-induced proteolysis
was also observed in whole retina, and calpain inhibitor SNJ-1945 ameliorated
degeneration of the GCL. However, involvement of calpain in damage to other
nerve fiber layers was not clear. Thus, the purpose of present study was to
determine where calpain causes degeneration and dysfunction in specific nerve
fiber layers.
Methods: IOP in rats was elevated to 110 mm Hg for 40 min. Oral calpain
inhibitor SNJ-1945 was administrated at 50 mg/kg 1 hour after ocular hypertension.
Retinal function was assessed by scotopic electroretinography (ERG). Histologic
damage was evaluated in thin sections of retina after H&E and immunostaining.
Results: One day after ocular hypertension, the ERG b-wave, arising primarily
from the on-bipolar cells, was lost and only minimally returned by day 7. The
thickness of the IPL+INL containing these bipolar cells became thinner. Calpain
protease inhibitor SNJ-1945 significantly ameliorated the loss of the b-wave as well
as the thinning of IPL+INL at 7 days. Interestingly, the changes in amplitude of the
b-wave were significantly correlated to changes in the thickness of IPL+INL. In
contrast, elevated IOP caused only mild losses in the a-wave arising from the PR,
the thickness of the OPL+OS containing the PR did not change, but SNJ-1945 did
significantly ameliorate IOP-induced losses of the a-wave.
Conclusions: Elevated IOP in rats predominantly damaged the IPL+INL.
Amelioration by the oral calpain inhibitor SNJ-1945 implicated calpain hydrolysis
in the degeneration of bipolar cells. Although much less obvious, calpain activation
may also damage PR cells.
Commercial Relationships: Rie Suzuki, Senju Pharmaceutical Co Ltd (E);
Takayuki Oka, Senju Pharmaceutical Co Ltd (E); Yoshiyuki Tamada, Senju
Pharmaceutical Co Ltd (E); Thomas R. Shearer, Senju Pharmaceutical Co Ltd

(C); Mitsuyoshi Azuma, Senju Pharmaceutical Co Ltd (E)
Support: None
Evaluating Retinal Toxicity of a New Heavy Intraocular Dye Using the Model
of Perfused and Isolated Retinal Cultures of Human Origin
Sebastian Mueller, Kai Januschowski, Martin Spitzer, Charlotte Schramm, KarlUlrich Bartz-Schmidt, Peter Szurman. Department of Ophthalmology I, Tuebingen,
Germany.
Purpose: During vitreoretinal surgery vital dyes such as Brilliant Blue (BBG) are
used to visualize anatomical structures. By adding deuteriumoxide (D2O) many
surgeons try to create a dye mixture heavier than water to facilitate staining of the
inner limiting membrane (ILM) without preceding fluid-air exchange. However,
the intraocular use of D2O is critical. This study investigated the effect of 0.4 ml
BBG (Brilliant Peel 0.25 mg/ml, Fluoron, Ulm, Germany) mixed with 0.13ml/ml
D2O on retinal function of a pseudo in vivo model using human whole mount
cultures.
Methods: Human retinas were isolated and superfused with an oxygen saturated
nutrient solution before the electroretinogram (ERG) was recorded. BBG mixed
with 0.13 ml/ml D2O was applied epiretinally for a staining period of 60 seconds.
ERG-recovery was monitored for 75 minutes. For obtaining a-waves1 mM
aspartate was added to the nutrient solution.
Results: Reductions of the a- and b-wave amplitude were found directly after
exposure with BBG in all test series. These effects on the electroretinogram were
rapidly and completely reversible within the recovery time for all exposure times.
Between the ERG-amplitudes no differences were found before and after dye
application at the end of the washout.
Conclusions: The clinically used mixture of BBG and 0.13 ml/ml D2O seems to be
safe for clinical use. Longer staining periods than 60 seconds were not
tested.
Commercial Relationships: Sebastian Mueller, None; Kai Januschowski,

None; Martin Spitzer, None; Charlotte Schramm, None; Karl-Ulrich BartzSchmidt, None; Peter Szurman, None
Support: None
Retinal Ganglion Cell Survival in Long-term Human Organotypic Retinal
Cultures
Marina Hopes1, Andrew Osborne2, David C. Broadway1, Julie Sanderson2.
1
Department of Ophthalmology, Norfolk and Norwich University Hospital,

Norwich, United Kingdom; 2School of Pharmacy, University Of East Anglia,
Norwich, United Kingdom.
Purpose: To investigate the long term survival of retinal ganglion cells (RGCs) in
human organotypic retinal cultures (HORCs) maintained for extended periods of up
to 4 weeks.
Methods: Donor eyes were obtained from the East Anglian Eye Bank within
24hours post mortem. The retina was dissected and five retinal explants (4mm in
diameter) were taken from the paramacular regions of the retina. The HORCs were
cultured for up to 4 weeks in DMEM/HamF12 +/- FCS or Neurobasal medium
supplemented with B27N2. RGC numbers were assessed by NeuN
immunohistochemistry and level of THY-1 expression (QRT-PCR). Apoptosis was
evaluated by TUNEL assay.
Results: Under all conditions basic retinal morphology and architecture were
preserved, with the nuclear layers remaining well defined over the 4 week culture
period. In all conditions there was a significant decrease in the number of NeuNlabelled RGC s over the culture period, with the greatest loss (approximately 60%
at 4 weeks) being seen in Neurobasal medium (n=4; p<0.05). There was a
corresponding increase in the number of TUNEL-positive NeuN-labelled cells
under all conditions, again with culture in Neurobasal medium resulting in the
highest proportion of apoptotic nuclei (almost 100%). DMEM/HamF12
supplemented with serum showed the best survival with no significant loss of
NeuN-labelled cells until the 3 week time point (n=4; p<0.05). Under all
conditions, THY-1 expression declined significantly by week 1, then remained at a
baseline level of approximately 20% for the 4 week culture period (n=4; p<0.05).
Conclusions: There was a decline in the number of RGCs in human organotypic
retinal cultures in long-term culture. Loss of RGCs in Neurobasal medium with
B27/N2 supplementation was greater than in SF DMEM/HamF12. Loss of RGCs
could be protected by serum supplementation. This long-term culture model might
be useful for investigation of neurodegeneration and neuroprotection of human
RGCs.
Commercial Relationships: Marina Hopes, None; Andrew Osborne,
None; David C. Broadway, None; Julie Sanderson, None
Support: The Humane Research Trust, Norwich Glaucoma Research Fund, The
Edith Murphy Foundation
Differences in GABA Receptor Contribution to Surround Inhibition Between
the ON and OFF Pathways in the Mammalian Retina
Ilya Buldyrev, W. Rowland Taylor. Casey Eye Institute, Oregon Health & Science
University, Portland, OR.
Purpose: Most retinal ganglion cells display concentric, centre-surround receptive
fields, which increase sensitivity to contrast borders. Amacrine cells in the innerplexiform layer are thought to contribute to surround suppression, however, the
relative role of the different GABA receptors remains uncertain.
Here we investigated the role of GABAergic transmission in providing surround
inhibition to the ON and the OFF pathways, using the ON and OFF brisk-sustained
ganglion cells (BSGCs) in the rabbit retina.
Methods: Extracellular and whole-cell voltage clamp recordings were performed
in whole-mount retinal preparations at 36 C. Spot stimuli of variable diameter were
centered in the BSGC receptive field. The spot intensity was square-wave
modulated at 1Hz with a peak-to-peak amplitude of 20% relative to a photopic
background. The strength of surround inhibition was determined by modeling the
stimulus size-response function as a difference of Gaussian integrals.
Results: Our recordings demonstrate that most of the GABAergic portion of
surround inhibition is presynaptic to the BSGCs, suppressing both excitatory inputs
from bipolar cells and inhibitory inputs from narrow-field glycinergic amacrine
cells. Extracellular recordings showed that this presynaptic inhibition almost
completely suppressed stimulus-evoked spike output in both ON (96 2.3%) and
OFF (98 2.0%) BSGCs. However, in the presence of GABA blockers, surround
stimulation suppressed responses by 6110% in ON and 573% in OFF BSGCs.
The residual surround inhibition likely originates in the outer plexiform layer. In
ON BSGCs, signaling via GABAC receptors was sufficient to account for the
GABAergic surround suppression. However, in OFF BSGCs, blocking both
GABAA and GABAC receptors was required to significantly increase the spike
output and synaptic excitation in response to wide-field stimuli. Although large
spots activated a small, GABAA receptor-mediated feed-forward inhibitory input
in OFF BSGCs, selectively blocking GABAA receptors did not increase OFF
BSGC responsiveness to wide-field stimuli, indicating that postsynaptic inhibition
did not play a major role in generating the surround.
Conclusions: Although GABAA receptors contribute to surround inhibition in
OFF BSGCs our results suggest that, GABAC receptor signaling at bipolar cell
terminals is central to surround inhibition in both the OFF and ON pathways.
Commercial Relationships: Ilya Buldyrev, None; W. Rowland Taylor, None
Support: EY014888

Erg Changes In A Triple Transgenic Mouse Model For Alzheimer`s Disease
Gabriela L. Ioshimoto1,2, Balazs V. Nagy1,2, Jan J. Kremers3,4, Dora F. Ventura1,2.
1
Experimental Psychology, University of Sao Paulo, Sao Paulo, Brazil;
2
Neuroscience and Behavior, Sao Paulo, Brazil; 3Dept of Ophthalmology,
University of Erlangen, Erlangen, Germany; 4Rhenovia Pharma, Mulhouse, France.
Purpose: To study the retinal function of the triple transgenic mouse model (3xTgAD) for Alzheimer`s disease (AD) by comparing retinal electrophysiological
responses in 3xTg-AD mice with those in the background control (b6;129-PS1).
The responses were measured between 2 and 12 months of age.
Methods: ERGs were recorded from 44 3xTg-AD mice and from 23 background
controls with a contact lens electrode on the cornea, a needle reference electrode on
the head and a ground on the tail. Recordings were obtained for: 1) Maximum
scotopic response (30cd.s/m2); 2) Light-adapted (30 cd/m2) flicker pulses (30
cd.s/m2) at 12, 18 and 30 Hz.
Results: 87% of control mice and 28% of the 3xTg-AD had very abnormal ERGs
with a large b-wave implicit time (111,73 22,56 ms) and no OPs. The others
displayed ERGs with OPs and with b-wave implicit times within the range (45,31
6,74 ms) expected from the literature. In the latter group, age dependent changes in
the flash ERG were found for the a- and b-wave amplitudes. While the control
group exhibited a mean decrease from 193,26 to 97,06 V in the a-wave amplitude
and a mean decrease from 452,4 to 230,71 V in the b-wave amplitude between 6
and 12 months, 3xTg-AD group presented a low and constant response (a-wave=
143,4 19,3 V; b-wave= 303,5 49,7 V) between 6 and 12 months of age.
Flicker amplitudes (1st harmonic after Fourier analysis) from the 3xTg-AD group
were significantly reduced compared to controls at 6 months, but not at 12 months,
both for the 12 Hz (p=0,0001) and for the 18 Hz (p=0,0001)stimuli.
Conclusions: Comparison of control and transgenic mice with ERGs with OPs and
implicit times within the normal range, revealed a physiological impairment of the
retina of AD mice. The a-wave amplitude decrease suggests that the effect involves
loss or impairment of photoreceptor function. We conclude that AD may also affect
the function of the retina.
Commercial Relationships: Gabriela L. Ioshimoto, None; Balazs V. Nagy,
None; Jan J. Kremers, None; Dora F. Ventura, None
Support: CAPES
A Novel Technique for Analysis of the Expression Profile of Genes in the
Human Retina
Ning Ma1A, Jeremy D. Rhodes1B, Martin C. Lott1C, Vincent Moulton1C, David C.
Broadway2, Julie Sanderson1A. ASchool of Pharmacy, BSchool of Biology, CSchool
of Computing Science, 1University of East Anglia, Norwich, United Kingdom;
2
Department of Ophthalmology, Norfolk and Norwich University Hospital, UK,
Norwich, United Kingdom.
Purpose: To develop a technique which yields high levels of good quality RNA
from retinal ganglion cells for analysis of gene expression profiles in the human
retina.
Methods: Human retina was obtained from the East Anglian Eye Bank within 24
hours post mortem. A 5x5mm section was dissected from the macula region of the
retina and was mounted for cryosectioning. Serial 20m sections were obtained in
the plane of the retinal nuclear layers. RNA was extracted (approximately 120360ng/section) and gene expression analysed by QRT-PCR. Markers used were
recoverin (RCVRN) for photoreceptors, calbindin (CALB) for horizontal cells,
choline acetyltransferase (CHAT) for amacrine cells and Thy -1, NeuN and Brn3a
(THY-1, RBFOX3 and POU4F1) for retinal ganglion cells. Profiling was carried
out for a minimum of 6 individual donors for each gene. Global gene expression
analysis (Illumina arrays) was carried out to compare retinal ganglion cell (RGC)
expression with expression in total retina (n=4). Immunohistochemistry was carried
out on fixed transverse sections of the human retina.
Results: Expression of known markers of retinal neurons was highly consistent
with their position in the retina: RCVRN expression was high in the outer layers
which subsequently declined to baseline levels; the second peak was for CALB and
the third for CHAT; THY-1 remained highest in the latter sections derived from the
inner retina. Two further retinal ganglion cell markers NeuN (RBFOX3) and Brn3a
(POU4F1) showed the same profile as THY-1. The profile was confirmed using
immunohistochemistry. Gene arrays, comparing the RGC layer to total retina,
showed good separation between RGC and photoreceptor genes. RGC markers
were amongst the set of most highly expressed genes and photoreceptor genes were
amongst those with the lowest expression.
Conclusions: The novel technique yielded high quality RNA in sufficient
quantities to carry out mRNA profiling and array analysis. The profiles of markers
for retinal neurons follow the expected localisation in the retinal nuclear layers. The
technique offers a powerful method for determining the expression profile of genes
of interest in the human retina.
Commercial Relationships: Ning Ma, None; Jeremy D. Rhodes, None; Martin
C. Lott, None; Vincent Moulton, None; David C. Broadway, None; Julie
Sanderson, None

Support: The Humane Research Trust, Norwich Glaucoma Research Fund
Concentration and Distribution of Various Metals in the Rat Retina Using
Inductively Coupled Plasma Mass Spectrometry (ICPMS) and Proton-induced
X-ray Emission (PIXE) Methodology
Marta Ugarte1, Geoff W. Grime2A, Melanie Bailey2B, Gillian Lord2B, Hannah
Farnfield2B, Neil I. Ward2B, Paul N. Bishop1, Neville N. Osborne3. 1School of
Biomedicine, University of Manchester, Manchester, United Kingdom; ASurrey Ion
Beam Centre, BICP-MS Facility, 2University of Surrey, Guildford, United
Kingdom; 3Fundacin de Investigacin Oftalmolgica, Instituto Oftalmolgico
Fernndez-Vega, Oviedo, Spain.
Purpose: To determine the concentration and distribution of various metals in the
rat neuroretina.
Methods: Retinas from adult male Sprague-Dawley rats (n=6) were immediately
frozen in liquid nitrogen. Inductively coupled plasma mass spectrometry (ICPMS)
(Agilent 7700 Series, Agilent Technologies, UK) was then used to quantify the
level of different metals in dried neuroretina from one eye. Dried frozen sections
(10 microns thick) of the fellow eye were used to determine the concentration of
metals within individual retinal layers. Here, the 3 proton microbeam facility on the
2MV Tandetron accelerator at the Surrey Ion Beam Centre was used. Trace and
minor element mapping of retinal sections was obtained with a beam of 3MeV
protons focused to 1 micrometer in diameter using particle-induced X-ray emission
(PIXE) combined with Rutherford backscattering spectrometry (RBS).
Results: The most abundant metal in the neuroretina was iron, followed by
aluminium, zinc and vanadium. Copper, nickel, manganese, chromium, cobalt,
selenium and cadmium were present in trace amounts. PIXE analysis yielded a
non-homogenous pattern distribution of metals in the retina. The highest amounts
(microg/g of dry retina) of iron were found in the RPE (121+32.9) and
photoreceptor inner segments (107+39). Zinc was most concentrated in the inner
nuclear layer (97+29.6) and the plexiform layers (OPL, 75+30.3; and IPL,
64+20.1). Copper was localised primarily in the photoreceptor inner segments
(16+5.4) and also in the plexiform layers (OPL, 17+1; and IPL, 13.25+3.4).
Conclusions: The present studies provide new information related to the
distribution and content of various metals in the mammalian retina.
Commercial Relationships: Marta Ugarte, None; Geoff W. Grime,
None; Melanie Bailey, None; Gillian Lord, None; Hannah Farnfield, None; Neil
I. Ward, None; Paul N. Bishop, None; Neville N. Osborne, None
Support: None
The Role of Hyperglycemia in Retinal Acidosis in the Rat Retina
Desmond L. Henderson1A, Jennifer C. Lau1B, Robert A. Linsenmeier1C.
A
Neurobiology, BChemical and Biological Engineering, CBiomedical Engineering
Dept, 1Northwestern University, Evanston, IL.
Purpose: To determine the extent of acidosis in normoglycemia and
hyperglycemia by measuring pH profiles in the rat retina.
Methods: Double-barreled ion-selective microelectrodes were made to measure
profiles throughout the retina of dark-adapted intact Long-Evans rats. Profiles were
measured in the normal retina, and in acute hyperglycemia in vivo by penetrating to
the choroid and then slowly withdrawing the electrode. The rats were anesthetized
during preparation with a 2.5-3% isoflurane/35% O2 mixture and during recordings
with urethane. The intraretinal electroretinogram (ERG) was evaluated to determine
the retinal depth of the electrode.
Results: Under normoglycemic conditions, rats had an average [H+] of 4.21E-08
3.78E-09 Moles/L (n=5 rats) at the choroid (pH= 7.37). The [H+] gradually
increased as the electrode was withdrawn from the choroid through the retina,
reaching a minimum pH of 7.10 in the outer nuclear later. The pH increased
through the inner retina, to a vitreal value of pH=7.20. Acutely hyperglycemic rats
(n=3, glucose 296.72 mg/dl) had an average choroidal, minimum outer retinal, and
vitreous pH of 7.33, 7.05, and 7.12 respectively.
Conclusions: Intraretinal rat pH profiles are similar to those in cats during
normoglycemia, and in acute hyperglycemia produced by a concentrated IV
injection of glucose (Padnick-Silver and Linsenmeier, 2002). The extracellular pH
in the retina, which is unusually acidic, acidifies still further under hyperglycemic
conditions, particularly in the inner retina.
Commercial Relationships: Desmond L. Henderson, None; Jennifer C. Lau,
None; Robert A. Linsenmeier, None
Vijayakumar Arumugam Ramamoorthy1A, Velpandian Thirumurthy1A, Biswas NR1B,

Menon V1C, Saxena R1C, Ghose S1D. AOcular Pharmacol & Pharm, BPharmacology,
C
Squint, Neuro-Ophthalmology & Glaucoma, DOphthalmology, 1All India Inst of
Med Sci, New Delhi, New Delhi, India.
Purpose: To study the protective effect of non-NMDA antagonist trimetazidine
against ethambutol induced retinal toxicity in rats using Electroretinogram (ERG).
Methods: White albino rats of either sex weighing 250-300gms (n=6) were
randomized into three groups: Group 1 received distill water and served as a
vehicle control. Group 2 received oral ethambutol at the dose of 200mg/kg/day and
served as control. Group 3 fed with oral ethambutol and injected with trimetazidine
intraperitonially at the dose of 3mg/kg/day. All treatments were continued for 21
days and ERG was recorded on 0, 1, 7, 14th and 21st day after the initiation of
experiment. ERG with gold electrodes (with green and white light) were recorded
after anesthesia using a standard procedure. Vitreous humor and blood samples
were subjected quantification of ethambutol using liquid chromatography coupled
tandem mass spectroscopy.
Results: Ethambutol induced ERG changes were observed from 7th day following
the initiation of treatment in rats. Differences in ERG were observed between white
and green light conditions suggesting the ethambutol toxicity on photoreceptors.
Trimetazidine pretreatment significantly reversed ERG changes induced by
ethambutol (as observed in both white (P=0.002) and green (P=0.004) light records
for a wave amplitude). Interestingly, trimetazidine pretreatment significantly
reversed b wave amplitude recorded in green light (P=0.007) whereas this effect
was insignificant on white light condition.
Conclusions: Trimetazidine showed a significant reversal of ethambutol induced
ERG changes in rats. This effect of trimetazidine was prominent in the ERG
observed with green light as compared white light. This study indicates the possible
beneficial role of trimetazidine in reversal of ethambutol-induced retinal toxicity.
Commercial Relationships: Vijayakumar Arumugam Ramamoorthy,
None; Velpandian Thirumurthy, None; Biswas Nr, None; Menon V,
None; Saxena R, None; Ghose S, None
Support: None
Dopamine Enhances the Potassium Inward Rectifier Current of Cultured
Retinal Horizontal Cells
Renate Pflug, Leo M. Schneider. Ctr for Physiology & Pharmacol, Medical
University of Vienna, Vienna, Austria.
Purpose: Retinal horizontal cells are second order neurons that modify
photoreceptor signals through a feedback circuit. They bear receptors for dopamine.
Dopamine release is triggered by light stimulation The aim of this study is to assess
the effects of dopamine on the inward rectifier current of cultured retinal horizontal
cells.
Methods: Retinal cell cultures have been prepared from 8-12 day old rabbit and
mouse retinae. Cultured rabbit A-type horizontal cells (HC) can be easily detected
by their characteristic shape. In cell cultures of mouse retinae identification of
putative HC based on their shape and their characteristic Ca 2+- currents has been
supported by calbindin staining after recording but has still to be regarded as
equivocal since similar criteria apply to at least one type of amacrine cell.
Dopamine effects on voltage gated currents were investigated by whole cell patch
clamp recording of cells that had been kept in culture for 3 to 14 days.
Results: Both, rabbit and putative mouse HC show a potassium inward rectifier
current (IKir) that can be blocked by Cesium. Adding 1 -10M dopamine to the
extracellular solution increased IKir (155%, average of 11 mouse HC; 141%,
average of 18 rabbit HC; 10M dopamine). Adding GTPS to the pipette solution
inhibited the effect of dopamine. Enhancement by dopamine could be blocked by a
1-10fold concentration of the D1-type antagonist SCH23390 or by an equal
concentration of the D2-type antagonist sulpiride. Dopamine effects could be
mimicked by a 0.1 fold concentration of the D2 agonist quinpirole but never by the
D1 agonist SKF38393.
Conclusions: Dopaminergic enhancement of the potassium current IKir might serve
to steepen the slope of HC light responses and enable them to follow high
frequencies of light

Evaluation of the Protective effect of AMPA/Kainate Receptor Antagonist on
Ethambutol Induced Retinal Toxicity Using ERG in Rats

stimulation.
Commercial Relationships: Renate Pflug, None; Leo M. Schneider, None

Support: None
Comparison of Dexmedetomidine Sedation vs. Propofol in Vitreoretinal
Surgery Under Local Block
Linda Y. Huang1A, Jing Jing Feng1A, Marcelino Potian1B, Anuradha Patel1B,
Catherine Schoenberg1B, Xiuru Sun1, Dennis Grech1B, Neelakshi Bhagat1A.
A
Ophthalmology and Visual Science, BAnesthesiology, 1Univ of Med & Dentistry
of New Jersey, Newark, NJ.
Purpose: To compare the efficacy of dexmedetomidine (dex) vs. propofol (prop) in
vitreoretinal surgery under local block.
Methods: An IRB approved double-masked, prospective randomized study.
Enrollment criteria include subjects between ages 18 and 65 years, ASA 1-3, with
good liver and renal function. All procedures were performed UMDNJ's outpatient
same day surgery center by the same surgeon (NB) under retrobulbar block
administered using sub-Tenon approach. Patients are randomized into group P
(prop) and group D (dex). Group P receives a of 1 mg/kg of prop intravenously
(IV) followed by an infusion of 25-100 ug/kg/min. Group D receives a bolus of 0.5
ug/kg IV of dex followed by an infusion of 0.2-0.7 ug/kg/hr. T-test and MannWhitney test were used for statistical analysis.
Results: 38 patients have been enrolled; 22 - group P, 16 - group D with one
patient excluded due to anxiety and subsequent conversion to general anesthesia.
Comparisons of parameters are outlined below.
Intraoperative blood pressure (BP) was similar in both groups, with Group P having
lower average systolic BP at times 5 and 10 minutes. Average heart rates (HR) for
all patients were between 50 and 89 beats per minutes. Intraoperative respiratory
rates (RR) for group D were lower than group P at all time points, but significant
only at 15 and 30 minutes.
In the Post-Anesthesia Recovery Unit (PACU), Group D had decreased systolic
and diastolic BPs at all time points during the two-hour follow-up period. Also, HR
in the PACU was always lower in group D compared to group P, but only
significant at 120 minutes. There was no difference in PACU RR, oxygen
saturation did not drop below 92% at any time.
Conclusions: In the 38 patients recruited, dex provided adequate sedation, patient
and surgeon satisfaction, and hemodynamic stability, with no difference in
incidence of adverse effects compared to prop. The only significant parameters
between groups P and D were in systolic and diastolic BP and HR in the PACU.
There were not any clinically significant events that warranted the use of rescue
medications for bradycardia or hypotension. No patients experience post-operative
nausea or vomiting and none required hospitalization. The mean patient and
surgeon satisfaction was between good and excellent in both groups.
Commercial Relationships: Linda Y. Huang, None; Jing Jing Feng,
None; Marcelino Potian, None; Anuradha Patel, None; Catherine Schoenberg,
None; Xiuru Sun, None; Dennis Grech, None; Neelakshi Bhagat, None
Support: Humira, Inc
Incidence of Steroid Induced Ocular Hypertension Following Vitreoretinal
Surgery With Difluprednate Versus Prednisolone Acetate
Jonathan L. Prenner1A, Daniel B. Roth2, Howard F. Fine3, Harold M. Wheatley1B,
Daniel Connors1A. ARetina Vitreous Center, BOphthalmology-UMDNJ, 1Robert
Wood Johnson Med Sch, New Brunswick, NJ; 2Ophthalmology, Robert Wood
Johnson Med School, New Brunswick, NJ; 3Ophthalmology, Robert Wood Johnson
Univ Hosp, Eatontown, NJ.
Purpose: To identify changes in intraocular pressure (IOP) after vitreoretinal
surgical procedures in eyes that received either difluprednate ophthalmic emulsion
0.05% (DP) or prednisolone acetate ophthalmic suspension 1% (PA).
Methods: A retrospective chart review compared a consecutive series of 100
patients who received DP with 100 patients who received PA after vitreoretinal
surgery. Data were collected for a three-month period from the time of surgery.
Results: A significantly higher number of patients treated with DP (34%, n=34)
developed increased IOP (>10mm Hg change from baseline and greater than 21)
compared with those receiving PA (21%, n=21), (p=0.04). The mean maximum
IOP in the DP cohort (28.0 mm Hg) was significantly higher than in the PA cohort
(24.3 mm Hg), (p=0.01). Additionally, the rise in IOP from baseline was
significantly higher in the DP treated cohort (9.6 mm Hg) than in the PA treated
cohort (6.7 mm Hg), (p=0.02).
Conclusions: Eyes treated with DP after vitreoretinal surgery were at increased

risk for developing clinically significant increases in IOP compared with those
receiving PA.
Commercial Relationships: Jonathan L. Prenner, None; Daniel B. Roth,
None; Howard F. Fine, None; Harold M. Wheatley, None; Daniel Connors,
None
Support: None
Oxytocin Regulation of Retinal Pigment Epithelium Cell Function
Patrick J. Halbach1A, Matti Asuma1B, Weinxiang Luo1B, De-Ann M. Pillers1C,
Bikash R. Pattnaik2. APediatrics/ERP, BPediatrics, CDepartment of Pediatrics, Eye
Research Institute, 1University of Wisconsin-Madison, Madison, WI; 2Pediatrics
Ophthal & Visual Science, Eye Research Institute, Univ of Wisconsin, Madison,
WI.
Purpose: Oxytocin (OT) is a neuropeptide that activates oxytocin receptor (OTR),
a rhodopsin family G-protein coupled receptor. OTR signals the cellular
phosphatidylinositol-calcium second messenger system. Within the retina, the cross
talk between retinal pigment epithelium (RPE) and photoreceptor neurons is
hypothesized to be mediated by one such phosphatidylinositol-calcium signaling
mechanism. Several molecules, particularly ATP, are proposed to mediate RPEretina communication. Since we have localized OTR transcripts to RPE, we were
interested in knowing whether RPE cells also utilize oxitocinergic signaling for
communication.
Methods: Human fetal RPE (hRPE) cells were cultured as a tight monolayer using
serum-free medium containing MEM alpha base medium, N1 supplement, nonessential aminoacids, taurine, hydrocortisone and triiodo-thryonin. Cultures were
maintained at 37C and 5% CO2 with a media change every 2-3 days. Cytoplasmic
content of isolated cells were harvested and analyzed by single-cell RT-PCR. We
used a two-step nested PCR method to amplify the transcripts. Immomix PCR mix
and appropriate primer pairs were utilized to amplify OTR and other RPE-specific
transcripts in individual cells. Localization of OTR to the RPE layer of monkey
frozen sections was determined by standard immunofluorescence methods.
Intracellular Ca2+ ([Ca2+]i) mobilization in response to OT and ATP was conducted
via live-cell imaging and FURA-2 ratiometric measurements.
Results: In four individual hRPE cells, a 256 bp transcript corresponding to the
OTR messenger was amplified. These cells also tested positive for RPE-specific
markers RPE65, Kir7.1, hBest1, NaKATPase, and ezrin transcripts. OTR was
localized to the posterior aspects of monkey RPE cells. hRPE cells in culture, when
stimulated by OT or ATP, exhibited a 70-120 nM increase of [Ca2+]i that was
completely reversible. However, while ATP induced an instantaneous increase in
Ca2+, OT induced a gradual rise, suggesting ATP and OT may be acting through
independent signaling mechanisms.
Conclusions: Localization of OTR in RPE is a novel finding that implicates an
important regulatory role of OT in the RPE-retina communication. The increase of
[Ca2+]i in response to OT stimulation, as well as previously established circadian
regulation of the RPE, suggests that the RPE may utilize oxytocinergic signaling as
one aspect of endocrine regulation of RPE function.
Commercial Relationships: Patrick J. Halbach, None; Matti Asuma,
None; Weinxiang Luo, None; De-Ann M. Pillers, None; Bikash R. Pattnaik,
None
Support: UW Pediatrics, Rebecca Meyer Brown Professorship of Retina Research
Foundation, Meriter Foundation
Comparison Of Macular Pigment In Patients With Age-related Macular
Degeneration And Healthy Control Subjects - A Study Using Spectral Fundus
Reflectance
Semira Kaya1A, Guenther Weigert1B, Berthold Pemp1B, Stefan Sacu1B, Rene M.
Werkmeister1C, Nikolaus Dragostinoff1C, Gerhard Garhofer1A, Ursula SchmidtErfurth1B, Leopold Schmetterer1A,1C. ADepartment of Clinical Pharmacology,
B
Department of Ophthalmology, CCenter for Medical Physics and Biomedical
Engineering, 1Medical University of Vienna, Vienna, Austria.
Purpose: Previous studies have reported an age-dependent decline of macular
pigment optical density (MPOD) as well as a relative lack of MPOD in age-related
macular degeneration (AMD). Results are, however, strongly dependent on the
technique used. In the present study we investigated the age-dependence of MPOD
using spectral fundus reflectance. In addition, we hypothesized that patients with
age-related macular degeneration (AMD) have a reduced MPOD as compared to
healthy controls.
Methods: A total of 85 healthy subjects and 96 patients with AMD were included
in this study. The healthy control subjects showed a wide range of ages (mean 51.6
years, range 21-79 years). Patients with AMD were significantly older (mean 71.2
years, range 50-89 years). Spectral fundus reflectance of the fovea was measured in
a 2.3 detection field with a custom built fundus reflectometer. Calculation of
MPOD was based on a previously published fundus reflectance model.
Results: Patients with AMD showed a reduced MPOD (0.35 0.12) as compared
to the healthy control group (0.39 0.12, p = 0.013 between groups). No age
dependence of MPOD (r = -0.14, p = 0.19) was found in the healthy control group.
In the AMD group, however, MPOD declined with age (r= -0.24, p = 0.019).
Conclusions: The present study indicates that MPOD is reduced in patients with
AMD. In addition, the data of the present study indicate that MPOD is age
dependent in AMD patients, but not in healthy controls. Taken together with data
indicating that lutein supplementation increases MPOD, this provides a rationale
for supplementation of the macular pigments in patients with AMD, although longterm clinical outcome data are lacking.
Commercial Relationships: Semira Kaya, None; Guenther Weigert,
None; Berthold Pemp, None; Stefan Sacu, None; Rene M. Werkmeister,
None; Nikolaus Dragostinoff, None; Gerhard Garhofer, None; Ursula SchmidtErfurth, None; Leopold Schmetterer, None
Support: Pharmaselect
Role of Prostanoid Receptors in the Excitatory Effect of Neuroprostanes on
Potassium Induced [3H]D-Aspartate Release in Isolated Bovine Retina
Catherine A. Opere1, Arnecia Flowers1, Namonique Floyd1, Edem Kegey1, Jamal
Jamil1, Thierry Durand2, Jean-Marie Galano2, Alexandre Guy2, Ya Fatou NjieMbye3, Sunny E. Ohia3. 1Pharmacy Sciences, Creighton University, Omaha, NE;
2
Facult de Pharmacie, Institut des Biomolcules Max Mousseron (IBMM),
Montpellier Cedex, France; 3College of Pharmacy & Health Sciences, Texas
Southern University, Houston, TX.
Purpose: There is evidence that neuroprostanes (nPs), a series of isoprostane
(IsoP)-like compounds that are spontaneously generated via free-radical catalyzed
peroxidation of long chain polyunsaturated fatty acids are elevated in
neurodegenerative conditions (Musiek et al., Brain Pathol. 15:149,2005). However,
their effect on excitatory neurotransmitter release in neuronal tissues has not been
fully elucidated. Purpose: In this study, we investigated the regulatory effect of the
eicosapentanoic aid (EPA)-derived epimer pair, 5(S)-F3t-IsoP (CO5-667) and 5-epi5-F3t-IsoP(CO5-668) and the docosahexaenoic acid (DHA)-derived nP, 4(S)-F4t-nP
(CO5-738) on K+-induced glutamate release (using [3H]D-aspartate as a marker) in
isolated bovine retina. We also examined the mechanism by which CO5-738
regulates this excitatory neurotransmitter release.
Methods: Freshly isolated bovine retinae were incubated in oxygenated Krebs
solution (pH 7.45; 37 C) containing 200nM of [3H] D-aspartate for 60 mins and
then prepared for studies of neurotransmitter release using the well established
superfusion method. Release of [3H]D-aspartate was evoked by iso-osmotic
concentration of K+ (50mM)-stimuli applied at 80-88 mins (S1) and 116-124 mins
(S2) after the onset of superfusion. When used, the antagonist was present before
and during S1 and S2.
Results: In the concentration range, 1 nM to 1 M, the nPs enhanced K+-induced
[3H]D-aspartate release from bovine retina without affecting basal [3H]D-aspartate
efflux. Of the EPA-derived epimer-pair examined, the cis-conformer, C05-668
exhibited a higher potency, achieving a maximal excitatory response of 80%
(p<0.01, n=4) at the 10 nM concentration of the nP. At an equimolar concentration
of 10 nM, the rank order of activity of the nPs was as follows: CO5-668> CO5738> CO5-667. Interestingly, the excitatory effect elicited by the DHA-derived nP,
CO5-738 (0.1 M) was completely reversed by the prostanoid TP/DP2-receptor
inhibitor, ramatroban (BAY-U3405) (10M).
Conclusions: In conclusion, the EPA- and DHA-metabolites enhance K+-induced
[3H]D-aspartate release in bovine retina. Moreover, prostanoid TP/DP2-receptors
mediate the excitatory action exhibited by the DHA-metabolite, CO5-738 on the
neurotransmitter release.
Commercial Relationships: Catherine A. Opere, None; Arnecia Flowers,
None; Namonique Floyd, None; Edem Kegey, None; Jamal Jamil,
None; Thierry Durand, None; Jean-Marie Galano, None; Alexandre Guy,
None; Ya Fatou Njie-Mbye, None; Sunny E. Ohia, None
Support: None
Endothelin B Receptors Contribute to Neurodegeneration in a Rodent Model
of Glaucoma
Alena Z. Minton, Nitasha R. Phatak, Hai-Ying Ma, Dorota L. Stankowska,
Shaoqing He, Brett H. Mueller, Raghu R. Krishnamoorthy. Cell Biology and
Anatomy, Univ of North Texas Hlth Sci Ctr, Fort Worth, TX.
Purpose: ETB receptors have gained increased attention for their
neurodegenerative role in glaucoma. The present study was aimed at investigating
changes in the expression of ETB receptors in vivo in a rodent model of glaucoma
and whether neurodegenerative changes following IOP elevation are attenuated in
ETB-deficient transgenic rats.
Methods: In one set of Brown Norway rats, IOP was elevated in one eye using the

Morrisons method (injection of hypertonic saline through episcleral veins), while
the corresponding contralateral eye served as control. After 2 or 4 weeks of IOP
elevation, animals were sacrificed. Retinal sections were obtained and stained for
ETB receptor expression by immunohistochemistry. Colocalization of ETB receptor
immunostaining was performed using an antibody to neuritin, a selective marker of
RGCs. In the second set of Brown Norway rats, retinal ganglion cells were
retrogradely labeled with Fluoro-gold following which IOP was elevated in one eye
using Morrisons method, while the contralateral eye served as control. After 2
weeks of IOP elevation, animals were sacrificed. Retinas were sectioned and
stained for ETB receptor expression by immunohistochemistry. In a separate study,
adult wild type and ETB- deficient transgenic Wistar rats were used for retrograde
labeling of retinal ganglion cells (RGCs) with Fluoro-gold. Following labeling, IOP
was elevated in one eye using the Morrisons method, while the contralateral eye
served as control. After IOP was elevated, rats were maintained for 4 weeks and
sacrificed. Fluoro-gold labeled retinas were isolated, flat-mounted, imaged in a
Zeiss LSM-510 confocal microscope, and labeled RGCs were counted. Optic
nerves were sectioned and stained with p-phenylenediamine (PPD).
Results: Immunohistochemical analysis showed that IOP elevation for both 2 and 4
weeks produced increased expression of ETB receptors in retinal ganglion cells.
After retrograde labeling and IOP elevation in Brown Norway rats, increased
colocalization of ETB immunostaining with Fluoro-gold was observed in retinal
ganglion cells. IOP elevation for 4 weeks in wild type transgenic rats caused
significant loss of RGCs and degeneration of axons in the optic nerve, whereas
significant neuroprotection of RGCs and optic nerve axons were observed in ETBdeficient transgenic rats.
Conclusions: Increased intraocular pressure caused increased ETB receptor
expression possibly through pressure-related mechanism. ETB receptors appear to
play a causative role in retinal ganglion cell death in the Morrisons rodent model
of glaucoma.
Commercial Relationships: Alena Z. Minton, None; Nitasha R. Phatak,
None; Hai-Ying Ma, None; Dorota L. Stankowska, None; Shaoqing He,
None; Brett H. Mueller, None; Raghu R. Krishnamoorthy, None
Support: EY019952-01
Transcriptional Regulation of Endothelin B (ETB) Receptor in Retinas of Rats
in Response to Elevated Intraocular Pressure
Shaoqing He, Alena Z. Minton, Dorota Stankowska, Raghu R. Krishnamoorthy.
Cell Biology and Anatomy, University of North Texas Hlth Sci Ctr, Fort Worth,
TX.
Purpose: Expression of the endothelin B (ETB) receptor is increased in retinas of
eyes with elevated intraocular pressure (IOP) in a rat model of glaucoma. The aim
of this study was to study transcription factors contributing to upregulation of ETB
receptor expression in retinal ganglion cells in rats.
Methods: Luciferase assays were employed to identify promoter elements involved
ETB gene expression in human non-pigmented ciliary epithelial cells, following
analysis of binding site using the software Promo 3. The Morrisons ocular
hypertension model in rats was developed by injection with hypertonic saline into
episcleral veins in the left eye to produce IOP elevation. Untreated right eye served
as the contralateral control. Immunofluorescent staining was used to determine cJun and C/EBP expression in retina sections from rats with elevated IOP. Realtime PCR was used to detect mRNA levels of c-Jun, C/EBP, ETA receptor and
ETB receptor from retinal ganglion cell layers obtained by laser capture
microdissection from cryosection of retinas.
Results: The -300 to -1 and -1200- to -600 bp upstream promoter regions of the
ETB receptor were found to be key elements that produced increased luciferase
expression. Using the Promo3 software, the 1200 bp promoter construct was found
to have six AP-1 binding sites. Immunostaining of c-Jun and C/EBP was
significantly increased in retinal ganglion cells in eyes with IOP elevation for two
weeks compared to the corresponding contralateral eyes. mRNA levels of c-Jun,
ETA receptor and ETB receptor were upregulated by 2.2-, 3.8- and 4.4-fold
respectively in retinal ganglion cell layer obtained from retinas of elevated IOP
eyes, compared to those from contralateral eyes.
Conclusions: The 1200 bp upstream promoter region is important for expression of
the human ETB gene. Increased expression and activation of c-Jun may contribute
to expression of ETB receptor retinas in response to elevated IOP. Six binding sites
of AP-1 in ETB receptor promoter may be important for inducible ETB expression
following IOP elevation. The direct role of c-Jun in controlling ETB receptor
expression is still under investigation, which will shed light on molecular
mechanisms underlying glaucomatous changes in rodent eyes with ocular
hypertension.
Commercial Relationships: Shaoqing He, None; Alena Z. Minton,
None; Dorota Stankowska, None; Raghu R. Krishnamoorthy, None

Properties And Bioavailability Of A Selective COX-2 Inhibitor In The Retina
After Optic Nerve Crush
Kathrin Bitz1A, Oliver W. Gramlich1A, Natarajan Perumal1A, Harald D. von Pein1B,
Anika Ziegler1A, Norbert Pfeiffer1A, Franz H. Grus1A. AExperimental
Ophthalmology, BDepartment of Neuropathology, 1University Medical Center
Mainz, Mainz, Germany.
Purpose: Celecoxib is a 382 Dalton (Da) selective cyclooxygenase 2 (COX-2)
inhibitor with potentially neuroprotective properties. Using the optic nerve crush
model, we investigate retinal ganglion cell (RGC) survival after topical
administration. Thereby, we prove a new mass spectrometry (MS) based approach
to analyze the bioavailability of Celecoxib in aqueous humor and retinal tissue.
Methods: Unilateral Optic nerve crush was performed in 20 Lewis rats. In ten
animals, 0.1% Celecoxib was applied topically (30 l, n=10) in the crushed eye
twice a day. The other 10 animals served as control without treatment. Fundus
photographies were taken through a surgical microscope regularly. After ten days,
neuronal survival was analyzed through cresyl stained retinal flatmounts
(cells/mm2), axon counts after toluidine blue staining or via Brn-3a
immunostaining in retinal cross-sections. Aqueous humor was collected post
mortem and biopsied retinal tissues were prepared in a raw and rapid extraction
method with chloroform for ESI- MS analysis via LTQ Orbitrap. Pure Celecoxib
was measured before the complex samples to assure Celecoxibs peak signature
and the exact weight.
Results: No major differences were visible in the fundi throughout the study. A
significant reduction of neuronal loss (p=0.005) and axon loss (p<0.001) was
detected in the topically treated group (2272636 cells/mm2; 548126 axons)
compared to the control (1985470 cells/mm2; 265128 axons). Thereby, the
analysis of Brn-3a labeled cells in cross-sections approved a reduction of RGC
around 10%. ESI-MS measurement of pure Celecoxib revealed a peak at 382.08
Da. In retinal tissues of topically treated eyes, a relatively low intensive peak of
exactly 382.08 Da was also available. The agent was absent in aqueous humor.
Conclusions: Topically applied selective COX-2 inhibitor promotes RGC survival
after optic nerve crush. Moreover, the availability of Celecoxib in retinal tissues
could be proven by MS. The reason for the relatively low intensity of the Celecoxib
peak is still unclear and might be due to the extraction method. Over all, this new
MS approach seems suitable for testing bioavailability of therapeutic agents in
ocular tissues, especially with further improvement of the extraction method and
later quantification.
Commercial Relationships: Kathrin Bitz, None; Oliver W. Gramlich,
None; Natarajan Perumal, None; Harald D. von Pein, None; Anika Ziegler,
None; Norbert Pfeiffer, None; Franz H. Grus, None
Support: None
The Multi-functional and Neuroprotective Peptide Derivative Carcinine Has
Strong Potential for Therapeutic Applications In Retinopathies
Anne Kasus-Jacobi1A, Huaiwen Wang1B, Feng Li1C, Kevin Wu1A, Mark
Babizhayev2, H. Anne Pereira1A. APharmaceutical Sciences, BMolecular Biology &
Cytometry Research, COphthalmology, 1Univ of Oklahoma Hlth Sci Ctr, Oklahoma
City, OK; 2Innovative Vision Products Inc., Moscow, Russian Federation.
Purpose: Carcinine (-alanyl histamine) combines antioxidant, peroxidase-like,
and 4-hydroxynonenal-scavenging activities and is a potent neuroprotector in
mouse retina subjected to light-induced oxidative damage. Our hypothesis is that it
could be beneficial for therapeutic applications in progressive retinopathies
involving oxidative damage.
Methods: Carcinine was administered topically through eyedrops to Balb/C mice
and pharmacokinetic and pharmacodynamic studies were performed. Carcinine was
quantified in dissected retinas by mass spectrometry. Mice were exposed to
damaging bright light (3,000 lux, 4h) and carcinine-mediated protection of retinal
structure was evaluated by optical coherence tomography and quantitative
histology. The mechanism of protection was investigated by immunoblot
quantification of 4-hydroxynonenal-modified protein, photoreceptor retinol
dehydrogenase 12 (RDH12), and hexokinase 1 (HK1).
Results: Following topical administration, carcinine reaches the retina in a doseand time-dependant manner. Retinal levels of carcinine reach a plateau starting at
0.2 M carcinine in eyedrops, suggesting a saturable transport mechanism. This
concentration is sufficient to maintain a normal retinal architecture and prevent rod
photoreceptor cells from apoptosis when carcinine is administered both during and
after exposure to bright light, but not if administered only during exposure.
Carcinine significantly decreased the amount of retinal 4-hydroxynonenal-modified
protein. It maintained high level of retinal RDH12, a detoxification enzyme that is
otherwise rapidly degraded following light-induced oxidative modification. Finally,
carcinine decreased the level of a putative retinal splicing variant of HK1.
Conclusions: Oxidative damage is an established aggravating factor in progressive
retinopathies such as age-related macular dystrophy, diabetic retinopathy and
retinitis pigmentosa. Thus, carcinine is a good candidate for therapeutic
applications in these retinopathies. This small molecule could be administered non-

invasively through eyedrops every day to protect photoreceptor cells and delay
vision loss associated with these retinopathies.
Commercial Relationships: Anne Kasus-Jacobi, None; Huaiwen Wang,
None; Feng Li, None; Kevin Wu, None; Mark Babizhayev, 5,792,784;
WO94/19325; 7,795,203 B2 (P), Innovative Vision Products Inc. (C); H. Anne
Pereira, None
Support: EY018907 and the Oklahoma Center for the Advancement of Science
and Technology
Safety and Pharmacokinetics of Simvastatin using intravitreal delivery routes
in the mouse retina
Inyoung Chung1A,2, Dennis Y. Tse1A, Feng He1B, Theodore G. Wensel1B, Jong Moon
Park2, Samuel M. Wu1A. AOphthalmology, BBiochemistry and Molecular Biology,
1
Baylor College of Medicine, Houston, TX; 2Ophthalmology, Gyeongsang National
University, Jinju, Republic of Korea.
Purpose: The objective of this study is to determine retinal toxicity and
pharmacokinetic profile after intravitreal injection of Simvastatin in mice.
Simvastatin is a potent statins that has been shown to protect retinal ganglion cells
(RGCs) from degenerative insults. Our long-term goal is to investigate the
neuroprotective actions of Simvastatin on RGCs in chronic and acute glaucoma
mouse models.
Methods: Simvastatin (1ul of 500uM) was injected into the vitreous of the right
eye of the mice, and the vehicle solution was injected into the left eye as a control.
Simultaneous bilateral dark-adapted electroretinography (ERG) was performed 1, 3
and 7 days following the injection. High-performance liquid chromatography
(HPLC) of the isolated mouse retina was used to determine drug concentrations at
6, 24, 48 hours after injection.
Results: The amplitude and kinetics of the ERG a- and b-wave exhibited no
statistically significant differences between the two eyes in the all mice tested.
Average retinal levels of Simvastatin (determined by HPLC) was 2.33 pmol (or
0.96 ng)/retina 6hrs after intravitreal injection, 9.55 pmol (3.99 ng)/retina 24 hrs,
and 10.05 pmo(4.2ng)/retina 48 hrs after injection.
Conclusions: Intravitreal injection of 500uM Simvastatin is safe in the mice, as the
retinas show no sign of dysfunction days after injection (no change in ERG a- and
b-wave). Simvastatin levels in the retina maintains for at least 48 hours, suggesting
intravitreal injection is a reasonable way to deliver this neuroprotective agent for
potential therapeutic treatment of retinal diseases such as glaucoma.
Commercial Relationships: Inyoung Chung, None; Dennis Y. Tse, None; Feng
He, None; Theodore G. Wensel, None; Jong Moon Park, None; Samuel M. Wu,
None
Support: Grants from NIH EY004446 and EY019908, NIH Vision Core EY02520,
the Retina Research Foundation (Houston), and Research to Prevent Blindness, Inc.
Retinal Toxicity Of Preservative-free FDA-approved Triancinolone
(Triesence) In Rabbit Eyes: A Morphologic And Electroretinographic Study
Leandro C. Zacharias1A, Priscilla S. Akamine1B, Gabriela L. Ioshimoto1C, Balsz
Nagy1C, Beatriz S. Takahashi1A, Cristiano N. Pessa1B, Mirella T. Barboni1C,
Walter Y. Takahashi1A, Dnia E. Hamassaki1B, Dora F. Ventura1C.
A
Ophthalmology, BHistology, CExperimental Psychology, 1University of Sao Paulo,
Sao Paulo, Brazil.
Purpose: To evaluate the effects of commercially available preservative free
triamcinolone acetonide (Triesence) on an in-vivo rabbit model.
Methods: 30 Dutch belted rabbits were assigned to 3 different intravitreal drug
concentrations of Triesence: 1, 4 or 8 mg. The animals were anesthetized and the
right eye received drug, while the left received balanced salt solution. After 30 days
the animals were sedated and electroretinogram (ERG) was recorded. After the
ERG, the animals were euthanized and the eyes were collected for morphological
analysis. 12 other rabbits had histology only analysis after 7 days of injection.
ERGs were recorded with the RETIport system (Roland Consult, Germany) with a
Ganzfeld Q450 SC stimulator. Stimulation protocol was : scotopic condition:
flashes at 0.01, 3.0, and 10 cd.s/m2 and photopic flicker of 30 cd.s/m2 at 12 18, 24,
and 30 Hz, with 30 cd.s/m2 background Amplitudes and implicit times of the awave and b-wave components and amplitudes of the FFT first harmonic were used
to analyze the responses. Wilcoxon signed rank test was used to compare related
samples.
Results: Dark-adapted ERGs did not show any difference between drug and
control groups. Light-adapted ERGs had smaller b-wave amplitudes in the 4 and 8
mg groups (p<0.05), but not in the 1mg group. There was a reduction in flicker
amplitude at 24 and 30 Hz in the 8mg group and at 30Hz in the 4mg group
(p<0.05). For 12 and 18Hz in the 8mg group p= 0.05. No difference was observed
in Hematoxylin-Eosin, Tunnel, Fluoro-Jade B and glial fibrillary acid protein
(GFAP) between any experimental and control retinas after 30-day injection.
However, after 7 days, retinas from the 8mg group showed GFAP-positive
processes of Muller glial cells at the bottom part of the retina, where the drug was
sited.
Conclusions: Rabbit retinas submitted to intravitreal injections of Triesence
show morphological, as well as ERG changes. GFAP-positive processes were
detected after 7 days, and disappeared after 30 days, suggestive of transient Muller
cell activation. No signs of apoptosis or necrosis were observed even at the highest
dose tested. The ERG results suggest retinal toxicity affecting the cone system with
[4mg] and [8mg] (four or eight times the clinical dose). The drug also affects
retinal mechanisms related to temporal processing at high frequencies.
Commercial Relationships: Leandro C. Zacharias, None; Priscilla S.
Akamine, None; Gabriela L. Ioshimoto, None; Balsz Nagy, None; Beatriz S.
Takahashi, None; Cristiano N. Pessa, None; Mirella T. Barboni, None; Walter
Y. Takahashi, None; Dnia E. Hamassaki, None; Dora F. Ventura, None
Support: FAPESP, CNPq
Bioavailability And Pharmacokinetics Of A Synthetic DHEA Analog, A Novel
Anti-apoptotic Agent, After IP Injection In Normal Rodents
Chrysanthi Tsika1A, Pavlina A. Tsoka2, Manolis Tzatzarakis1B, Paschalis
Efstathopoulos1C, Sophia Antimisiaris3, Ioannis Charalampopoulos1C, Achilleas
Gravanis1C, Miltiadis K. Tsilimbaris1D. ADepartment of Ophthalmology,
B
Department of Forensic Sciences and Toxicology, CDepartment of Pharmacology,
D
Ophthalmology-Research Acct, 1University of Crete, Heraklion, Greece;
2
Neurology & Sense Organs, University of Crete, Heraklion, Crete, Greece;
3
Department of Pharmacy, University of Patras and FORTH-Institute of Chemical
Engineering, Patras, Greece.
Purpose: To investigate the bioavailability of a synthetic Dehydroepiandrosterone
(DHEA) analog, a novel anti-apoptotic agent, after intraperitoneal (IP)
administration in normal rodents.
Methods: The pharmacokinetics in the blood were evaluated in C57BL/6 mice,
after IP injection of the molecules solution at concentration C=10mg/ml. The
blood was collected from the orbital sinus of 5 animals per time point, at time
points 0, 30, 60, 120, 240 and 360mins, 12hrs and 24 hrs. The retinal
bioavailability was evaluated after transcardial perfusion with Ringer-Lactate
solution for 15 min in 5 Sprague-Dawley rats, 2hrs after IP administration
(C=10mg/1ml). The molecule was also administered in 5 Sprague Dawley rats in a
cyclodextrin solution of the same concentration. The bioavailability in the retina
was also evaluated after 2 hrs. The quantification was performed with HPLC
LC/MS.
Results: The molecule follows first order kinetics in the blood (k=0.54,
t1/2=1.2hrs), while second order kinetics have been measured in the retina tissue in
our previous experiments (ARVO 2011). The mean concentration in the retina after
perfusion was found 104ng/ml (SD=50.3). The mean concentration of the
cyclodextrin-solution in the retina tissue was 194ng/ml (SD= 61.2).
Conclusions: The blood bioavailability of this DHEA analog proved to follow first
order kinetics after IP administration in C57BL/6 mice. The difference in kinetics
between blood and retina tissue together with the detection of the substance in the
retinal tissue after perfusion provide convincing proof of the presence of the
substance in the rat retina after intraperitoneal administration. Identification of the
substance in the retina after administration of the cyclodextrin formulation is an
encouraging outcome for the development of alternative pharmaceutical forms of
this anti-apoptotic agent.
Commercial Relationships: Chrysanthi Tsika, None; Pavlina A. Tsoka,
None; Manolis Tzatzarakis, None; Paschalis Efstathopoulos, None; Sophia
Antimisiaris, None; Ioannis Charalampopoulos, None; Achilleas Gravanis,
None; Miltiadis K. Tsilimbaris, None
Support: None
Characterization of the Dark Adaptation Curve of the Domestic Pig
Gil Ben-Shlomo1A, Jason W. Ross1B. AVeterinary Clinical Sciences, BAnimal
Science, 1Iowa State University, Ames, IA.
Purpose: The full field electroretinogram (fERG) is a very useful, objective, noninvasive tool to assess retinal function used extensively for research and in various
animal models. The fERG is used in various porcine models of retinal diseases. To
date, despite scientific need, there is lack of data regarding the properties of the
normal porcine ERG and dark adaptation curve. The purpose of this study is to
evaluate the dark adaptation curve of the domestic pig by means of full field
electroretinogram.
Methods: The electroretinographic responses were recorded bilaterally from six
healthy female pigs, 6 months old using a contact lens electrode and a miniGanzfeld electroretinographic unit. The pigs were anesthetized and the ERG was
recorded in response to 4 low intensity (10 mcd.s/m2) light stimuli given at a
frequency of 0.1 Hz at each time (T) point: T= 5, 10, 15, 20, 25, 30, 40, 50 and 60
min of dark adaptation. Off-line analysis of the ERG was then performed.

Results: Mean b-wave amplitude of the full-field ERG increased continuously
from 5 to 25 min of dark adaptation (peak amplitude at T(25) = 204V), and then
reached a plateau until the end of the study at T = 60. No distinct a-wave was
observed during the testing time.
Conclusions: Evaluation of porcine rod function or combined rod/cone function by
means of full-field ERG should be performed after a minimum 25 min of dark
adaptation.
Commercial Relationships: Gil Ben-Shlomo, None; Jason W. Ross, None
Support: ISU, Competitive Research Grant
The involvement of neuroglobin in mitochondrial integrity: the case of the
Harlequin Mice
Christophe Lechauve1A, Aicha Bouaita1A, Sebastien Augustin1A, Helene CwermanThibault1, Jose A. Sahel, Jr.1B, Marisol Corral-Debrinski1A. AInserm, BUMR-S 968,
1
Institut de la Vision, Paris, France.
Purpose: Harlequin (Hq) mouse exhibits a 80% decrease of AIF (Apoptosis
Inducing Factor) which is essential for respiratory chain complex I activity. Thus,
the mitochondrial impairment induced leads to progressive retinal neuron loss and
ultimately blindness. Neuroglobin (Ngb) is a monomeric hemoprotein of the globin
family that is mainly expressed in neurons of the central and peripheral nervous
systems. We demonstrated that Ngb function is a reliable marker of mitochondrial
activity, since its downregulation in retinal ganglion cells (RGC) is correlated to
complex I and III defects and cell loss. Moreover, in rat retinas a significant amount
of the protein localizes to mitochondria. Our main objectives were to extensively
define gene expression changes in Hq mice and to determine whether Neuroglobin
overexpression could protect RGC integrity in these mice. For the latter we used an
optimized AAV2 vector containing the Ngb ORF with its entire 5 and 3 UTRs.
Methods: Morphological and functional examinations of retinal damage were
performed in Hq mice over time. Mitochondria and oxidative stress PCR Array
profiles were performed to study the expression of genes involved in mitochondrial
biogenesis and function (electron transport chain, oxidative phosphorylation
complexes) and retinal oxidative stress. Protein expression, microglial activation,
and reactive gliosis were evaluated by immunohistochemistry on retinal crosssections.
Results: First signs of RGC and nerve fiber decrease were evidenced in mice aged
of 4 months leading to visual performance decline at 6-8 months. We show
differences of gene expression between Hq and control mice correlate to retinal
degeneration. For instance, Ngb expression decrease of 50% in Hq compare to
control and is associated with a decrease in the Ngb intensity of labeling. These
changes permit us to consider an AAV therapeutic gene therapy to prevent RGC
loss.
Conclusions: We demonstrate major change of gene expression reliable with
mitochondrial activity and intimately associated with RGC survival in Hq mice.
Thus these mice represent a faithful genetic model which mimics human retinal
degeneration due to mitochondrial impairment.
Commercial Relationships: Christophe Lechauve, None; Aicha Bouaita,
None; Sebastien Augustin, None; Helene Cwerman-Thibault, None; Jose A.
Sahel, Jr., None; Marisol Corral-Debrinski, None
Support: ANR, CNRS, INSERM, SANOFI-FOVEA
Conclusions: Our data suggest that ischemic retinopathy maybe mediated by

aberrant Zn homeostasis brought about by ZnT8 downregulation. In addition, YC-1
treatment plays a crucial role in impacting the ZnT8 expression level and Zn
homeostasis.
Commercial Relationships: Michael DeNiro, None; Falah H. Al-Mohanna,
None; Futwan Al-Mohanna, None
Support: None
Assessment of Macular Hole Dynamics Utilizing Intraoperative Optical
Coherence Tomography
Justis P. Ehlers, David Xu, Gina Smith, Sunil K. Srivastava. Cole Eye Institute,
Cleveland Clinic, Cleveland, OH.
Purpose: To assess the microarchitectural changes of macular hole (MH) anatomy
following surgical manipulations with intraoperative optical coherence tomography
(iOCT) and an automated segmentation algorithm.
Methods: A consecutive case series of ten eyes with full-thickness MH were
analyzed with iOCT at multiple points during surgical repair. All eyes underwent
vitrectomy and peeling of the internal limiting membrane (ILM). Pre-peel and postpeel iOCT images were obtained with a microscope-mounted spectral domain OCT
device. An automated software algorithm was developed to segment the boundaries
of the MH in consecutive B-scans allowing for high resolution measurements of
numerous dimensions of the MH architecture. A three-dimensional representation
of the MH was generated from the composite segmentation (Figure 1).
Results: Ten of 10 eyes were successfully imaged with iOCT at numerous time
points and analyzed utilizing the segmentation algorithm. Following ILM peeling,
there was a significant increase in mean MH volume (0.18 vs 0.14 mm3, p<0.005)
and base diameter (1334 vs 1073 microns, p<0.02). There was a trend towards
increased MH height (504 vs 462 microns, p=0.09) and decreased apical diameter
(540 vs 647 microns, p=0.06). Additionally, there was a significant reduction in the
apical/basal diameter ratio following ILM peeling (0.62 to 0.37, p<0.005).
Conclusions: MH anatomy undergoes significant changes during surgical repair.
Assessment with iOCT suggests significant increase in MH volume and basal
diameter following peeling of the ILM. Automated segmentation of MH is feasible
with resulting volumetric measurements. This high-resolution three-dimensional
measurement system combined with iOCT may help to identify MH features that
impact surgical outcomes and

Targeting Zinc Transporter-8 as a Therapeutic Approach in Ischemic
Retinopathy
Michael DeNiro1, Falah H. Al-Mohanna2A, Futwan Al-Mohanna2B. 1Research,
King Khaled Eye Specialist Hosp, Riyadh, Saudi Arabia; AComparative Medicine,
B
Cell Biology, 2King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi
Arabia.
Purpose: The Zinc (Zn) transporter encoded by SLC30A8, ZnT8, plays a
particularly important role in Zn homeostasis in beta cells. Previous studies have
indicated that an acute decrease in ZnT8 levels impairs beta cell function and Zn
homeostasis and may contribute to the pathophysiology of diabetes. The link
between ZNT8 and ischemic retinopathy has not been determined yet. It was the
aim of this study to address the impact of ischemic retinopathy on the expression of
ZnT-8 in vivo and in vitro.
Methods: We used an array of assays to evaluate the effects of YC-1, a small
molecule inhibitor of HIF-1 and a novel NO-independent activator of soluble
guanylyl cyclase (sGC) on ZNT8 expression; in vivo and in vitro.
Results: We show that hypoxic/ischemic insult mediates a significant
downregulation of ZnT8 at the message and the protein levels in the rat
neurosensory cells (in vitro), and the ischemic retina (in vivo). Our data indicate
that ZnT8-depleted cells are primarily localized in the neurosensory retina. Dualinjection regimen of YC-1 (100 M) into the neovascular retinas, at post-natal day
12 (P12) and (P15) restores the ZnT8 levels to basal homeostatic level, which was
comparable to those of the nontreated normoxic retinas.

management.
compared to baseline (P < 0.01). Histopathological analysis of eyes demonstrated

both inner and outer retinal atrophy and necrosis.
Conclusions: Our findings indicate that the intravitreal injection of polyethylene
glycol-400 is toxic to retina and it should not be used as a vehicle in the eye.
Commercial Relationships: Kasra Attaran-Rezaei, None; Toma Hassanian,
None; Stephen J. Kim, None
Support: 1. Unrestricted Grant from Research to Prevent Blindness to the
Vanderbilt University School of Medicine Dept. of Ophthalmology and Visual
Sciences. 2. Vanderbilt Vision Research Center (P30EY008126)
The Treatment Of Chronic Central Serous Chorioretinopathy With Reduced
Power Pdt
Mayumi Kaida1A, Takeya Kohno1B, Michiko Hirabayashi1A, Kunihiko Shiraki2.
A
Ophthalmology, BOphthalmology & Visual Science, 1Osaka City Univ Grad Sch
of Med, Osaka, Japan; 2Ophthalmology, Osaka City Univ Graduate Med, Abeno
ku, Japan.
Purpose: To evaluate the efficacy of reduced power photodynamic therapy (PDT)
for chronic central serous chorioretinopathy (CSC).
Methods: We reviewed the medical records of seven eyes of 7 patients with
chronic CSC, on who were reduced power PDT performed and followed up for
more than 6 months after the treatment. They were six males and one female, and
the average age was 58.0 years old. All of them were suffered from CSC for more
than three months. The total PDT energy was reduced to about a half power by
usage of the contact lens with 1.44 magnifying power. The visual acuity and the
degrees of central macular thickness (CMT) were compared before and after the
treatment. The resolution and the recurrence of serous retinal detachment (SRD)
and pigment epithelial detachment (PED) were also accessed by optical coherence
tomography.
Results: The average of visual acuity before the treatment was 0.187 in logMAR.
PED and/or SRD were observed in all cases. At one month after the treatment, the
average of visual acuity was 0.188, and both PED and SRD were disappeared in all
cases. The average of CMT was 312 micrometers before PDT and reduced to 190.6
micrometers at one month after the treatment. The average of visual acuity at 6
months after PDT was 0.023, one of CMT was 193 micrometers, and no recurrence
of PED or SRD was observed in any cases.
Conclusions: The reduced power PDT is effective in chronic CSC to diminish the
thickness of retina, extinguish both PED and SRD and suppress the recurrence of
them. The good visual acuity is able to be maintained even six months after the
treatment.
Commercial Relationships: Mayumi Kaida, None; Takeya Kohno,
None; Michiko Hirabayashi, None; Kunihiko Shiraki, None
Support: None
503 Gene Therapy and Delivery II
Thursday, May 10, 2012, 8:30 AM - 10:15 AM
Room 114 Paper Session
Commercial Relationships: Justis P. Ehlers, None; David Xu, None; Gina

Smith, None; Sunil K. Srivastava, None
Support: None
Retinal Toxicity of Polyethylene Glycol (PEG)-400
Kasra Attaran-Rezaei1A, Toma Hassanian1A, Stephen J. Kim1B. AOphthalmology &
Visual Science, BOphthalmology, 1Vanderbilt University, Nashville, TN.
Purpose: Polyethylene glycol (PEG)-400 is a clear, colorless, viscous liquid that
readily dissolves many hydrophobic drugs. It is a widely used commercial solvent
and vehicle for many pharmaceutical and cosmetic products and safe for human
oral consumption and topical application. To our knowledge, however, there are no
reports on the intraocular safety of PEG-400. The purpose of this study is to
investigate the intraocular safety of PEG 400 to determine its suitability as a
potential vehicle for intraocular delivery of hydrophobic drugs.
Methods: Six healthy, male, Dutch Belted rabbits, weighing between 2.0 and 3.0
kg, were used for this experiment. The left eye of each animal received a single
0.1ml intravitreal injection of PEG-400. Complete eye exams and dark- and lightadapted electroretinograms (ERG) of both eyes were obtained at baseline and at 1
and 4 weeks after injection. After the 4-week ERG was completed, animals were
euthanized and eyes were enucleated and analyzed with histology and transmission
electron microscopy (TEM).
Results: Ophthalmic examinations demonstrated signs of retinal necrosis in all 6
left eyes injected with PEG-400 which was apparent at 1 week but more evident by
4 weeks. Photopic and scotopic ERG studies of left eyes injected with PEG-400
showed significantly reduced b-wave amplitudes at 1 and 4 weeks after injection

A Comparative Evaluation Of Translational Read-through Inducing Drugs
For Treatment Of Ush
Kerstin Nagel-Wolfrum1, Tobias Goldmann1, Fabian Mller1, Nora Overlack1,
Valery Belakhov2, Timor Baasov2, Uwe Wolfrum1. 1Cell and Matrix Biology,
Johannes Gutenberg University of Mainz, Mainz, Germany; 2Edith and Joseph
Fischer Enzyme Inhibitors Laboratory, Schulich Faculty of Chemistry, TechnionIsrael Institute of Technology, Haifa, Israel.
Purpose: Translational read-through inducing drugs (TRIDs), promote readthrough of nonsense mutations, placing them in the spotlight of current gene-based
therapeutic research. We compare the efficacies of different TRIDs as a treatment
option for nonsense mutation-based retinal disorders, focusing on the human Usher
syndrome (USH). USH is the most frequent cause of inherited combined deafblindness. It is clinically and genetically heterogeneous, assigned to three clinical
USH types and 11 known gene loci. So far no effective treatment for the
ophthalmic component of USH exists. In USH, nonsense mutations account for
~11% of all disease-causing mutations.
Methods: We assessed retinal toxicities of TRIDs in mouse and human retinal
explants by TUNEL-assays and immunofluorescence analyses applying molecular
markers for retinal integrity. We generated constructs coding for disease-related
nonsense mutations in USH1C and USH2A and quantified TRIDs induced readthrough on the different USH causing nonsense mutations in HEK293T cells,
mouse retinal explants and in vivo in mice.
Results: In comparison with classical aminoglycoisdes (gentamicin), designer
aminoglycosides and PTC124 exhibits significant better retinal biocompatibility.

We observed a dose-dependent read-through of the USH nonsense mutations by
TRIDs in transfected cultured cells. We demonstrate the functionality of the
recovered proteins. In addition, we show TRIDs induced read-through in
transfected retinal explants and in vivo in murine retinas.
Conclusions: The high biocompatibility combined with the good read-through
efficacies of TRIDs emphasizes the potential of designer aminoglycosides and
PTC124 in treating nonsense mutation-based retinal disorders, in particular for
USH.
Commercial Relationships: Kerstin Nagel-Wolfrum, None; Tobias Goldmann,
None; Fabian Mller, None; Nora Overlack, None; Valery Belakhov,
None; Timor Baasov, None; Uwe Wolfrum, None
Support: Deutsche Forschungsgemeinschaft (GRK1044), European Community
FP7/2009/241955 (SYSCILIA), FAUN-Stiftung, Nuremberg, Foundation
Fighting Blindness (FFB), US-Israel Binational Science Foundation, Germ
Gene Therapy For Choroideremia - Initial Report On A New Clinical Trial
Robert E. MacLaren1,2, Markus Groppe1, Alun R. Barnard1, Tanya Tolmachova3,
Matthew J. During4, Susan M. Downes5, Andrew J. Lotery6, Graeme C. Black7,
Andrew R. Webster8,2, Miguel C. Seabra3. 1Nuffield Laboratory of Ophthalmology,
University of Oxford, Oxford, United Kingdom; 2Moorfields Eye Hospital NHS
Foundation Trust, London, United Kingdom; 3Molecular Medicine, Imperial
College London, London, United Kingdom; 4Ohio State University Medical
Center, Columbus, OH; 5Oxford Eye Hospital, Oxford University Hospitals NHS
Trust, Oxford, United Kingdom; 6Ophthalmology - Eye Unit, Southampton General
Hospital, Southampton, United Kingdom; 7Genetic Medicine, University of
Manchester, Manchester, United Kingdom; 8UCL Institute of Ophthalmology,
London, United Kingdom.
Purpose: To report the initial findings from a new clinical trial using gene therapy
with an adeno-associated viral (AAV) vector encoding Rab escort protein-1 (REP1)
to treat patients suffering from choroideremia (NCT01461213).
Methods: An AAV2 vector encoding human REP1 driven by a CBA promoter
with a woodchuck hepatitis virus post-translational regulatory element (WPRE)
was made to Good Medical Practice (GMP) standards and approved for clinical use
by the UK Medicines and Healthcare products Regulatory Authority (MHRA). The
final vector solution was 1 x 10e12 genome particles (gp) per ml, in 0.001% PF68
surfactant. Two cohorts each of six patients will receive a total dose of 1 x 10e10
gp or 1 x 10e11 gp delivered by subretinal injection. Disease status is measured at
baseline using microperimetry, electroretinography, autofluorescence and spectral
domain optical coherence tomography (OCT). The primary end point is delivery of
vector without complications and the secondary endpoint is measurement of
choroidal shrinkage compared to the control eye.
Results: Preclinical testing revealed that surfactant was essential to prevent loss of
AAV particles in the plastic injection system. In the first patient to be treated, the
retina overlying a residual central island of choroid was detached in a controlled
two step procedure, which created a space into which 0.1 ml of the AAV vector
suspension was injected through a 41 gauge teflon needle on a 23 gauge cannula.
Subretinal fluid was evident at post-operative day one, but had reabsorbed by one
week. By one month best corrected visual acuity had returned to at least baseline
levels.
Conclusions: In choroideremia the retina can be detached for subretinal injection
of an AAV vector expressing REP1 with no immediately obvious detrimental
effects on structure or function. The global deficiency of the RPE and choroid does
not appear to inhibit reabsorption of subretinal fluid. Choroideremia appears to be a
good candidate disease for gene replacement therapy. High resolution clinical
imaging tests will require several months to determine whether or not sight loss can
be prevented.
Commercial Relationships: Robert E. MacLaren, Named inventor on UK

patent application 1103062.4 filed on behalf of the University of Oxford
(P); Markus Groppe, None; Alun R. Barnard, None; Tanya Tolmachova, None;
Matthew J. During, Named inventor on UK patent application 1103062.4 filed on
behalf of the University of Oxford (P); Susan M. Downes, None; Andrew J.
Lotery, None; Graeme C. Black, None; Andrew R. Webster, None; Miguel C.
Seabra, Named inventor on UK patent application 1103062.4 filed on behalf of the
University of Oxford (P)
Support: Wellcome Trust, UK Department of Health, NIHR Biomedical Research
Centres, Health Innovation Challenge Fund, Fight for Sight, Royal College of
Surgeons of Edinburgh
Adenoviral and Lentiviral Vectors for Efficient Gene Transfer to Mouse
Retina
Agostina Puppo1, Giulia Cesi1, Donna Palmer2, Pasquale Piccolo1, Robin J.
Parks3, Philip Ng2, Nicola Brunetti-Pierri1, Alberto Auricchio1,4. 1TIGEMTelethon Institute of Genetics and Medicine, Naples, Italy; 2Dept. of Molecular and
Human Genetics, Baylor College of Medicine, Houston, TX; 3Ottawa Hospital
Research Institute, Ottawa, ON, Canada; 4Dept. of Pediatrics, Medical Genetics,
"Federico II" University, Naples, Italy.
Purpose: Photoreceptors (PR) are important targets of gene therapy as these are
affected cells in inherited retinal degenerations (IRD). Several forms of IRD are
due to defects in large genes which cannot be accommodated into AAV vectors,
which so far have demonstrated the greatest potential in preclinical animal models
and human clinical trials. PR are refractory to transduction by the most studied
Adenoviral (Ad) and Lentiviral (Lv) vectors, namely Ad5 and Lv-VSVG which
have the unique ability to transfer large DNA sequences. The use of Ad and Lv
vectors would allow to either transfer therapeutic genes with large coding
sequences or to deliver whole genomic loci including their endogenous regulatory
regions. Here, we aimed at the identification of Lv and Ad vectors with high PR
tropism and transduction efficiency in preparation for further testing in animal
models of severe inherited PR diseases.
Methods: We collected 16 different Ad serotypes which were either isolated as
naturally infectants of humans and chimpanzees or genetically capsid modified
Ad5. We have also collected seven different Lv pseudotypes with heterologous
envelope proteins. The vector containing either a CMV-eGFP or lacZ expression
cassette were injected in adult C57/Bl6 or Cd1 mice and harvested at either four or
14 days post-injection. Transgene expression was evaluated by indirect
ophthalmoscopy or by analysis of retinal sections to visualize eGFP or lacZ
expression.
Results: We identified five vectors either based on Ad serotypes or on Lv
pseudotypes with PR transduction efficiency higher than Ad5 and Lv-VSVG
respectively. Since the promoter used is ubiquitous, RPE, PR, and cells from the
inner nuclear layer were transduced. Some vectors achieved substantial levels and
extension of PR transduction of up to 40-50% of the retinal sections.
Conclusions: We have identified five vectors either based on Ad serotypes or on

Lv pseudotypes that appear promising for efficient murine PR transduction. To
better define the potential of these vectors for retinal gene therapy, we are currently
performing experiments using an expression cassette including the PR-specific
rhodopsin promoter. In addition, we will test these promising vectors in the pig
retina, a large model retina which is highly cone-enriched.
Commercial Relationships: Agostina Puppo, None; Giulia Cesi, None; Donna
Palmer, None; Pasquale Piccolo, None; Robin J. Parks, None; Philip Ng,
None; Nicola Brunetti-Pierri, None; Alberto Auricchio, None
Support: TAAMT9NIHG
procedure
Conclusions: We describe here a novel minimally invasive, non-viral gene therapy
method for the transfection of RPE cells without any retinal detachment. Further
analysis will be performed to optimize cell specific targeting and long-term
expression.
Commercial Relationships: Francine F. Behar-Cohen, inventor (P); Elodie
Touchard, Inventor (P); Berdugo Marianne, None; Michle Savoldelli,
None; Marie-Christine Naud, None; Jean-Claude Jeanny, None
Support: Fondation Recherche Mdicale

Suppression of the Neurodegeneration of Experimental Optic Neuritis by
Single-subunit Yeast NADH-Ubiquinone Oxidoreductase (NDI1)
Venu Talla, Sacide S. Ozdemir, Tsung-Han Chou, Vittorio Porciatti, John Guy.
Ophthalmology, Bascom Palmer Eye Institute, University of Miami, Miami, FL.
Purpose: Mitochondrial dysfunction mediates neurodegeneration in optic neuritis
and multiple sclerosis (MS) that contributes to permanent loss of function. This
dreaded sequelae has no remedy. We rescued retinal ganglion cells (RGCs) and
optic neuropathy in experimental optic neuritis using gene therapy with the single
subunit yeast complex I (NDI1).
Methods: Experimental autoimmune encephalomyelitis (EAE) was induced in
female DBA/1J mice (n=20). Mice got intravitreal scAAV-NDI1 (n=10) and
controls received an scAAV that contained the 23 amino acid COX8 mitochondrial
targeting sequence appended to the N terminus of cherry. An additional 10 mice
received scAAV-Cox8-mcherry but were unsensitized. RGC's were assessed by
serial PERG and OCT at 1, 3 and 6 months post injection (MPI). 6MPI mice were
sacrificed, for histolopathology. An additional 6 mice that received NDI1 (OD) and
GFP (OS) were sacrificed 3MPI and the retinas were assessed by TUNEL, DHE
and DCFDA staining. Expression of the NDI1 in the retina and ONs were
evaluated at 15d PI by RT-PCR, immunofluorescence (IF) and western blotting
(WB).
Results: Expression: IF revealed punctate and perinuclear expression of NDI1,
colocalized with GRIM19 and Thy1.2 indicating expression in mitochondria of
RGCs. RT-PCR and WB confirmed the NDI1 expression in the retina and ONs.
Rescue: PERG analysis at 3M and 6MPI showed a significant reduction, in
amplitude of EAE-mcherry compared to control mcherry (42%, 45%) (p=0.0035,
p=0.0004), whereas NDI1 rescued mice showed a 13% and 9% reduction in the
amplitude (p>0.05). NDI1 significantly rescued PERG amplitude by 67% and 80%
in EAE (p=0.029,p=0.0015). PERG latency was delayed by 15% and 21% in EAEmcherry compared to mcherry control (p<0.05), whereas the NDI1 injected mice
rescued this delay by 100% and 56% (p<0.05) at 3M and 6MPI. OCT images
showed a significant thinning in EAE-mcherry compared to mcherry control at 3M
(16%) and 6MPI (15%) p<0.05. Whereas NDI1 rescued mice showed thickening by
16% and 12% (p<0.05). There was 14.3% TUNEL + cells in RGC layer of GFP
control compared to 4% in NDI1 injected eyes (p=0.02). In addition, DHE and
DCFDA staining indicated more mitochondrial stress in retina and ONs of GFP
control vs NDI1. Ultrastructural analysis of the EAE ONs demonstrated different
levels of degeneration in axon, myelin and connective tissues whereas NDI1 ONs
showed relatively healthy axons with continuous myelin organized microtubules
and healthy looking mitochondria.
Conclusions: NDI1 gene therapy is a good candidate for suppression of
mitochondrial induced neurodegeneration in optic neuritis and multiple sclerosis.
Commercial Relationships: Venu Talla, None; Sacide S. Ozdemir,
None; Tsung-Han Chou, None; Vittorio Porciatti, None; John Guy, None
Support: EY07982

Progeny Of Pronuclear Injections Of Mutant Human Mitochondrial Genes
Hong Yu1, Tsung-Han Chou1, Vittorio Porciatti2, William W. Hauswirth3, Vince
Chiodo3, Sanford L. Boye3, John Guy1. 1Ophthalmology, Bascom Palmer Eye Inst,
Univ of Miami, Miami, FL; 2Bascom Palmer Eye Inst, Univ of Miami Miller Sch
Med, Miami, FL; 3Ophthalmology, University of Florida, Gainesville, FL.
Purpose: To generate a transgenic mouse with human mutated G11778A ND4
mtDNA.
Methods: The mutant human ND4 gene with a FLAG epitope tag followed by
mitochondrial encoded mCherry under control of the mitochondrial promoter (scHSP-ND4G1019AmtmCherry) was packaged with mito-targeted (COX8) scAAV
that was injected into fertilized oocytes using a standard pronuclear microinjection
protocol. Briefly, hybrid (C57BL/6J x DBA/2J) F1 mice, referred to as B6D2F1,
were obtained for production of fertilized oocytes. Superovulation was stimulated
by intraperitoneal injection of B6D2F1 females with 5 IU pregnant mare serum
gonadotropin followed 46-48 h later by injection of 5 IU human chorionic
gonadotropin. Following HCG injection, females were mated with B6D2F1 stud
males and oocytes harvested the following day. A concentrated AAV virus stock
(1.06E+12 VG/ml) was microinjected into the pronuclei of fertilized oocytes using
a continuous flow injection mode. Surviving eggs were implanted into the ampulla
of pseudopregnant females. After weaning the resulting offspring were analyzed for
the presence of human ND4 in isolated mouse mitochondria was assessed by PCR,
sequencing and immunohistochemistry. Visual function was monitored by pattern
electroretinography (PERG).
Results: PCR amplification of mitochondrial DNA, isolated from different organs
of transgenic mice and their progeny including retina, optic nerve, brain, liver, heart
and muscle, revealed expected 500bp bands. The corresponding DNA sequences
confirmed that PCR products are human ND4 and mCherry. Fluorescence of
mCherry could be visualized in retina and optic nerve head of live mice by using a
laser scanning ophthalmoscope. Longitudinal retina sections revealed ND4FLAG
in RGC layer and co-localization with the mitochondrial membrane protein VDAC
porin. Pattern electroretinogram (PERG) showed that the amplitudes decreased
dramatically with aging (p=0.006) in transgenice mice and transgenic mice had
significantly lower PERG amplitude compared to normal mice of the same strain
(p=0.02).
Conclusions: Despite multiorgan dissemination of mutant G11778A ND4, our
transgenic mice developed only visual loss, as also seen in LHON patients.
Pronuclear injection of mutant human mitochondrial genes is an innovative
biotechnical advance for evaluating LHON and likely other devastating
neurodegenerative disorders with mitochondrial etiology and their treatment.
Commercial Relationships: Hong Yu, None; Tsung-Han Chou, None; Vittorio
Porciatti, None; William W. Hauswirth, None; Vince Chiodo, None; Sanford L.
Boye, None; John Guy, None
Support: R01 EY017141

A Novel Method To Transfect Retinal Pigment Epithelial Cells Without
Detaching The Retina
Francine F. Behar-Cohen1,2A, Elodie Touchard2B, Berdugo Marianne2C, Michle
Savoldelli1, Marie-Christine Naud2A, Jean-Claude Jeanny2A. 1Ophthalmology,
Hotel Dieu de Paris, Universite Paris Descartes, Paris, France; APhysiopathology
ocular diseases, BPhysiopathologie of ocular dseases, CPhysiopathology of ocular
diseases, 2Inserm UMRS872, Paris, France.
Purpose: Sub retinal route of viral vectors is the validated method to transfect
retinal cells at the site of retinal detachment. A safer method would benefit to
macular targeting. We have evaluated a novel method that combines the
administration of plasmid DNA in the suprachoroidal space of rat eyes with scleral
electroporation procedure.
Methods: Optimal injection, current electroporation parameters and electrode
geometry were evaluated using a plasmid encoding Lac-Z. Safety of the procedure
was monitored using fluorescein angiography, electroretinography and histology.
Results: Suprachoroidal injection induces a large area of diffusion. Scleral
electroporation using external electrodes efficiently transfects not only choroidal
cells but also retinal pigment epithelial cells and photoreceptors using a CMV
promoter. No histologic or functional resulted from this minimally invasive

Increased Longevity of Rescue of Light-Induced Retinal Damage in an Adult
Mouse Using Peptide for Ocular Delivery (POD) as a Gene Transfer Vector
Rajendra Kumar-Singh, Christina Binder, Siobhan Cashman. Ophthalmology,
Tufts University, Boston, MA.
Purpose: Nonviral gene delivery is a promising approach to gene therapy for
ocular diseases, such as Retinitis Pigmentosa (RP) and Age-Related Macular
Degeneration (AMD). The use of nonviral systems has been hampered by limited
efficiency of gene transfer and short-lived duration of expression. Using a
pegylated cationic peptide, PEGPOD, to deliver a transgene expressing GDNF
(glial-derived neurotrophic factor), we have previously shown rescue of lightinduced retinal degeneration in an adult mouse up to 18 days post-transgene
delivery. In this study, we determined factors necessary to significantly increase
duration of expression and rescue.
Methods: PEGPOD was complexed with a plasmid expressing luciferase and
different amounts of the complex injected into the subretinal space of Balb/c mice.
Luciferase expression was quantified up to 15 weeks post-injection. Once the
optimal amount of plasmid was determined, PEGPOD was complexed with a
GDNF-expressing plasmid and injected into the subretinal space of Balb/c mice.
Mice were subjected to 450-nm blue light at 14, 30, and 70 days post-injection.
Electroretinograms (ERG) and thickness of outer nuclear layer (ONL) were

measured 7 days post-light treatment.
Results: Reduced dose of the PEGPOD/DNA complex resulted in expression of
the luciferase up to 10 weeks post-injection. When PEGPOD was complexed with
GDNF (PEGPOD/GDNF) and injected 30 days prior to light treatment, significant
functional and anatomical rescue was observed. A- and B- wave amplitudes of
mice injected 30 days prior to light treatment were increased by 77% and 65%,
respectively, relative to control-injected mice. While functional rescue of mice
injected with PEGPOD/GDNF 70 days prior to light treatment was not significant,
ONL thickness of eyes injected with PEGPOD/GDNF was significantly increased
by 25% relative to control-injected eyes.
Conclusions: PEGPOD/GDNF complexes show biologically significant expression
in retinas of a mouse model of light-induced degeneration up to 70 days postinjection. This study illustrates the potential of PEGPOD/GDNF to mediate longterm rescue of retinal diseases such as RP and AMD, and asserts the need for
consideration of this nonviral gene transfer system as a clinically viable approach
to treatment of these devastating diseases.
Commercial Relationships: Rajendra Kumar-Singh, Tufts University
(P); Christina Binder, None; Siobhan Cashman, Tufts University (P)
Support: NIH 1R01EY021805, NIH 5R01EY013837, The Ellison Foundation,
Research To Prevent Blindness, Mass Lions Foundation
531 Inflammation and Infection
Thursday, May 10, 2012, 8:30 AM - 10:15 AM
An Antimicrobial Peptide Can Enhance the Activity of a Fluoroquinolone in
Reducing Colony Counts of Fluoroquinolone-Resistant MRSA in the NZW
Rabbit Keratitis Model
Eric G. Romanowski, Kathleen A. Yates, Francis S. Mah, Y J. Gordon, Regis P.
Kowalski. The Charles T. Campbell Laboratory, UPMC Eye Center, University of
Pittsburgh, Pittsburgh, PA.
Purpose: Fluoroquinolone-resistant, methicillin-resistant Staphylococcus aureus
(FQR-MRSA) ocular infections are increasing in number. Unfortunately, there are
no new antibiotics in development to treat these ocular infections. An approach to
solve this problem is to enhance the activity of older antibiotics with additional
agents to make a combination more effective against resistant pathogens than either
alone. To determine whether the activity of a highly resistant antibiotic could be
enhanced, we tested whether combination therapy with ciprofloxacin (CIP) and an
antimicrobial peptide (Nisin [NIS], a 34 amino acid amphiphilic antimicrobial
peptide used as a food preservative) will have enhanced activity compared to either
alone in reducing FQR-MRSA colony counts in the NZW rabbit keratitis model.
Methods: A total of 48 rabbits were inoculated intrastromally in both eyes with
~1000 CFU of FQR-MRSA. After 4 h, the rabbits were divided into 4 treatment
groups (n=12/group): I) 0.075% NIS; II) 0.3% CIP; III) 0.075% NIS + 0.3% CIP;
IV) PBS. Topical treatment was instilled in both eyes every 15 min. for 5 hours.
One hour after the final dose, the corneas were harvested, homogenized, and colony
counts were performed. Colony count data were Log10 transformed and analyzed
statistically using a one-way ANOVA with Fishers pairwise comparisons. The
data is expressed as mean sd Log10 CFU/ml.
Results: CIP treatment alone (6.76 0.35 CFU/ml) demonstrated no difference in
the number of colony counts compared to PBS (6.87 0.30 CFU/ml) (p>0.05). NIS
alone (4.90 1.84 CFU/ml) significantly decreased the number of colony counts
compared to PBS and CIP (p<0.05). NIS + CIP (3.84 2.01 CFU/ml) significantly
decreased the number of colony counts compared to NIS alone, CIP alone, and PBS
(p<0.05).
Conclusions: Combination therapy with 0.075% NIS and 0.3% CIP demonstrated
a significant decrease in FQR-MRSA colony counts compared to the PBS control
and either drug alone. This study provides proof of principle that in vivo
enhancement of topical antimicrobial agents can be achieved and may be evaluated
using the NZW rabbit keratitis model.
Commercial Relationships: Eric G. Romanowski, Alcon, Inspire (C), Novartis
(F); Kathleen A. Yates, Novartis (F); Francis S. Mah, Alcon, Inspire (C),
Novartis (F); Y. J. Gordon, Alcon (C), Novarits (F); Regis P. Kowalski, Alcon,
Inspire (C), Novartis (F)
Support: Novartis, NIH CORE Grant EY08098, Eye & Ear Foundation of
Pittsburgh, RPB
Pharmacokinetic Parameters Determined For A Fluoroquinolone In The Eye
Of Rabbits And Multiple Dosage Regimens Prediction
Juan Carlos Rivera-Castro, Sr., Jos Rubn Tornero-Montao, Juan de Dios

Quintana-Hau, Humberto Figueroa-Ponce, Abida Carrillo-Guzmn, Luis Gerardo
Urquidez-Espinoza. R&D, Laboratorios Sophia SA de CV, Zapopan, Mexico.
Purpose: Determine pharmacokinetic parameters of the compound in study after a
single dose. Use the single dose pharmacokinetic parameters to predict the steadystate of multiple dosage regimens for the determination of an appropriate dosage
interval.
Methods: There were used 54 male New Zeland white Rabbits with a weight
between 2.0 and 3.0 kg, using 9 rabbits per time, the dosage administered was 30
L of quinolone solution 0.3%, the Rabbits were sacrificed at the corresponding
time 15, 30, 60, 90, 120 and 180 minutes, following the extraction of the humor
aqueous. The concentration of the quinolone was determined using HPLC coupled
to an UV detector. The pharmacokinetics parameters were determined using first
order reaction equations and one compartment model. For the multiple dosage
prediction was applied the principle of superposition which allows to project the
concentration-time curve of a drug after several consecutive doses based on the
drug concentration-time curve obtained after a single dose.
Results: Pharmacokinetics parameters were obtained: absorption constant
ka=1.70008hr-1, elimination constant k=0.3078hr-1, fraction of dose absorbed
F=0.002. Using this parameters concentration curves for different dosage intervals
were constructed.
Conclusions: The MIC90 for most of the quinolones is between 100-2000 ng/mL.
An appropriate dosage interval for reaching therapeutic levels is every 4 hours in
which the steady-state predicted is between Cmin=362 ng/mL and Cmax=751 ng/mL,
this steady-state was reached after the fourth
dosage.
Commercial Relationships: Juan Carlos Rivera-Castro, Sr., Laboratorios

Sophia SA de CV (F); Jos Rubn Tornero-Montao, Laboratorios Sophia SA de
CV (F); Juan de Dios Quintana-Hau, Laboratorios Sophia SA de CV (F);
Humberto Figueroa-Ponce, Laboratorios Sophia SA de CV (F); Abida CarrilloGuzmn, Laboratorios Sophia SA de CV (F); Luis Gerardo Urquidez-Espinoza,
Laboratorios Sophia SA de CV (F)
Support: None
In Vitro Activity of ACH-0139586, a Novel Isothiazoquinolone, Moxifloxacin
and Gatifloxacin Against Clinical Isolates, Including Methicillin and
Fluoroquinolone Resistant
Aron Shapiro1, Laura Belen1, Andy Whitlock1, Dan Sahm2. 1Ora, Inc., Andover,
MA; 2Eurofins Medinet, Chantilly, VA.
Purpose: To evaluate the in vitro activity of a novel isothiazoquinolone, ACH0139586, against common ocular pathogens (S. aureus, S. epidermidis, S.
pneumonia, H. influenza, M. catarrhalis, and P. aeruginosa) compared with
moxifloxacin and gatifloxacin. Isothiazoloquinolones are a new class of antiinfectives proven to inhibit bacterial DNA primase, in addition to DNA gyrase and
topoisomerase IV, targeted by fluoroquinolones. Added inhibition of DNA primase
increases antimicrobial efficacy and decreases chance for resistance compared to
fluoroquinolones.
Methods: Non-duplicate, non-consecutive clinical isolates (including ocular) were

selected from the Eurofins Medinet Bacterial Repository. Isolates with
fluoroquinolone and methicillin resistance were preferentially selected to challenge
ACH-0139586, which is reported to maintain potency against fluroquinolone
resistant isolates. S. aureus with MRSA and FQ-R phenotypes, S. epidermidis with
MRSE and FQ-R phenotypes, S. pneumoniae with MDR, Pen-R, and FQ-R
phenotypes, H. influenzae with Beta-lactamase-positive and FQ-R phenotypes, M.
catarrhalis with Beta-lactamase-positive phenotypes, and P. aeruginosa with
Imipenem-R, FQ-R, and MDR phenotypes were included. Isolates were tested by
broth microdilution in accordance with CLSI M7-A8 and Eurofins Medinet SOP as
appropriate.
Results:
Conclusions: Against the evaluated isolates, ACH-0139586 was consistently more

potent in vitro relative to gatifloxacin and moxifloxacin, regardless of methicillin
and fluroquinolone resistance. The potency advantage was most apparent against
evaluated gram-positive pathogens. ACH-0139586 proved to have increased
potency against fluroquinolone resistant isolates, in particular S.aureus, where
fluoroquinolone resistance is relatively common. These results show the ocular
anti-infective therapeutic potential of ACH- 0139586.
Commercial Relationships: Aron Shapiro, Ora, Inc., Andover, MA (E); Laura
Belen, Ora, Inc., Andover, MA (E); Andy Whitlock, Ora, Inc., Andover, MA (E);
Dan Sahm, Eurofins Medinet, Chantilly, VA (E)
Support: None
A Novel Antiviral Protein RC28
Naihong Yan1, Frank Piraino2, Xuyang Liu1. 1Ophthalmic Laboratories, Chengdu,
China; 2Department of Ophthalmology and Visual Sciences, University of
Wisconsin Medical School, WI.
Purpose: Herpes simplex keratitis is a major cause of adult eye disease, and may
lead to chronic inflammation of the cornea. An antiviral protein, RC28, with antiherpes virus activity was extracted from Rozites caperata (Cortinarious caperata).
The goal of this study was to test the activity of RC28 in a murine herpes simplex
virus (HSV)-1 keratitis model.
Methods: RC28 was isolated and purified from R. caperata mushroom. RC28
protein was sequenced from the amino terminus using the Edman degradation
procedure. RC28 cDNA fragment was obtained using 3- RACE method and
expressed in E.coli and purified by affinity chromatography. The in vitro antiviral
activity of RC-2 against HSV-1 was determined by yield reduction and viral
inactivation assays. The virus keratitis in mice animal models had established to
study the role of anti-virus RC28.
Results: RC28 with anti-herpesvirus activity was purified from a PBS extract of
Rozites caperata by acetone precipitation followed by gel filtration and ion
exchange chromatography. The molecular weight of this protein was 28.251kDa as
measured by MALDI-TOF mass spectrography. RC28 inhibited HSV-1 replication
in vitro with an IC(50) value of 0.078mg/ml and a therapeutic index >32. The first
30 amino acid residues of RC28 were sequenced by Edman degradation to be
MLTYR GKLNW YNYAV NEGFT LILPG XELKV. Based on that sequence, the
RC28 cDNA fragment was cloned by 3'-RACE, and the rest of the amino acid
sequence was inferred from the cDNA sequence. RC28 was cloned into pET26b(+)
vector and expressed of protein in Escherichia Coli. Cytotoxicity test showed that
the RC28 is very weak cytotoxicity and RC28 can inhibit HSV-1 in culture. The
animal model results showed that RC28 delayed the occurrence of stromal keratitis
and alleviated the severity of the disease.
Conclusions: RC28 is an antiviral protein with multi-functional activities that
interfere with both early and late HSV-1 viral functions, a remarkable and clinically
important characteristic of an antiviral drug.
Commercial Relationships: Naihong Yan, None; Frank Piraino, None; Xuyang
Liu, None
Support: NSFC Grant 30901635
Clinical utility of Ophthalmic Antimicrobial Susceptibility Measurement Plate
Norihiko Tou1, Ryohei Nejima2, Yoshifumi Ikeda3, Yuichi Hori4, Kaoru Sasaki5,
Masako Sakamoto6, Kazunori Miyata2, Yoshitsugu Inoue3, Akihiko Tawara1,
Hiromitsu Fujiwara7. 1Ophthalmology, Univ of Occup & Environ Health,
Kitakyushu-shi, Japan; 2Miyata Eye Hospital, Miyazaki, Japan; 3Department of
Ophthalmology, Tottori Univ Faculty of Medicine, Yonago, Japan;
4
Ophthalmology, Toho University Sakura Medical Center, Sakura, Japan; 5Ideta
Eye Hospital, Kumamoto, Japan; 6Ophthalmology, The Research Foundation for
Microbial Diseases of Osaka University, Osaka, Japan; 7Department of Clinical
Laboratory, Tottori University Hospital, Yonago, Japan.
Purpose: To evaluate the clinical utility of SG17 (Ophthalmic Antimicrobial
Susceptibility Measurement Plate:Fig1), newly developed plate to measure the
minimum inhibitory concentration (MIC) for bacterial isolates of ocular infection.
Methods: Antimicrobial susceptibility was measured using 96 strains detected
from 78 patients diagnosed as ocular infection in five institutes. The clinical utility
of SG17 was evaluated to compare MIC measured by SG17 to susceptibility
measured by routine method at each institute.
Results: Of the 96 strains, 85(88.5%) were gram positive strains, those of
34(35.4%) were coagulase-negative Staphylococci, 22(22.9%) were
Corynebacterium spp. and 15(15.6%) were Staphylococcus aureus. SG17 measured
MIC up to higher concentration actually used for topical treatment at ocular surface
than systemic treatment. MIC90 of each strain was showed in Table 1. The 78
patients received one or more drugs among 11 antimicrobial eye drops or ointment.
The rate that can measure the susceptibility of actually used drugs was 100% in
SG17 and30.8% in routine method at each institute. In 54(69.2%) patients, the
clinical utility of SG17 was better than that of routine method at each institute.
Conclusions: These results suggest that SG17 can measure the drug susceptibility
of antimicrobial eye drops and it is useful for the treatment of ocular infection.
Commercial Relationships: Norihiko Tou, None; Ryohei Nejima,

None; Yoshifumi Ikeda, None; Yuichi Hori, None; Kaoru Sasaki,
None; Masako Sakamoto, None; Kazunori Miyata, None; Yoshitsugu Inoue,
None; Akihiko Tawara, None; Hiromitsu Fujiwara, None
Support: None
Prevalence and risk factors of methicillin-resistant Staphylococcus aureus
nasal carriage among ophthalmology outpatients in Puerto Rico
Maria H. Berrocal1, Victoria Lpez2, Luis A. Acab3, Alexandra Acab4.
1
Ophthalmology, University of Puerto Rico, San Juan, PR; 2Bryn Mawr College,
Bryn Mawr, PA; 3U. of Puerto Rico, San Juan, PR; 4U. Puerto Rico, San Juan, PR.
Purpose: To determine the prevalence and risk factors for nasal colonization of
methicillin-resistant Staphylococcus aureus (MRSA) and its antibiotic

susceptibilities among Hispanic patients in a general ophthalmology clinic in
Puerto Rico.
Methods: Two hundred randomly selected patients in a general ophthalmology
clinic were included in this study. Ages ranged from 20 to 96 with a mean age of
68 years. Nasal swabs of the patients were cultured in a chromogenic media and
antimicrobial identification and susceptibility performed. Associated risk factors
evaluated included age, sex, diabetes, hypertension, hemodyalisis, hospitalizations
and antibiotic use.
Results: Twenty-eight of the 200 patients evaluated were colonized with MRSA
(14%). The number of colonies resistant to different antibiotics were as follows:
tobramycin-8/28 (28.6%), moxifloxacillin- 6/28 (21.4%), vancomycin- 2/28
(7.1%), besifloxacillin- 0/28 (0%). The two patients that had MRSA resistant to
vancomycin had been hospitalized during the past year. The only factor
independently associated with increased risk of MRSA colonization was antibiotic
use during the past year.
Conclusions: The prevalence of nasal MRSA colonization in this population was
14%. The only risk factor associated with MRSA colonization was recent antibiotic
utilization. Resistance to vancomycin was associated with prolonged
hospitalization. MRSA colonization among ophthalmology clinic patients could
present a potential problem when intraocular surgery is performed
Commercial Relationships: Maria H. Berrocal, Alcon (R); Victoria Lpez,
None; Luis A. Acab, None; Alexandra Acab, None
Support: None
A Comparative Study in the Clinical and Microbial Efficacy of Topical
Besifloxocin Ophthalmic Suspension 0.6% with Erythromycin Ophthalmic
Ointment 0.5% for Management of Acute Blepharitis
George John. VA Medical Center, Louisville, KY.
Purpose: To evaluate the relative efficacy of Besifloxocin and Erythromycin in the
management of symptomatic acute blepharitis. Specifically, the clinical response in
terms of graded signs and symptoms as well as the microbiology and bacterial
eradication rates using these two antibiotics to treat blepharitis were studied.
Methods: Thirty patients with symptomatic anterior blepharitis, as documented by
eyelash crusting along with other signs of lid inflammation, were randomized to
receive either topical Besifloxocin 0.6% or Erythromycin 0.5%, bid along with
standard lid hygiene measures. This was an open label, unmasked comparison and
all patients were graded using a scoring system for signs and symptoms of
blepharitis over a period of four weeks. Cultures and sensitivities to a panel of
antibiotics were obtained at the first visit prior to starting therapy and again after
two weeks of therapy.
Results: All patients demonstrated similar improvement in both groups based on
scored symptoms of common blepharitis complaints as well as a grading of
conjunctival, lid and corneal findings. Mean subjective scores improved from 7.60
to 3.53 in the Besifloxocin (B) group as compared to 7.26 and 3.13 in the
Erythromycin (E) group. Average scoring of conjunctival injection improved as
follows: 1.20 to 1.07 (B), 1.27 to 1.00 (E). Lid inflammation scores improved as
follows: 4.73 to 3.00 (B), 4.27 to 2.67 (E). Average corneal staining improved from
0.60 to 0.47 (B) and 1.13 to 0.60 (E). Rosacea was a known diagnosis in 5/15
patients in each group.
The largest notable difference was in the cultures post-treatment with 6/13 showing
no growth in the Besifloxocin group and 0/15 in the Erythromycin group. Of
particular note was the fact that 7/15 cultures in the Erythromycin group showed
either increased growth of coagulase negative staphylococcus or growth of new
organisms.
Conclusions: All patients treated with standardized lid hygiene measures and a
topical antibiotic demonstrated similar rates of improvement in terms of signs and
symptoms of acute blepharitis. Based on culture results, Besifloxocin was able to
eliminate organisms in all cases with the exception of light growth of the skin
organism coagulase negative staphylococcus in some cases. Erythromycin treated
patients demonstrated overgrowth of coagulase negative staphylococcus or new
growth of other organisms. Overall, cultures of patients with symptomatic anterior
blepharitis were positive in 16/30 patients in this study with 6/16 demonstrating
resistance to erythromycin and ciprofloxacin.
Commercial Relationships: George John, Bausch and Lomb (R)
Support: Bausch and Lomb Independent Research Grant
Clinical Trial: http://www.clinicaltrials.gov, SAIRB-11-0007
Effect Of Different Excipients In Tobramycin Sulfate Watery Solutions
Degradation Rate
Abida Carrillo-Guzman, Luis David Torres-Pedraza, Jos Rubn TorneroMontao, Humberto Figueroa-Ponce. R & D, Laboratorios Sophia SA de CV,
Zapopan, Mexico.
Purpose: Evaluate the effect of different excipients in tobramycin sulfate watery

solutions degradation rate.
Tobramycin sulfate is an aminoglycoside antibiotic widely used in ophthalmic
solutions. In acid solutions main degradation mechanism is hydrolysis. In solutions
with a pH value near to neutrality main degradation mechanism is oxidation. One
of first signs of degradation in tobramycin solutions is development of yellow
coloration.
Methods: We evaluated the effect in tobramycin degradation of different
commonly used ophthalmic solutions excipients (glycerin, sodium chloride, borate
buffer at three different concentrations, edetate disodium, sodium sulfite,
polysorbate 80 and cyclodextrins). Tobramicyn sulfate watery solution was used as
control.
Solutions were stored during 28 days at 70C to increase degradation velocity.
Samples were analyzed at 0, 7, 14, 21 and 28 days. Parameters evaluated were pH
variation, yellow coloration development and tobramycin assay.
Results: Samples with better behavior during evaluation were samples containing
glycerin, cyclodextrins and sodium sulfite.
A big increase in tobramycin degradation rate was observed in sample containing
borate buffer at a concentration higher than 0.5%; a significant degradation was
observed also in samples containing Sodium chloride and polysorbate 80 also
increased tobramycin degradation but effect was lower compared with borates
buffer and edetate disodium.
A big increase in yellow coloration development occurred in presence of
polysorbate 80, probably for the additive effect of oxidative degradation products
generated from polysorbate 80 and tobramycin.
Conclusions: Tobramycin is degraded by hydrolysis and oxidation. Edetate
disodium is a chelating agent that reduces oxidation caused by metal ions,
nevertheless it increases tobramycin degradation, for that reason it is preferable to
use sodium sulfite to reduce tobramycin oxidation. When is required to use a borate
buffer it is better to use it at a concentration not higher than 0.5%. Glycerin and
cyclodextrins seems to reduce tobramycin degradation.
Commercial Relationships: Abida Carrillo-Guzman, Laboratorios Sophia SA
de CV (F); Luis David Torres-Pedraza, Laboratorios Sophia SA de CV (F); Jos
Rubn Tornero-Montao, Laboratorios Sophia SA de CV (F); Humberto
Figueroa-Ponce, Laboratorios Sophia SA de CV (F)
Support: None
Effect of Simultaneous Treatment of Quinolones and Antifungal Drugs on
Fungal-Bacterial Coculture
Diana Gabriela Ponce-Angulo, Jr.1A, Maria de los Angeles Martinez-Rivera, Sr.2,
Victor Manuel Bautista-de Lucio, Sr.1A, Aida Veronica Rodriguez-Tovar, Sr.2,
Concepcion Santacruz-Valdez, Sr.1B, Alejandro Climent-Flores, Sr.1B, Atzin RoblesContreras, Jr.1A, Cesar Diaz-Godinez, Jr.2, Ernesto Felix Diaz-Parga, Jr.2,
Herlinda Mejia-Lopez, Sr.1A. AResearch Unit / Microbiology and Proteomics,
B
Cornea service, 1Institute of Ophthalmology, Mexico, D.F., Mexico; 2Laboratory
of Medical Mycology, Department of Microbiology, National School of Biological
Sciences (IPN), Mexico, D.F., Mexico.
Purpose: The most common complications of keratomycosis is superinfection,
patients are often treated concomitantly with quinolones and antifungal drugs.1 In a
retrospective study conducted at Institute of Ophthalmology of Mexico City, in
samples of patients with keratitis or corneal ulcer, was found that Gram-positive
bacterial infections are 20% higher risk of infection by fungus compared those
Gram negative.2 The aim of the present work was to evaluate in vitro, simultaneous
treatment effect with quinolones and antifungal drugs in fungal-bacterial
cocultures.
Methods: Were isolated and identified strains from patients with keratitis.
Cocultures were performed between Fusarium oxysporum and Aspergillus
fumigatus with S. aureus or S. epidermidis in presence of gatifloxacin or
moxifloxacin and amphotericin B, natamycin or itraconazole at minimal inhibitory
concentrations (MICs) determined for every microorganism studied.
Results: The untreated cocultures showed effects of competition between fungi and
bacteria. Amphotericin B inhibited the growth of both fungi. The growth of A.
fumigatus was also affected by natamycin. In the simultaneous treatment of the
cocultures, quinolones interfere with the effect antifungal except for treatment with
itraconazole. In all cases of simultaneous treatment, was observed collaboration
between bacteria in the growth of fungus.
Conclusions: The MICs for quinolones used in this work interferes with the
antifungal effect. Simultaneous quinolone-antifungal treatment represents stress
conditions inducing inter-specie association.
References. 1. Parmar P et. al. Curr Opin Infect Dis 2006;20:129-141). 2. MejaLpez, H et. al. ARVO Meeting fort Lauderdale,Florida, EUA; Abril-Mayo 2010.
Commercial Relationships: Diana Gabriela Ponce-Angulo, Jr., None; Maria
de los Angeles Martinez-Rivera, Sr., None; Victor Manuel Bautista-de Lucio,
Sr., None; Aida Veronica Rodriguez-Tovar, Sr., None; Concepcion SantacruzValdez, Sr., None; Alejandro Climent-Flores, Sr., None; Atzin Robles-

Contreras, Jr., None; Cesar Diaz-Godinez, Jr., None; Ernesto Felix DiazParga, Jr., None; Herlinda Mejia-Lopez, Sr., None
Support: This work was supported by "Conde de Valenciana" Foundation and
CONACyT 126779
Lacritin, a Novel Tear Glycoprotein, is an Effective Topical Antimicrobial
Agent in an Animal Model
Alireza Hosseini1, Frank A. Lattanzio, Jr.1, Sandeep S. Samudre1, John D.
Sheppard, Jr.2, Gordon W. Laurie3, Robert L. McKown4, Patricia B. Williams1.
1
Physiological Sciences, Eastern Virginia Medical School, Norfolk, VA; 2Virginia
Eye Consultants, Norfolk, VA; 3Cell Biology, University of Virginia,
Charlottesville, VA; 4Integrated Science & Technology, James Madison University,
Harrisonburg, VA.
Purpose: Due to the development of resistance, new and safe antimicrobial agents
are always needed. Lacritin, a natural tear glycoprotein, increases tear flow in
animal models and has bactericidal effects at low micromolar concentrations in
vitro (both gram positive & negative). In this study, the antimicrobial effect of
topical lacritin N-65 and pLac was evaluated in a rabbit keratitis model of
Pseudomonas aeruginosa infection.
Methods: Anesthetized White New Zealand rabbits were injected midstromally in
the cornea of one eye (10 L, containing 1000 CFU of Pseudomonas aeruginosa).
16 hours after injection, 50 L aliquots of N-65 lacritin or pLac (both at 250
g/mL), 0.3% gatifloxacin (positive control) or saline (negative control) were
administered as follows: 5 doses / every 15 min, followed by 14 doses / every 30
min. 1 hr after the last dose, animals were graded via semi-quantitative slit lamp
examination for inflammation, chemosis, iritis, hypopyon, corneal infiltrate,
anterior chamber fibrin and corneal edema and then were euthanized. Aqueous
humor and corneas were then extracted to remove bacteria, extracts cultured and
colonies counted, permitting the four treatments to be compared.
Results: The infection was localized to the cornea, with virtually no measurable
bacterial activity in the aqueous humor. N-65 treatment significantly reduced slit
lamp scores compared to saline control (p<0.007) and was not significantly
different from the reduction by gatifloxacin treatment (p<0.4). pLac treatment did
not significantly reduce the slit lamp scores compared to saline control.
N-65 treatment was not significantly effective in reducing bacterial loads. pLac did
significantly reduce corneal bacterial loads compared to saline controls (p<0.05).
The commercial gatifloxacin preparation reduced bacterial loads significantly more
than pLac. The gatifloxacin effect may have been enhanced by 100 fold greater
molar concentration compared to pLac and also by the bactericidal effects of
0.005% BAK, used as a preservative in this commercial preparation.
Conclusions: We speculate that the lower molecular weight N-65 may be a more
effective anti-inflammatory agent due to its readily diffusing into the anterior
chamber, but the larger pLac may be more stable to degradation and therefore have
a more pronounced antimicrobial effect.
Commercial Relationships: Alireza Hosseini, None; Frank A. Lattanzio, Jr.,
None; Sandeep S. Samudre, None; John D. Sheppard, Jr., None; Gordon W.
Laurie, None; Robert L. McKown, None; Patricia B. Williams, None
Support: NIH EY020044-01
Susceptibility Of Methicillin-resistant Staphylococci Clinical Isolates To
Netilmicin And Other Antibiotics Commonly Used In Ophthalmic Therapy
Anna Rita Blanco1A, Andrea Sudano Roccaro1A, Vincenzo Papa1B, Maria Grazia
Mazzone1C. APharmaco Biology Unit - BU Pharma, BMedical Marketing - BU
Pharma, CProduct Portfolio Development - BU Pharma, 1SIFI SPA, Catania, Italy.
Purpose: The aim of this study was to test the activity of selected antimicrobial
agents against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin
resistant Staphylococcus epidermidis (MRSE) isolates.
Methods: The agar disk diffusion test, performed according to the National
Committee for Clinical Laboratory Standard guidelines, was used to test the
activity of six antibiotics commonly used in the treatment of ocular infections. In
particular, netilmicin, tobramycin, azithromycin, levofloxacin, moxifloxacin and
chloramphenicol were tested against 20 (8 MRSA and 12 MRSE) strains. The
isolates, among a total of 43 staphylococci from respiratory tract and ocular
infections, were previously phenotypically and genotipically characterized for
methicillin resistance using three different methods: Epsilometer test (E-test),
polymerase chain reaction for mecA gene detection and PBP2 latex agglutination
test.
Results: All (100%) of MRSE and 87,5% of MRSA isolates tested were netilmicin
sensitive. Except for chloramphenicol, a great percentage of MRSA resulted
resistant to all of the other antibiotics. In particular, 75%, 87% and 100% of the
isolates resulted resistant to fluoroquinolones (levofloxacin and moxifloxacin),
azitrhomycin and tobramycin respectively. As for the MRSE group, 25% of the
strains tested resulted resistant to chloramphenicol and moxifloxacin while 33%,
42% and 58% of the strains were resistant to levofloxacin, azithromycin and
tobramycin respectively (Table I).
Conclusions: Netilmicin, third-generation amminoglycoside, among the antibiotics
used in this study, showed the best susceptibility profile against the MRSA and
MRSE clinical isolates tested. Probably, the exclusive topical use of this antibiotic
for the treatment of ocular infections limits the emerging, spreading and persisting
of resistant microorganisms.
Commercial Relationships: Anna Rita Blanco, S.I.F.I. SpA (E); Andrea

Sudano Roccaro, S.I.F.I. SpA (E); Vincenzo Papa, S.I.F.I. SpA (E); Maria
Grazia Mazzone, S.I.F.I. SpA (E)
Support: None
Clinical Efficacy and Safety of Azithromycin 1.5% versus Tobramycin 0.3 %
Eye Drops in the Treatment of Children Bacterial Conjunctivitis
Dominique Bremond-Gignac1, Frederic Chiambaretta2, Hachemi Nezzar2, Bruno
Mortemousque3, Claude Speeg-Schatz4, Solange Milazzo5, Azithromycin Study
Group. 1Ophthalmology, St Victor Center, CHU Amiens, Picardie University,
Amiens, France; 2Ophthalmology, CHU Clermont Ferrand, Clermont Ferrand,
France; 3Ophthalmology, CHU Bordeaux, Bordeaux, France; 4Ophthalmology,
CHU Strasbourg, Strasbourg, France; 5Ophthalmology/Saint Victor Center, CHU
Amiens, University Jules Verne, Amiens, France.
Purpose: Bacterial infection is a common cause of conjunctivitis and accounts for
70% to 80% of all cases of conjunctivitis in children. The aim of the study was to
evaluate safety and efficacy of topical azithromycin 1.5% in bacterial conjunctivitis
in children.
Methods: A phase III randomized masked study, approved by ethics committee,
included 283 children presenting purulent bacterial conjunctivitis in 8 countries.
Patients received 1 drop of azithromycin twice a day for 3 days (146 in group A) or
1 drop of tobramycin every 2 hours (up to 8 times per day) for 2 days, then 1 drop 4
times daily for 5 days (137 in group T). Ocular examinations were performed at
D0, D3, D7 with conjunctival swabbing at D0 and D7. Patients range was: 148
patients (0- <2 yo), 44 patients (2- <4 yo), 77 patients (4- <12 yo) and 13 patients
(12- <18 yo). Efficacy was evaluated with the clinical cure (based on bulbar
conjunctival injection and conjunctival purulent discharge) on D3 for patients with
D0 positive cultures. Clinical cure assessment at D7 and conjunctival swabbing at
D0 and D7 was also performed. Safety data were also recorded.
Results: Among patients with a positive culture on D0, clinical cure in the worse
eye on D3 the azithromycin group 47.1% was significantly superior compared with
tobramycin group 28.7% (p=0.013). At D7, azithromycin (89.2% of patients cured)
was non-inferior to tobramycin (78.2% cured). The global efficacy assessed by the
investigator, at D3, was rated as "very satisfactory" for 60.6% of patients group A
and for 42% of patients group T (p=0.011), at D7: 76.8% for azithromycin group
versus 55% for tobramycin group (p=0.003). Bacteriological resolution on Day 7
was observed for 89.8% of patients in the T1225 group and 87.2% in the
Tobramycin group.Similar efficacy findings were observed in the younger
subgroup (0-2yo). No significant adverse event was observed during the study and
acceptability assessments by the investigator were significantly better for
azithromycin than for tobramycin (p=0.013).
Conclusions: In the study, Azithromycin 1.5% eye drops appears non inferior to
Tobramycin with a good safety profile for the treatment of purulent bacterial
conjunctivitis in paediatric patients. Azithromycin therapy, with a short duration
treatment twice a day, provided a significant clinical cure on D3 and leads to a
better quality of life for children and parents.
Commercial Relationships: Dominique Bremond-Gignac, Alcon (C, R), Thea
(C, R); Frederic Chiambaretta, Alcon (C, R), Thea (C, R); Hachemi Nezzar,
Thea (R); Bruno Mortemousque, Alcon (C, R), Thea (C, R); Claude SpeegSchatz, None; Solange Milazzo, Alcon (R)
Support: None

Increased Antibiotic Resistance Of Ocular Surface Flora After Repeated Use
Of Prophylactic Topical Fluoroquinolone Post Intravitreal Injection For
Neovascular Age-related Macular Degeneration (amd)
Vivian T. Yin1, Daniel Weisbrod1,2, Efrem Mandelcorn1,3, Carol Schwartz1,2, Radha
Kohly1,2, Ken Eng1,2, Wai-Ching Lam1,3, Peter Kertes1,2. 1Department of
Ophthalmology, University of Toronto, Toronto, ON, Canada; 2Sunnybrook Health
Sciences Center, Toronto, ON, Canada; 3Toronto Western Hospital, University
Health Network, Toronto, ON, Canada.
Purpose: To determine if repeated use of prophylactic topical fluoroquinolone post
monthly intravitreal injections for AMD will change the antibiotic resistance
profile of the ocular surface flora over time.
Methods: This was a prospective, multicenter, case-control study of patients 65
years and older with neovascular AMD undergoing treatment with monthly
intravitreal injections of ranibizumab. Patients were excluded if they had active
ocular or systemic infection, previously received intravitreal injections or were
treated with topical or systemic antibiotics in the past three months. Patients were
divided into two groups, those that received topical moxifloxacin for 3 days post
injection (antibiotic group) and those that did not (no antibiotic group). Cotton
tipped swabs of the inferior fornix were taken at baseline and at months 1, 2 and 3
prior to each injection. Samples were cultured and antibiotic sensitivity was
recorded by MIC50 levels. The data was analyzed for differences in antibiotic
resistance.
Results: Of 177 patients included in the study, 82 received antibiotics post
injections and 93 did not. The mean age was 81 years. The culture positive rate at
baseline was 28.0% in the antibiotic group and 14.5% in the no antibiotic group. In
the antibiotic group, the culture positive rate increased monthly to 34.7% at month
1, 38.0% at month 2 and 41.8% at month 3. In the no antibiotic group, the culture
positive rate was 22.2% at month 1, 8.8% at month 2 and 22.2% at month 3. The
most common organism cultured was coagulase negative staphylococcus (75%)
followed by diphtheroid (22.9%), staphylococcus aureus (6.3%) and streptococcus
viridians (4.9%). In the antibiotic group, there was a significant change in MIC50
for moxifloxacin from 0.105 at baseline, to 0.549 at month 1, 0.184 at month 2 and
1.184 at month 3 (p = 0.0106). In the no antibiotic group, the MIC50 for
moxifloxacin was 0.439 at baseline, 0.469 at month 1, 0.0052 at month 2 and 0.687
at month 3 (p = 0.102). There were no cases of endophthalmitis.
Conclusions: Patients treated with repeated use of topical moxifloxacin post
intravitreal ranibizumab showed a significant increase in antibiotic resistance of the
ocular surface flora at 3 months compared to those not given prophylactic topical
antibiotics.
Commercial Relationships: Vivian T. Yin, None; Daniel Weisbrod, None;
Efrem Mandelcorn, Bauch and Lomb (R); Carol Schwartz, None; Radha Kohly,
None; Ken Eng, None; Wai-Ching Lam, Alcon (R), Allergan (R, S), Novartis (S),
Pfizer (F); Peter Kertes, Alcon (R), Allergan (R), Arctic Dx (I), Bauch and Lomb
(C), Bayer/Regeneron (F, R), Novartis (F, R)
Support: Physician Services Inc Foundation
Multicenter Comparison of Loteprednol 0.5% vs Prednisilone Acetate 1% in
patients Post-Phacoemulsifaction with IOL implants
Carlos Buznego1, Gabriela Perez2, William Trattler3, Joseph A. Khell4, Bonnie
Henderson5. 1General & Surgical Ophthal, Center for Excellence in EyeCare,
Miami, FL; 2Ctr for Excellence in Eye Care, Miami, FL; 3Cornea, Center For
Excellence in Eye Care, Miami, FL; 4Ophthalmology/Cornea, Center for
Excellence in Eyecare, Miami, FL; 5Boston Eye Surgery and Laser Center, Boston,
MA.
Purpose: The study was a prospective, multicenter comparative trial evaluating the
effectiveness of Loteprednol 0.5% vs Prednisilone Acetate 1% for patients
undergoing cataract surgery
Methods: The study was conducted as a prospective, masked evaluation of cataract
surgery patients who received either Loteprednol 0.5% or brand-name Pred Forte
(Prednisilone Acetate 1%). The patients were prescribed either loteprednol or
prednisilone acetate, along with brand name bromfenac BID and besivance 0.6%
BID starting 3 days before surgery. The topical steroid as well as topical NSAID
was continued for 4 weeks postop. Besifloxacin was continued for 10 days postop.
Outcome measures included change in CCT, UCVA in patients who ended up on
target for distance (Spherical equivelant of -0.5 to +0.5, with 0.75 D of astigmatism
or less, BSCVA, AC cell scores, IOP at 1 day, 1 week, and 1 month post-Op, as
well as macular thickness as measured by high resolution OCT.
Results: 31 subjects were enrolled in group A of the study and 34 were in enrolled
in group B. 36% of the subjects in group A were male while 50% were male in
Group B. Compared to preop, subjects in group A experienced an increase in CCT
of 58, 15, and 4 microns at 1 day, 1 week and 1 month, respectively. Subjects in
group B experienced an increase in CCT from pre-Op of 25, 16, and 5 microns at 1
day, 1 week and 1 month, respectively. 79% and 78.6% of subjects in group A and
90% and 94.4% of subjects in group B had a UCVA of 20/30 or better at 1 week
and 1 month, respectively. 100% of patients in group A and 96.7% of patients in
group B had a BSCVA of 20/30 or better at 1 week. At 1 month, 100% of patients
in both groups had a BSCVA of 20/30 or better. Average AC cell scores at 1 week
averaged 0.95 cells in group A and 0.82 cells in group B. Elevations in IOP (>21
mmHg) were noted in one patient only in group B at 1 month.
Conclusions: Subjects in groups A and B fared equally in regards to BSCVA of
20/30 or better at 1 week and 1 month post-Op. Patients in group B had a lower
increase corneal thickness at 1 day, and achieved a higher percentage of eyes with
an UCVA of 20/30 or better at 1 week and 1 month post-Op.
Commercial Relationships: Carlos Buznego, Alcon (E, C), Allergan (E, C),
Bausch & Lomb (E, C), ISTA (E, C); Gabriela Perez, None; William Trattler,
None; Joseph A. Khell, None; Bonnie Henderson, Bausch & lomb (F)
Support: Bausch & Lomb
Prostaglandin E2 Sensitive Receptors Reduce Leukocyte Infiltration And
Protein Exudation In Lps Induced Uveitis In Rats
Neil poloso, Chau Vu, David F. Woodward. Biological Sciences, Allergan Inc,
Irvine, CA.
Purpose: Anterior uveitis can lead to potentially vision threatening conditions.
Several models of uveitis in rodents have been used to characterize potential
therapeutics. In this study we aimed to characterize the contribution of prostanoids
to inflammation in an LPS-induced uveitis model in rats.
Methods: We employed the rat EIU(endotoxin-induced uveitis) model. All drugs
and vehicle were applied to each eye in 10l of volume. Prostanoid receptor
agonists or vehicle were applied 1 hr before model induction (injection of LPS in
the hind rear footpad) and applied twice during uveitis development (2 hr and 5hr).
Rats were sacrificed at 18 hrs and aqueous humor was collected and pooled
bilaterally. Inflammatory markers (Leukocyte infiltration and protein exudation)
were measured using a hemocytometer and protein concentration, repectively.
Additionally, cytokines present in the aqueous humor were measured as an another
indicator of inflammation by Luminex.
Results: The peak of leukocyte infiltration was determined experimentally to be
18-20 hrs. Given this data receptor agonists were given TID, with one dose being a
pre-treatment. Initial studies showed no effect of the NSAID, Ketorolac (Acular).
Based on this data we hypothesized that receptor agonists and not antagonists
might be active in this model. PGE2 and selective receptor agonists Sulprostone
(EP1,3), Butaprost (EP2), S(5)-[1 E.3S)-3-hydroxy-4-phenylbut-1-en-1-yl]-1 -[6(1H-tetrzol-5-yl)hexyl] pyrrolidin-2-one (EP4) were applied topically at 0.1% as
described above. All EP receptor agonists as well as PGE2 suppressed leukocyte
infiltration in this model. However, only the EP 1,3 receptor agonist, Sulprostone,
inhibited leukocyte infiltration, protein exudation and inflammatory cytokines
(MCP-1, MIP-1, RANTES, IL-6, and TNF-).
Conclusions: Although the rat EIU model has been in use for decades, very little
published data exists on the activity of prostanoid agonists or antagonists in this
model. Surprisingly, we found that EP receptor agonists (even PGE2 itself), not
antagonists are active in inhibiting multiple inflammatory markers induced in this
model. This highlights that this model of uveitis is a useful tool in screening
prostanoid agonists with potential anti-inflammatory properties.
Commercial Relationships: Neil poloso, Allergan (E); Chau Vu, Allergan (E);
David F. Woodward, Allergan (E)
Support: None
Retinal Damage in Severe Chemical Burn and the Use of Infliximab Therapy
Fabiano Cade1,2, Eleftherios Paschalis1, Caio V. Regatieri3,2, Reza Dana1,3, Claes
H. Dohlman1. 1Cornea and Refractive Surgery, Massachusetts Eye & Ear
Infirmary, Harvard Medical School, Boston, MA; 2Ophthalmology, Federal Sao
Paulo University, Sao Paulo, Brazil; 3Schepens Eye Research Institute, Harvard
Medical School, Boston, MA.
Purpose: Severe alkali burns can lead not only to corneal opacity, but also to hardto-treat glaucoma and/or traction retinal detachment. The aim of this study was to
identify the mechanisms of damage to the retina, and its prevention by reducing the
inflammatory response after chemical injury.
Methods: A 20 second burn was performed by applying a 3mm filter paper soaked
with 1N NaOH to the central cornea of anesthetized Balb/c mice, followed by
continuous irrigation for 15 minutes. The animals were randomly divided into two
groups. Group 1 received an intra-peritoneal (i.p.) injection of (anti-TNF)
infliximab, and Group 2 received the same amount of isotype-matched IgG control
i.p.. The mice were clinically evaluated at days 1, 3, 5, 7, 10, and 14.
Neovascularization of the cornea was measured and compared between groups.
TUNEL assay was performed to assess retina damage.
Results: TUNEL assay showed damage to the ganglion cell layer in Group 2, but

not in the infliximab-treated Group 1. Although no statistically significant
difference was found at days 1 and 3, subsequently corneal neovascularization
invasion into the cornea was significantly less in the infliximab group 1 compared
to the control group 2. There was no difference in terms of opacification of the
cornea.
Conclusions: This study demonstrates damage to the ganglion cell layer early after
severe alkali burn. Additionally, the data suggest suppression of TNF can
drastically reduce both corneal and retinal damage.
Commercial Relationships: Fabiano Cade, None; Eleftherios Paschalis,
None; Caio V. Regatieri, None; Reza Dana, None; Claes H. Dohlman, None
Support: Boston Keratoprosthesis Research Grant
Topical Treatment With A Selective COX-2 Inhibitor Promotes Retinal
Ganglion Cell Survival After Optic Nerve Crush
Oliver W. Gramlich1A, Harald D. von Pein1B, Anika Ziegler1A, Kathrin Bitz1A,
Norbert Pfeiffer1A, Franz H. Grus1A. AExperimental Ophthalmology, BDepartment
of Neuropathology, 1University Medical Center, Mainz, Mainz, Germany.
Purpose: Our previous studies revealed that a selective cyclooxygenase 2 (COX-2)
inhibitor promotes neuronal cell survival in cell culture. Now, the study aim was to
examine if a COX-2 inhibition with Celecoxib promotes retinal ganglion cell
(RGC) survival after optic nerve crush and could reduce inflammation, especially
microglial activation.
Methods: 30 Lewis rats underwent unilateral optic nerve crush (ONC) under
ketamine/xylazine anesthesia. Animals received Celecoxib orally (50mg/kg; n=10)
or topically (0.1%, 30 l; n=10) twice a day. 10 animals served as control with no
medical treatment. In five animals per group, neuronal density in the ganglion cell
layer was evaluated on cresyl stained retinal flatmounts after ten days.
Correspondingly, axon counts of optic nerve sections were performed via toluidine
blue staining. In the other five animals per group, cellular infiltrates and optic nerve
architecture were examined after H&E and Luxol Fast blue staining in serial
horizontal optic nerve sections. Microglia was investigated in detail via anti-Iba 1
immunostaining. Amounts of infiltrates were graded by a 0 (no infiltrates) to 3
(strong infiltration) scoring and microglia was evaluated semi-quantitative.
Results: No changes in cell and axon density were detected in untreated eyes. At
the crushed site, cell and axon density differed not in the oral treated group
(1938372 cells/mm; 30383 axons) and the controls with no medication
(1922455 cells/mm; 278135 axons). Therefore, the topical treated group showed
a significantly increased RGC density and axons (2156587 cells/mm, p=0.04;
595161 axons, p<0.001). In this group, the laminar structure of myelin in the optic
nerves was predominantly intact and accompanied by a significantly reduction of
cell infiltrates (p=0.01). At least, microglial activity was decreased in animals with
topical administration.
Conclusions: A selective COX-2 inhibition reduces infiltration and microglial
activity after optic nerve crush and promotes RGC survival. The topical modulation
of the cellular immune response by COX-2 inhibition might be a new and sufficient
feature for neuroprotection and seems more suitable than a general systemic
modulation.
Commercial Relationships: Oliver W. Gramlich, None; Harald D. von Pein,
None; Anika Ziegler, None; Kathrin Bitz, None; Norbert Pfeiffer, None; Franz
H. Grus, None
Support: None
Twenty-Eight Day Microbial Preservative Efficacy of Loteprednol Etabonate
Ophthalmic Ointment, 0.5%; an Unpreserved Ointment with Low Water
Activity
Brien C. David, Lynne S. Gearinger, June Klingensmith, Heleen H. Decory. R&D
Microbiology, Bausch & Lomb, Rochester, NY.
Purpose: Loteprednol etabonate ophthalmic ointment, 0.5% (Lotemax ointment)
does not contain a preservative due to its low water activity. The purpose of this
study was to conduct a Preservative Efficacy (PE) test on Lotemax Ointment to
confirm that the unpreserved ointment demonstrates inherent biostatic properties.
Methods: Three lots of Lotemax ointment (including one expired lot) were
evaluated for PE testing following USP <51> guidelines against 5 USP PE
microorganisms (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia
coli, Candida albicans, Aspergillus brasiliensis), 2 field isolates (Serratia
marcescens, Bipolaris australiensis), 1 clinical isolate (Stenotrophomonas
maltophilia), and 1 American Type Culture Collection (ATCC) fungal strain
(Fusarium solani) representing a total of 5 bacterial and 4 fungal strains. Product
was heated to not more than (NMT) 44C, inoculated with ~10 6 CFU/mL challenge
microorganism, and incubated at 20-25C. At Day 7, 14 and 28 post-inoculation,
the test products were removed from incubation and heated to NMT 44C. Aliquots
were transferred into similarly heated isopropyl myristate, and dilutions thereof
were filtered for quantitative membrane colony count recovery.
Results: A reduction in microorganism levels was observed for all challenge

microorganisms at day 7, 14, and 28 for all three lots evaluated.
Conclusions: The results of this study confirm that the inherent low water activity
(<0.6) of the Lotemax Ointment does not provide an environment for microbial
growth.
Commercial Relationships: Brien C. David, Bausch + Lomb (E); Lynne S.
Gearinger, Bausch + Lomb (E); June Klingensmith, Bausch + Lomb (E); Heleen
H. Decory, Bausch + Lomb (E)
Support: None
A Novel Peptide from Adiponectin Suppresses LPS-induced Pro-inflammatory
Signaling in Macrophages by Inducing Interleukin-10 Expression
Huiyi Jin, Xiaolu Yang, Xun Xu, Kun Liu. Shanghai First People's Hospital,
Shanghai, China.
Purpose: Adiponectin (APN), an adipocyte-derived protein, is known to have
potent anti-inflammatory properties. It is present in a full length (fAPN) and a
globular form (gAPN). Previous studies observed that gAPN was more potent than
fAPN in decreasing inflammatory responses. Therefore, we identified a novel
peptide (named YH15, Y137~H151) from gAPN and investigated its antiinflammatory properties in lipopolysaccharide (LPS)-induced RAW264.7 cells.
Methods: Cells were pretreated with YH15 (1M, 10M, 50M), a control peptide
(50M) or dexamethasone (1M) for 18 h, and then stimulated with 100 ng/mL
LPS for 24 h. The protein levels of TNF-, IL-6 and PGE2 in the medium were
assessed with ELISA kits. LPS-stimulated NF-B activation in RAW264.7 cells
was examined using western blot analysis. The effects of YH15 on induction of IL10 expression and activation of possible signaling pathways in RAW264.7 cells
were determined in the absence of LPS.
Results: Pretreatment with YH15 significantly inhibited the productions of TNF-,
IL-6 and PGE2 in LPS-induced RAW264.7 cells (Fig. 1). YH15 pretreatment dosedependently suppressed LPS-mediated IB degradation and NF-B p65 nuclear
translocation. In addition, we demonstrated that YH15 peptide (50M) initially
increased the release of IL-10 in RAW264.7 cells after 18 h incubation. Western
blotting indicated that the peptide time-dependently increased phosphorylation of
ERK 1/2 rather than P38 MAPK or JNK within 1 hour. Treatment with U0126, a
selective inhibitor, abolished YH15-stimulated IL-10 induction.
Conclusions: Taken together, YH15 probably exerts anti-inflammatory effects by
stimulating the expression of IL-10 via ERK1/2 activation and subsequently
suppressing pro-inflammatory mediator expressions through the down-regulation of
NF-B
activity.
Commercial Relationships: Huiyi Jin, None; Xiaolu Yang, None; Xun Xu,
None; Kun Liu, None
Support: National Natural Science Foundation of China No. 30930097 and No.
30872827
Amelioration Of Endotoxin-induced Uveitis Treated With An Ikb Kinase
Inhibitor, Imd-0354 In Rats
Anton Lennikov1A, Nobuyoshi Kitaichi1A,2, Kosuke Noda1A, Ryo Ando1A, Zhenyu
Dong1A, Kenichi Namba1A, Kenichi Namba1A, Shigeaki Ohno1B, Susumu Ishida1A.
A
Laboratory of Ocular Cell Biology and Visual Science, Department of
Ophthalmology, BDepartment of Ocular Inflammation and Immunology, 1Hokkaido
University, Sapporo, Japan; 2Department of ophthalmology, Health Sciences
University of Hokkaido, Sapporo, Japan.
Purpose: Endotoxin-induced uveitis (EIU) is an animal model for acute ocular
inflammation. Nuclear factor-kappa B (NF-B) plays a key role to induce
inflammation. Inactive NF-B complexed with inhibitor of kappa B (IB) and
activated by degradation of IB by IB kinases (IKKs). IMD-0354 is one of the
IKK inhibitors which down-regulates NF-B activation. In the present study, we
examined whether administration of new IKK inhibitor IMD-0354 has therapeutic

effects on EIU in rats.
Methods: Six-week-old male Lewis rats were used. EIU was induced by
subcutaneous injection of 200 g of LPS from Escherichia Coli. Immediately, rats
were administered 30, 10, 3 or 0 mg/kg of body weight of IMD-0354
intraperitoneally. At 24 hours after LPS injection, the aqueous humor was
collected. Total aqueous protein concentrations were quantified and cell numbers
were counted. Also eyes were fixed with PFA 4% via intracardial injection and
immunohistochemically stained with anti-NFB antibodies.
Results: Aqueous protein concentrations were 92.55.3, 101.511.7, 112.63.2
and 117.33.0 g/ml in rats treated with 30, 10, 3 or 0 mg/kg of IMD-0354,
respectively. It was significantly lower in rats treated with 30 mg/kg and 10 mg/kg
of IMD-0354 compared with controls (P<0.01). Mean aqueous protein
concentration was 21.54.7 in nave rats. The numbers of inflammatory cells in
aqueous humor were 46.416.8, 68.2530.1, 128.4154.9, and 133.344.0 in rats
treated with 30, 10, 3 or 0 mg/kg of IMD-0354, respectively. Significantly fewer
aqueous inflammatory cells were counted in rats treated with 30 and 10 mg/kg of
IMD-0354 than controls (P<0.05). NF-Bp65 positive nuclei (yellow) in ciliary
body were detected as 249.027.8 in untreated eyes (Image 1,B), but only
146.63.0 in 30 mg/kg IMD-0354-treated rats (Image 1,A) (P<0.01). No NF-Bp65
positive nuclei were detected in Nave eyes (Image 1,C).
Conclusions: It was demonstrated that acute intraocular inflammation was
ameliorated by inhibition of IKK/NF-B pathway. IMD-1041, a prodrug for IMD0354, also showed similar anti-inflammatory effect with oral application in EIU
model (Data is not shown).Therefore IKK phosphorylation inhibitor might be a
promising substance for treatment of uveitis and other intraocular inflammation.
Commercial Relationships: Anton Lennikov, None; Nobuyoshi Kitaichi,

None; Kosuke Noda, None; Ryo Ando, None; Zhenyu Dong, None; Kenichi
Namba, None; Kenichi Namba, None; Shigeaki Ohno, None; Susumu Ishida,
None
Support: Japan Society for the Promotion of Science (JSPS); Ministry of
Education, Culture, Sports, Science, and Technology (MEXT); and the Mishima
Memorial Foundation, Japan.
Lutein-rich Marigold Extract Induces Gene Expression Of Phase II
Antioxidants In The PC12D Neuronal Cells
Seiji Miyake1A,2, Noriko Takahashi1A, Mariko Sasaki1,1A, Saori Kobayashi2, Kazuo
Tsubota1, Yoko Ozawa1,1A. ALaboratory of Retinal Cell Biology, 1Department of
Ophthalmology, Keio University School of Medicine, Tokyo, Japan; 2Wakasa
Seikatsu Co., Ltd., Kyoto, Japan.
Purpose: A carotenoid, lutein, has biological effects as an antioxidant as well as a
blue light filter. In the retina of disease models, such as an endotoxin-induced
uveitis model and a diabetes model, the levels of reactive oxygen species (ROS) are
reduced by lutein administration, as we have recently reported. However, it remains
to be elucidated whether lutein acts directly on ROS as a scavenger or indirectly
through activating phase II antioxidant enzymes and its related genes which can
promote ROS catalysis. In this study, we analyze whether lutein affects expression
of phase II antioxidants in a neuronal cell line, the PC12D cells.
Methods: The PC12D cells were cultured in Dulbeccos modified Eagles medium
(DMEM) supplemented with 10% fetal bovine serum (FBS) and 10% horse serum.
Cells were incubated with 50 ng/ml of nerve growth factor (NGF) together with
0.5% FBS for differentiation to neurons for 2 days. After removal of NGF, cells
were applied with lutein-rich marigold extract or vehicle for 6, 9, 12 and 24 hours,
and total RNA or whole cell lysate were prepared. Gene expression of phase II
antioxidants was analyzed using real-time PCR. Moreover, protein expression of
these molecules was measured using immunoblotting.
Results: Treatment with lutein-rich marigold extract induced gene expression of
catalase and Pi isoform of Glutathione S-transferase, GSTP1, in the neuronal cell
line. The latter was gradually upregulated in a time-dependent manner. However,
there was no effect on expression of other antioxidants such as superoxide
dismutase 1, superoxide dismutase 2 or 4-Nitroquinoline 1-oxide. Protein
expression of GSTP1 was also elevated.
Conclusions: Lutein-rich marigold extract induced the expression of phase II
antioxidants, catalase and GSTP1, suggesting that a pathway through which lutein
indirectly reduces ROS can be involved in the luteins effect in reducing ROS.
Commercial Relationships: Seiji Miyake, Wakasa Seikatsu Co., Ltd.
(E); Noriko Takahashi, None; Mariko Sasaki, None; Saori Kobayashi, Wakasa
Seikatsu Co., Ltd. (E); Kazuo Tsubota, None; Yoko Ozawa, Wakasa Seikatsu
Co., Ltd. (F)
Support: None
Ocular and Systemic Pharmacokinetics of Loteprednol Etabonate Gel (0.5%)
following Topical Ocular Administration to Rabbits
Shellise Glogowski, Joel W. Proksch. Drug Metabolism & Pharmacokinetics,
Global Pharmaceutical R&D, Bausch & Lomb, Rochester, NY.
Purpose: Loteprednol etabonate (LE) is a potent corticosteroid currently used to
treat ocular inflammation in a variety of suspension formulations and in an
ointment formulation. An ophthalmic LE gel formulation has been developed
which is non-settling, and contains a lower concentration of BAK at a more
physiological pH. This study investigated the ocular and systemic
pharmacokinetics of LE following a single topical ocular dose of LE gel to rabbits.
Methods: Male Dutch Belted rabbits (n=40) received a single 35-L topical ocular
instillation of the test formulation into each eye, corresponding to an LE dose of
175 g/eye. Over a 24-h period after dosing, animals were euthanized at predetermined time intervals and selected ocular tissues were collected from each
animal. Additionally, serial blood samples were also collected from one cohort of
animals for plasma analysis. Concentrations of LE in ocular tissues and plasma
were determined by mass spectrometry.
Results: LE was rapidly absorbed and distributed within the eye, with measurable
concentrations observed in ocular tissues within 5 min after dosing. Maximal
concentrations of LE were achieved within 0.5 h in ocular tissues and 1.5 h in
plasma following a single, topical ocular dose. Maximum concentrations of LE
were highest in tear fluid (1560 g/g), followed by bulbar conjunctiva (4.03 g/g),
cornea, (2.18 g/g), iris/ciliary body (0.162 g/g), and aqueous humor (0.0138
g/mL). Concentrations of LE remained measurable in all ocular tissues through at
least 12 h after dosing. In plasma, low but variable levels of LE were measurable
through 4 h following dosing.
Conclusions: Topical ocular administration of an LE gel formulation provided
rapid and sustained exposure to LE in ocular tissues with low systemic exposure in
rabbits. These data are consistent with the efficacy results from Phase 3 studies of
LE gel in postoperative inflammation and pain.
Commercial Relationships: Shellise Glogowski, Bausch & Lomb (E); Joel W.
Proksch, Bausch & Lomb (E)
Support: None
Topical Application Of Infliximab (Remicade) In The Treatment Of Corneal
Caustication
Fabio Bignami1A, Giulio Ferrari1A,2, Chiara Giacomini1A, Stefano Franchini1,
Paolo Rama1B. ACornea Unit - Eye Repair Lab, BOphthal-Cornea and Ocular
Surface Unit, 1San Raffaele Scientific Institute, Milan, Italy; 2Bietti Eye
Foundation, Rome, Italy.
Purpose: TNF-alpha is involved in various ocular diseases. Different therapeutic
options are available to inhibit TNF-alpha. Infliximab (Remicade), a recombinant
humanized monoclonal IgG1 antibody, has been administered systemically to treat
corneal peripheral ulcers and ocular pemfigoid. This pilot study was designed to
evaluate the safety and efficacy of topical Infliximab in the treatment of corneal
neovascularization induced by alkali burn in mice.
Methods: 20 C57BL/6 mice were used for this study. The left eye of 10 mice was
causticated with NaOH. Infliximab was applied topically four times a day (10 l) at
5 mg/ml dilution. Mice were divided in two groups: 1) instilled with Infliximab and
2) instilled with saline. The other 10 non causticated mice were divided in two

groups: 1) instilled with Infliximab and 2) with saline. After 7 days of treatment,
the eyes were removed for immunohistochemical analyses to evaluate corneal
penetration, using a goat anti-human IgG labeled with Alexa Fluor 546 dye. Safety
and efficacy endpoints were the following: 1) slit-lamp examination at 7 days to
evaluate corneal opacity and edema using a published scale, 2) corneal neovascular
area and 3) leukocyte infiltration determined on whole mounted corneas by
immunofluorescence, using anti-CD31 and anti-CD45 monoclonal antibodies,
respectively.
Results: Infliximab immunoreactivity was only found in the superficial corneal
epithelium. No pathological changes were detected in the epithelium, stroma, or
endothelium in normal corneas treated with either saline or Infliximab. Alkali
burned eyes receiving Infliximab didnt show a significant increase in corneal
transparency compared to the saline-treated group (p=0.35). In treated eyes,
neovascular area was similar to the vehicle treated group (p=0.50) and there was no
significant difference in CD45+ cell infiltration (p=0.35). The Mann-Whitney U
test was used for comparisons.
Conclusions: Our data suggest that Infliximab eye drops at 5 mg/ml is not toxic to
the normal cornea. We did not notice any difference in corneal neovascularization,
corneal opacity and CD45+ leukocyte infiltration in the Infliximab versus vehicle
groups. This could be due to the fact that Infliximab 5mg/ml 4 times a day does not
appear to penetrate the corneal epithelium. Studies are underway to see whether
different dosages and/or administration routes may achieve a better Infliximab
penetration in the corneal stroma.
Commercial Relationships: Fabio Bignami, None; Giulio Ferrari,
None; Chiara Giacomini, None; Stefano Franchini, None; Paolo Rama, None
Support: None
Identification of The Anti-Inflammatory Annexin-A1 Protein in Tears of
Normal Subjects and Association of its Cleaved-Inactive Form with Active
Vernal Keratoconjunctivitis Patients
Samia Yazid1, Andrea Leonardi2, Virginia Calder1, Roderick Flower3. 1Molecular
Therapy, UCL, Institute of Ophthalmology, London, United Kingdom; 2Medicine
School, University of Padua, Padua, Italy; 3Biochemical Pharmacology, QMUL,
William Harvey Research Institute, London, United Kingdom.
Purpose: Annexin-A1 (Anx-A1) is a glucocorticoid-regulated 37kDa protein with
powerful anti-inflammatory actions: enhanced release from target cells occurs
following addition of the anti-allergic cromone drugs. Anx-A1 is inactivated by
proteolytic cleavage of the N-terminus and increased amounts of the cleaved 33kDa
product correlate with inflammatory responses. We investigated if Anx-A1 is
detectable in human tear specimens from patients with vernal keratoconjunctivitis
(VKC).
Methods: Tear specimens were collected from patients with VKC (n=23), and noninflammatory control tear specimens from healthy volunteers (n=17) who gave
informed consent. Anx-A1 protein levels were measured by ELISA and by Western
blotting.
Results: In cell-free tear specimens from healthy donors, the concentration of AnxA1 was 433.6 54.3 pg/ml (n=17) and >90% was in the intact form. In tears from
VKC patients however, total Anx-A1 increased to 1908 319.3 pg/ml (n=23;
p<0.05) but only 48% (921.5 193.5 pg/ml) of this was the intact biologically
active species. Proteolytic cleavage of the protein was reduced in the group treated
with Lodoxomide (>80% is intact form, n=11, p<0.01).
Conclusions: Anx-A1 is constitutively present in normal human tears and is
proteolytically cleaved to inactivation during chronic allergic disease. Lodoxomide
treatment decreased the level of cleaved protein in VKC patients, and this is
perhaps related to its therapeutic action.
Commercial Relationships: Samia Yazid, None; Andrea Leonardi,
None; Virginia Calder, None; Roderick Flower, None
Support: None
HC-HA but not High Molecular Weight HA Polarizes LPS-Activated
Macrophages toward M2 Phenotype via CD44-Mediated Suppression of TLR4
Signaling
Hua He1, Scheffer C. Tseng2. 1TissueTech and Ocular Surface Center, Miami, FL;
2
Ocular Surface Center, Ocular Surface Res & Educ Fndtn, Miami, FL.
Purpose:
Human amniotic membrane (AM) and AM extract (AME)
exert anti-inflammatory actions by inducing neutrophil apoptosis and
suppressing macrophage activation. We attempt to elucidate the mechanism by
comparing HC-HA complex purified
from AME to high molecular weight hyaluronic acid (HA).
Methods:
LPS-stimulated RAW264.7 cells were treated with soluble or immobilized
HC-HA. The cell viability, proliferation, apoptosis, and adhesion were
determined. Macrophage M1 and M2 phenotypes were assessed by qPCR,

proteomic
arrays, cytokine ELISAs, immunostaining, and Western blot. The effects of HCHA
on apoptosis of activated neutrophils and phagocytosis of apoptotic neutrophils
by macrophages were also determined.
Results:
In solution, the cell viability was dose-dependently
inhibited by HC-HA at 5-100 g/ml but by HA only at 100 g/ml (p <0.05). The
decreased viability was attributed to reduced cell adhesion and elevated
apoptosis. Cells adhered on immobilized HC-HA but poorly on immobilized HA
via
CD44 and TLR4 receptors, and expressed significant lower pro-inflammatory M1
markers (TNF-, IL-12,) but higher anti-inflammatory M2 markers (IL-10, SPHK1,
LIGHT). IRF5, a transcriptional factor essential for polarizing toward M1
phenotype, is downregulated and excluded from nuclei. Compared to PBS and HA,
both
soluble and immobilized HC-HA significantly promoted apoptosis of activated
neutrophils and phagocytosis of apoptotic neutrophils. Antibody blocking of CD44
binding eliminated the M2 polarization on immobilized HC-HA.
Conclusions:
Polarization of LPS-stimulated macrophages by
immobilized HC-HA toward M2 phenotype is mediated by CD44 receptor resulting
in
suppression of TLR4 signaling.
Commercial Relationships: Hua He, TissueTech, Inc. (E); Scheffer C. Tseng,
TissueTech, Inc. (I, P)
Support: NIH, NEI, R44 EY017497 and R43 EY021045
Genetically Engineered IL-30 (IL27p28) Suppresses Experimental
Autoimmune Uveitis
Ren-Xi Wang, Cheng-Yong Yu, Rashid Mahdi, Charles Egwuagu. Laboratory of
Immunology, NEI, Bethesda, MD.
Purpose: IL-12 family cytokines have emerged as an important family of proteins
in host immunity. IL-27 (IL27p28/EBI3) promotes the differentiation of Th1 cells
while antagonizing Th17 development. On the other hand, IL-6 is a major inducer
of Th17 differentiation. Both IL-27 and IL-6 exert their effects through binding and
activating gp130. In this study, we have genetically engineered IL-30 (IL-27p28)
and investigated whether this IL-27 subunit protein is a dominant negative
regulator of gp130 signaling. Because both Th17 and Th1 cells are implicated in
the development of experimental autoimmune uveitis (EAU), we also examined
whether IL-30 can ameliorate EAU.
Methods: Full-length mouse IL-30 cDNA fragment was cloned into insect
expression vector pMIB. The IL-30 protein was expressed in insect cells, purified
by combination of Ni-NTA chromatography and differential centrifugation on
centricon filtration units and characterized by polyacrylamide gel electrophoresis,
immunoprecipitation and western blot. Biological effects of IL-30 were analyzed
by RT-PCR, Thymidine incorporation, Annexin-V staining and intracellular
cytokine staining assays. The effect of IL-30 on the development and progression
of EAU was monitored by Fundoscopy, Flow cytometry and histopathology.
Results: IL-30 inhibited the proliferation of CD4+T cells and transcription of genes
coding for pro-inflammatory genes (TNF-, IFN- and IL-17a) and pro-apoptotic
gene, ICE. IL-30 also suppressed the development and progression of EAU, in part,
by inhibiting the differentiation and expansion of Th1 and Th17 cells and
promoting IL-10 production.
Conclusions: IL-30 suppressed the development and progression of EAU,
suggesting that it can be used to treat uveitis and other autoimmune diseases.
Commercial Relationships: Ren-Xi Wang, None; Cheng-Yong Yu,
None; Rashid Mahdi, None; Charles Egwuagu, None
Support: None
Viscoelastic And Sedimentation Characterization Of Loteprednol Etabonate
Ophthalmic Gel, 0.5%
Martin J. Coffey, Stephen R. Davio. Pharmaceutical Product Development, Bausch
and Lomb, Rochester, NY.
Purpose: An ideal suspension formulation for consistent ophthalmic dosing is one
which requires no shaking to resuspend drug particles but is administered as a drop.
Loteprednol etabonate ophthalmic gel, 0.5% (LE gel), was designed to exhibit nonsettling characteristics yet be expressed as a drop. This study examines the
viscoelastic properties which are the basis for this unique behavior.
Methods: Yield point values were measured on an AR 2000 Rheometer (TA
Instruments) fitted with a vane rotor and cup containing 30 mL undiluted product at

25 oC. Oscillatory rheology was conducted on the AR 2000 using a 40-mm parallel
plate apparatus at an oscillatory frequency of 1 rad/s with variable oscillatory
stress. Sedimentation behavior was characterized using a LUMiSizer dispersion
analyzer (L.U.M. GmbH) at 1000 rpm (116-145 g) for up to 24 hr.
Results: Rotational rheology experiments on LE gel spanned the range of 1-100 Pa
of shear stress. At high shear stress (100 Pa) viscosity is <0.1 Pa-s. As shear stress
decreases viscosity increases. Viscosity approaches infinity near 4 Pa of shear
stress indicating this value as the yield point for this formulation. Oscillatory
rheology studies confirm the solid-fluid behavior of the product, dependent on
shear stress. At low oscillatory stress the elastic modulus, G' (~38 Pa) greatly
exceeds the viscous modulus, G" (2 Pa). As oscillatory stress increases, G'
decreases and eventually crosses over G" at ~6 Pa, demonstrating the transition
from a gel- to a sol-state. The transition from sol at high shear back to gel at low
shear occurs rapidly (within ~15 sec). The effect of these rheological properties is
seen in sedimentation studies. Sedimentation analysis of LE gel and Lotemax
using the LUMiSizer dispersion analyzer shows that LE gel does not sediment over
24 hr at 1000 rpm. Lotemax is a low viscosity ophthalmic suspension and
sediments almost immediately. We have observed visually and confirmed by HPLC
analysis that LE gel does not sediment over 16 months under ambient conditions.
Conclusions: The rheologic data indicate loteprednol etabonate ophthalmic gel,
0.5%, to be a shear-thinning material with solid-like properties at low shear and
fluid-like properties above shear stress of ~6 Pa. This results in a gel at rest which
allows no sedimentation of drug particles, and a fluid under applied shear stress
such as occurs when the product is expressed through a dropper tip. Because the
sol-gel transition occurs quickly, drug particle sedimentation does not occur during
the use of the product. By eliminating the need to shake the product, inconsistent
patient compliance to shaking instructions will not affect the delivered dose of
loteprednol etabonate ophthalmic gel, 0.5%.
Commercial Relationships: Martin J. Coffey, Bausch and Lomb (E, P);
Stephen R. Davio, Bausch and Lomb (E)
Support: None
A Novel Peptide Inhibits Inflammation in Endotoxin-induced Uveitis by
Suppressing NF-kappaB and MAPK Signaling Pathway
Xiao lu Yang, Hui yi Jin, Xun Xu. Ophthalmology, Shanghai First People's
Hospital, Shanghai, China.
Purpose: The aim of the present study was to investigate the effect of a novel
peptide derived from C-type lectin-like domain of human PAP on endotoxininduced uveitis (EIU) in rats.
Methods: EIU was induced in male Wistar rats by a footpad injection of
lipopolysaccharide. Peptides or dexamethasone were injected intravitreally an hour
before LPS was administered. The rats were killed 3 or 24 hours after LPS
injection. Eyes were enucleated for histologic examination, aqueous humor (AqH)
was collected, and the number of infiltrating cells, protein concentration, and
inflammatory marker levels were determined. The ocular tissue levels of nuclear
factor (NF)-kB p65 and phosphorylation levels of p38, ERK 1/2 and JNK were
evaluated by Western Blot.
Results: We assessed the anti-inflammatory effect of PAPep on EIU rats and
demonstrated that intravitreal pretreatment of PAPep concentration-dependently
attenuated clinical manifestation of EIU rats, reduced protein leakage and cell
infiltration into the AqH, suppressed TNF-, IL-6 production in AqH (Figure1),
and improved histopathologic manifestation of EIU (Figure2). Furthermore,
western blot analysis revealed that the possible mechanism for this antiinflammatory effect of PAPep may depend on its ability to inhibit the activation of
NF-kB and MAPK signaling pathway.
Conclusions: Our studies provide the first evidence that a novel peptide derived
from PAP could inhibit inflammation and the peptide may be a promising
candidate for the management of ocular inflammatory
diseases.
Commercial Relationships: Xiao lu Yang, None; Hui yi Jin, None; Xun Xu,
None
Support: National Natural Science Foundation of China No. 30930097 and No.
30872827
Anti-inflammatory Effects Of Glucocorticoids On Endotoxin-induced Uveitis
In Rats : Effects Of The Mode Of Administration
Pierre-Paul Elena, Nicolas CIMBOLINI, Sophie ANTONELLI, Hlne DUBOS,
Laurence FERAILLE, Philippe MARGARON. Iris Pharma, La Gaude, France.
Purpose: This study examined the outcomes of glucocorticoids administered
topically, systemically, or subconjunctivally on endotoxin-induced uveitis (EIU) in
rats.
Methods: EIU was induced in male Lewis rats by footpad injection of
lipopolysaccharide (LPS, 200 g). Animals were randomized in four groups. The
first two groups received either a single intravenous dose of 2.5 mg/kg
dexamethasone phosphate (immediately after LPS inoculation) or multiple
instillations of 0.1% dexamethasone (1h before and 1h, 2h and 3h after induction).
The third group received a single subconjunctival dose of 1 g methylprednisolone.
A non-treated induced group was used as control of induction. Treatment effects
were evaluated 24h after induction using clinical scoring, leucocyte and protein
infiltration and cytokine production using multiplex analysis in aqueous humor
(AH).
Results: Both intravenous and topical administrations of dexamethasone markedly
decreased clinical signs of EIU, inflammatory cell counts, protein concentration,
and levels of IL1beta, TNFalpha, IL6 and IL12 in AH. Sub-conjunctival
administration of methylprednisolone also decreased the symptoms of EIU but to a
lesser extent than dexamethasone.
Conclusions: In conclusion topical administration of dexamethasone allows for a
therapeutic effect on the anterior segment of the eye in the rat EIU model, and may
present a viable alternative to systemic administration of glucocorticoids.
Commercial Relationships: Pierre-Paul Elena, IRIS PHARMA (E); Nicolas
Cimbolini, IRIS PHARMA (E); Sophie Antonelli, IRIS PHARMA (E); Hlne
Dubos, IRIS PHARMA (E); Laurence Feraille, IRIS PHARMA (E); Philippe
Margaron, IRIS PHARMA (E)
Support: None

Clinical Experience With Sustained-Release Intravitreal Corticosteroid
Implants: A Comparison Between The Fluocinolone Acetonide (Retisert) And
Dexamethasone (Ozurdex) Implants In Uveitis
Cheryl A. Arcinue1, C. Stephen Foster1, Olga Ceron1, Lama Almulki2. 1Uveitis and
Ocular Immunology, Massachusetts Eye Research & Surgery Institution,
Cambridge, MA; 2Ophthalmology, Massachusetts Eye Res and Surgery Inst,
Cambridge, MA.
Purpose: To evaluate the efficacy of the fluocinolone acetonide (Retisert) implant
compared with the dexamethasone (Ozurdex) implant in patients with
noninfectious uveitis.
Methods: Chart review of patients at the Massachusetts Eye Research and Surgery
Institution (MERSI) was done and patients were selected and matched according to
age, sex, and type of uveitis. Twenty-seven eyes received either the fluocinolone
acetonide (n=16) or dexamethasone (n=11) implant. The primary efficacy outcome
was a comparison of the recurrence rate of uveitis after implantation, with followup ranging from 6 months to 2 years.
Results: There were no significant differences in the average age (p=0.56), sex
(p=1.0), and pre-implantation lens status (p=0.10) of patients in the two groups.
The diagnosis of CME (p=1.0) and glaucoma (p=0.31) were also not different
between the two groups. Majority of cases were idiopathic panuveitis, with 36.4%
in the Ozurdex group and 31.3% in the Retisert group. Recurrence rate in the
Retisert group was 1.8 per 100 person-months or 18 per 1000 person-months while
it is 7 per 1000 person-months in the Ozurdex group. Those in the Retisert group
were 2.6 times more at risk of recurrence than those in the Ozurdex group,
however, this was not statistically significant (p=0.41).
Conclusions: The dexamethasone intravitreal implant seems comparable to the
fluocinolone acetonide intravitreal implant in preventing recurrence of
noninfectious uveitis. Although the findings suggest that those in the Retisert group
were 2.6 times more at risk of recurrence than those in the Ozurdex group, this was
not found to be statistically significant.
Commercial Relationships: Cheryl A. Arcinue, None; C. Stephen Foster,
None; Olga Ceron, None; Lama Almulki, None
Support: None
Cytokine Levels In The Vitreous Fluid Of Patients With Ocular Sarcoidosis
And Patients With Diabetic Retinopathy
Kenji Nagata1, Kazuichi Maruyama1, Kazuhito Yoneda1, Takeru Yoshimura2, Kohhei Sonoda3, Shigeru Kinoshita4. 1Ophthalmology, Kyoto Prefectural Univ of Med,
Kyoto, Japan; 2Ophthalmology, Kyushu University, Fukuoka, Japan;
3
Ophthalmology, Yamaguchi University, Ube, Japan; 4Ophthalmology, Kyoto
Prefectural Univ of Med, Kamigyo-Ku, Japan.
Purpose: The purpose of this present study was to compare the levels of 27
cytokines that exist in the vitreous fluid between patients with ocular sarcoidosis
and patients with diabetic retinopathy.
Methods: This study involved 11 patients with sarcoidosis and 9 patients with
diabetic retinopathy. Undiluted vitreous samples were collected from each patient
during vitrectomy. Multiplex bead analysis was then used to investigate the levels
of 27 cytokines existing in those samples; i.e., measurement of the levels of platelet
derived growth factor (PDGF) bb, interleukin (IL)-1b, IL-1ra, IL-2, IL-4, IL-5, IL6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, eotaxin, fibroblast growth
factor (FGF) basic, granulocyte colony stimulating factor (G-CSF), granulocyte
macrophage colony stimulating factor (GM-CSF), interferon gamma (IFN-), IFN inducible protein (IP)-10, monocyte chemoattractant protein (MCP) 1,
macrophage inflammatory protein (MIP)-1a, MIP-1b, regulated on activation
normal T-cell expressed, and secreted (RANTES), tumor necrosis factor alpha
(TNF-), and vascular endothelial growth factor (VEGF).
Results: Vitreous concentrations of IL-1ra, IL-4, IL-6, IL-8, IL-10, IFN-, IP-10,
MCP-1, MIP-1b, and RANTES were found to be significantly higher in the ocular
sarcoidosis samples than in the diabetic retinopathy samples. Vitreous
concentrations of PDGFbb and VEGF were significantly higher in the diabetic
retinopathy samples than in the ocular sarcoidosis samples.
Conclusions: The results of this study show that anti-inflammatory therapy is
useful for patients with ocular sarcoidosis. Moreover, anti-VEGF treatment is
useful for patients with diabetic retinopathy. However, proinflammatory cytokine
levels were also high in the patients with diabetic retinopathy. These findings
indicate that diabetic retinopathy requires not only anti-VEGF treatment but also
anti-inflammatory treatment.
Commercial Relationships: Kenji Nagata, None; Kazuichi Maruyama,
None; Kazuhito Yoneda, None; Takeru Yoshimura, None; Koh-hei Sonoda,
None; Shigeru Kinoshita, None
Support: None

Human Tears Reveal Insights Into Corneal Neovascularization
Nadia Zakaria1A,2, Siggi Van Grassdorff1A, Kristien Wouters1B, Jos Rozema1A,
Nathalie Cools1C, Viggo Van Tendeloo1D, Zwi Berneman1D, Marie-Jose
Tassignon1A. AOphthalmology, BStatistics, CHematology, DCenter for Cell Therapy
and Regenerative Medicine, 1University Hospital Antwerp, Antwerp, Belgium;
2
Center for Cell Therapy and Regenerative Medicine, Antwerp University Hospital,
Antwerp, Belgium.
Purpose: The main aim of this study is to identify the factors crucial to the
development of corneal neovascularization in humans.
Methods: A total of 12 patients were enrolled in this study along with 10 healthy
volunteers constituting the control group. Basal tears along with reflex tears from
the inferior fornix, superior fornix and diluted corneal tears (using a corneal bath)
were collected along with blood serum samples. Ocular surface photographs (OSP)
from the vascular group were also taken. Flow cytometry was used to determine
levels of Interleukin 6 (IL-6), Interleukin 8 (IL-8), Vascular Endothelial Growth
Factor (VEGF), Monocyte Chemoattractant Protein 1 (MCP-1) and Fas Ligand
(FasL) levels in blood and tear samples which were then correlated with OSPs.
Results: Our results indicate that pro-angiogenic cytokines are present in tears of
normal control subjects at significantly higher levels than in serum(fig.1), with
highest concentrations found in basal tears. Localized corneal tear levels displayed
a significant rise in IL- 6, IL-8 and VEGF in patients with vascularized corneas
(fig.2). Of the five analytes examined, four positively correlated with the
percentage area of corneal vascularization, IL-8 being the exception although it was
positively correlated up until 10ng/ml. MCP-1 and IL-6 both showed positive
correlation with the highly angiogenic VEGF implicating them in its positive
feedback loop.
Conclusions: The results are encouraging and this is the first study of its kind
which has succeeded in highlighting significantly higher levels of pro-angiogenic
cytokines IL-6, IL-8 and VEGF in tears from patients with vascular
corneas.
Commercial Relationships: Nadia Zakaria, None; Siggi Van Grassdorff,

None; Kristien Wouters, None; Jos Rozema, None; Nathalie Cools, None; Viggo
Van Tendeloo, None; Zwi Berneman, None; Marie-Jose Tassignon, None
Support: Funds for Research in Ophthalmology, (FRO), Belgium


Errors In Measuring VEGF Concentrations In The Presence Of Anti-VEGF
Antibodies By Using ELISA
Hidenori Takahashi1,2, Yujiro Fujino1,2, Yasuo Yanagi2. 1Ophthalmology, Tokyo
KoseiNenkin Hospital, Tokyo, Japan; 2Ophthalmology, University of Tokyo,
Tokyo, Japan.
Purpose: Several reports have investigated the intraocular concentration of
vascular endothelial growth factor (VEGF) after administration of anti-VEGF
antibodies. Enzyme-linked immunosorbent assay (ELISA), which is widely used
for measuring VEGF concentration, uses specific antibodies. Therefore, if antiVEGF antibodies are already present in the samples, competition between these
antibodies would introduce errors in measuring the concentration of VEGF in the
samples. To confirm these hypothesis, we measured VEGF concentration in the
presence of anti-VEGF antibody by using ELISA.
Methods: We used the R & D Systems Quantikine Human VEGF ELISA kit
(Minneapolis, MN, USA) for this study. Ranibizumab, bevacizumab, pegaptanib,
and mouse control immunoglobulin G (IgG) were added to 1000, 500, 250, 125,
63, 31, 16, and 7.8 pg/ml VEGF. Using the previously investigated half-life of
these compounds, we calculated the each antibody concentration in the vitreous
cavity 28 days after the antibody injection, which showed that the concentration of
ranibizumab, bevacizumab, pegaptanib, and IgG was calculated as 0.11, 13, 8.6,
and 13 g/ml respectively.
Results: VEGF was measured at 540, 290, 120, 58, 29, 12, 6.4 pg/ml and
undetectable in the presnce of ranibizumab. VEGF was measured at 31 and 10
pg/ml and undetectable from then on in bevacizumab group. VEGF was measured
at 1000, 480, 230, 110, 55, 22, 11 pg/ml and undetectable in pegaptanib group.
VEGF was measured at 1000, 650, 300, 150, 73, 32, 13, and 7.2 pg/ml in IgG
group.
Conclusions: The VEGF concentration measured by ELISA was lower than the
total VEGF concentration when anti-VEGF antibodies were present. It was much
lower when the antibodies had a low dissociation constant or when there was a high
concentration of antibodies. Caution should be exercised when interpreting the
VEGF concentration measured by ELISA in the presence of anti-VEGF antibody.
Commercial Relationships: Hidenori Takahashi, None; Yujiro Fujino,
None; Yasuo Yanagi, None
Support: None
BDNF and Forskolin act on Retinal Development in a Ganglion Cell-free
Novel Explant System of the Chick Embryo
Alexander Greif, Julia Wiedemann, Gopenath Thangaraj, Paul G. Layer.
Entwicklungsbiologie, Technische Universitaet Darmstadt, Darmstadt, Germany.
Purpose: After its retrograde transport from the optic tectum, BDNF is strictly
required to ensure survival of ganglion cells in the vertebrate retina. Not much is
known, however, about i) possible further functions of this cytokine on
development of the other retinal layers, and ii) whether such functions could be
independent of the presence of ganglion cells. These two topics were approached
by applying a novel explant system (1), where ganglion cells die off quickly in
vitro, but otherwise differentiation of the retina pursues normally for 2-3 weeks.
Methods: The preparation of explants from E6 chicken embryonic retina has been
described (see ref.). At onset of cultures, explants were supplied with 10 ng/ml
BDNF and/or 5 M forskolin, a plant diterpene increasing intracellular levels of
cAMP. Cellular and laminar differentiation were documented
immunohistochemically, using cell type-specific antibodies, e.g. Brn3a for
ganglion cells, choline acetyltransferase (ChAT) for cholinergic amacrine cells,
CERN 901 for rhodopsin; cell proliferation was studied by BrdU uptake and
apoptosis by TUNEL assay.
Results: Treatment with BDNF or forskolin led to an extended survival of ganglion
cells, as shown by Brn3a. BrdU studies showed strongly increased rates of
proliferation in the future INL and ONL. Apoptosis was reduced in the GCL, but
not much in other retinal layers. Noticeably, differentiation of the inner plexiform
layer (IPL) was much advanced, as documented by ChAT. Similarly advanced was
the differentiation of rod photoreceptors. Some of these effects were particularly
pronounced after combination of both agents.
Conclusions: This study provides evidence that BDNF not only supports the
survival of ganglion cells, but rather has pronounced retina-internal functions, e.g.
IPL and photoreceptor differentiation. Further, it also shows that these processes
can occur in the absence of ganglion cells. References: 1. Thangaraj G, Greif A,
Layer PG (2011). Simple explant culture of the embryonic chicken retina with
long-term preservation of photoreceptors. Exp Eye Res 93(4), 556-564.
Commercial Relationships: Alexander Greif, None; Julia Wiedemann,
None; Gopenath Thangaraj, None; Paul G. Layer, None
Support: DFG-La379/20-1

Tear Cytokine Profile as a Non-invasive Biomarker of Inflammation for
Multicenter Clinic Trials of Ocular Surface Diseases
Yi Wei, Karen Fernandez, Neha Gadaria, Peter Dentone, Brittlyn Pearlman, Penny
Asbell. Ophthalmology, Mount Sinai School of Med, New York, NY.
Purpose: To evaluate the use of inflammatory cytokine profile of tears of human
subjects as a noninvasive biomarker of ocular surface inflammation and for
multicenter clinic trials.
Methods: Tears from normal subjects (n=13), contact lens wearers (n=10) and dry
eye disease patients (n=18) were collected from local and distant clinic sites.
Inflammatory cytokine concentrations in tear samples were measured using high
sensitive human cytokine multiplex kit from Millipore (Luminex).
Results: The repeatability was evaluated in three ways: (1) Pooled human tears
were divided into 3 repeats, each with 5 different dilutions. A total of
11inflammatory cytokines (IL-1, -2, -4, -5, -6, -8, -10, -12, -13, INF- and TNF-)
were analyzed. Theres no statistically significant difference either within the
repeats or among the dilutions. (2) Dilution effect: Since, in most dry eye subjects
and some normal subjects, only limited tear volume can be collected, dilution of
samples with assay buffer is required. To determine a reliable dilution range,
pooled tear samples with serial dilutions (from 3 to 156-fold) were evaluated.
Similar results were obtained when dilution factors were within 3-20
(corresponding to 15-2.5 L of tear sample per assay). (3) Standard operating
procedure (SOP) was developed using cytokine standards of known concentrations.
No statistically significant difference was observed among 6 repeats. The spike
recovery was within 80-120% range for all of the cytokines assayed, satisfying the
manufacturers standards of quality control criteria for Luminex analysis of
multiple cytokines. The feasibility was evaluated with tear samples from both local
(n=18, all DE) and distant (n=20, 10 contact lens wearers and 10 controls) clinic
centers in a masked fashion. At least 5-10 different cytokines, including IL-1, IL6, TNF-, were simultaneously detected from each sample. DE subjects treated
with anti-inflammatory agents for 3 months showed a decrease in IL-1 (4-fold)
and TNF- (2-fold), but an increase in IL-6 (3-fold) in comparison with subjects
randomized to placebo.
Conclusions: We have established a standard operating procedure in our institute
for multicenter clinic trials on human tear cytokine assessment. This study
demonstrated that tear inflammatory cytokines, such as IL-1, IL-6, TNF-, can be
used as biomarkers of inflammation in multicenter clinic trials of ocular surface
diseases such as DED. Cytokine biomarkers may provide critical information to fill
the gap between clinical treatment modalities and the mechanism(s) involved in
DED treatment.
Commercial Relationships: Yi Wei, Martin and Toni Sosnoff Foundation,
Research to Prevent Blindness Foundation (F); Karen Fernandez, Martin and Toni
Sosnoff Foundation, Research to Prevent Blindness Foundation (F); Neha
Gadaria, Martin and Toni Sosnoff Foundation, Research to Prevent Blindness
Foundation (F); Peter Dentone, Martin and Toni Sosnoff Foundation, Research to
Prevent Blindness Foundation (F); Brittlyn Pearlman, Martin and Toni Sosnoff
Foundation, Research to Prevent Blindness Foundation (F); Penny Asbell, Martin
and Toni Sosnoff Foundation, Research to Prevent Blindness Foundation (F)
Support: This study was supported by NIH fund (1R34EY017626-01A2), the
Reasearch to Prevent Blindness (RPB) Foundation, and the Martin and Toni
Sosnoff Foundation.
Cytokine Profile In Active Ocular Toxoplasmosis
Amanda Rey Torrente, Blanca Molins, Victor Llorens, Laura Pelegrn, Marina
Mesquida, Marc Figueras, Alfredo Adn Civera. Ophthalmology, Hospital Clinic
Barcelona, Barcelona, Spain.
Purpose: Toxoplasma gondii infection is an important cause of ocular disease.
Although parasite-mediated host cell lysis is probably the principal cause of tissue
destruction in immunodeficiency states, hypersensitivity and inflammatory
responses may underlie severe disease in otherwise immunocompetent individuals.
Methods: Using a multiplex assay, we determined the serum concentrations of 26
cytokines (Eotaxin, G-CSF, GM-CSF, IFN 2, IFN, IL-1 , IL-1, IL-2, IL-3, IL4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, IL-17, IP-10,
MCP-1, MIP-1 , MIP-1, TNF , TNF) obtained from patients with ocular
toxoplasmosis (n=41), and from an age-matched healthy control group (n=17).
Cytokine profiles were correlated with disease activity, complications, visual
prognosis, and demographical patient data.
Results: Levels of IL-8 were significantly reduced in patients with ocular
toxoplasmosis compared to the control group (20.13.0 vs. 48.112.2 pg/ml,
P<0.05). Active ocular toxoplasmosis was associated to increased levels of G-CSF
(52.29.4 vs. 31.1pg/mL, P<0.03) and decreased levels of MCP-1 (422.37 vs.
55234, P<0.02) compared to age-matched controls. Moreover, MCP-1
significantly decreased in active ocular toxoplasmosis compared to inactive disease
(422.37 vs. 51729, P<0.03). Spanish patients with ocular toxoplasmosis had
significantly higher levels of IL-8 compared to South American patients (25.24.6

vs. 10.42.7, P<0.02). Large interindividual variations were observed and no
significant correlations were found with specific cytokine profiles and
ophthalmoscopic findings or visual prognosis.
Conclusions: Decreased serum levels of MCP-1 and IL-8 and increased levels of
G-CSF were associated to active ocular toxoplasmosis. Thus, serum cytokine
profiling may contribute to the understanding of the physiopathology processes
underlying retinal damage in ocular toxoplasmosis and provide tools for new
targeted treatments and diagnosis.
Commercial Relationships: Amanda Rey Torrente, None; Blanca Molins,
None; Victor Llorens, None; Laura Pelegrn, None; Marina Mesquida,
None; Marc Figueras, None; Alfredo Adn Civera, None
Support: None
Results: Macular edema was most rapidly improved by IVB; however, decrease in
chorioretinal blood flow was seen in some cases after IVB treatment. Plasma nitrate
level before and 1 month after the treatment was 10829mol/L (meanSD) and
4210mol/L, respectively; and this reduction was significant (P<0.05, paired ttest). Laser treatment for avascular area was also effective for macular edema
without significant changes in ocular blood flow.
Conclusions: Intravitreal injection of bevacizumab is effective for the treatment of
macular edema from RVO; however, bevacizumab may cause a reduction of
chorioretinal blood flow compared to other treatments.
Commercial Relationships: Teruyo Kida, None; Hidehiro Oku,
None; Tsunehiko Ikeda, None
Support: None
557 Blood Flow

Thursday, May 10, 2012, 11:15 AM - 1:00 PM

Presentation Time: 11:15 AM - 1:00 PM
The Diameter Response To L-lactate And The Prostaglandin Analogue
U46619 Is Different In Porcine Retinal Arterioles And Capillaries In Vitro
Simon M. Pedersen, Toke Bek. Dept of Ophthalmology, Aarhus University
Hospital, Aarhus, Denmark.
Purpose: Disturbances in the regulation of retinal blood flow are involved in the
pathogenesis of vision threatening diseases such as diabetic retinopathy. These flow
disturbances are due to changes in the tone regulation of retinal arterioles, but
recent evidence suggests that disturbances in the regulation of retinal
microcirculation, such as lactate induced vasodilatation during retinal ischemia, are
also involved in the disease pathogenesis. Therefore, it is necessary to introduce in
vitro methods for studying the retinal microcirculation.
Methods: A special tissue chamber was developed for perfusion of a porcine
hemiretina while controlling flow, oxygenation, pH, and ambient metabolite
concentrations. The diameter of retinal capillaries were quantified by monochrome
microscopy video recordings during intraluminal perfusion with a fluorescent
dextran solution. The linear flow was quantified by counting the passage per time
unit of fluorescence labelled erythrocytes. In preliminary experiments changes in
the diameter of small retinal vessels were measured during intraluminal perfusion
with L-lactate and with the prostaglandin analogue U46619.
Results: Intraluminal perfusion with L-lactate had a significant dilating effect on
retinal arterioles from (meanSEM, n=4) 48.59.6m to 63.19.6m (p=0.01), and
a significant contractile effect on retinal capillaries from 7.90.6m to 6.40.5 m
(p=0.0004), whereas U46619 had a significant contracting effect on arterioles from
69.48.5m to 48.59.6 m (p=0.01), but no effect on the diameter of retinal
capillaries from 7.90.6m to 7.00.4m (p=0.32).
Conclusions: L-lactate and U46619 have different effects on the diameter of
arterioles and capillaries in the porcine retina in vitro. This emphasizes that the
mechanisms underlying flow regulation in retinal arterioles differs from the
regulation of retinal microcirculation. This may have implications for the
development of new treatments to normalize blood flow in retinal disease.
Commercial Relationships: Simon M. Pedersen, None; Toke Bek, None
Support: None

Coronary And Retinal Reactivity To Hyperoxia In Prediabetes And Type 2
Diabetes
Mary E. Lott1A, Bruce Smith1A, Julia E. Slocomb1B, Vikram Shivkumar1B, Kerstin
Bettermann1B. AHeart and Vascular Institute, BNeurology, 1Penn State Milton S
Hershey Med Ctr, Hershey, PA.
Purpose: To determine whether changes in coronary reactivity are associated with
changes in retinal blood vessel diameter and reactivity using a similar stimulus in
individuals with prediabetes and type 2 diabetes. We hypothesized that individuals
with prediabetes and type 2 diabetes would be associated with impaired coronary
reactivity and that this would be associated with impaired retinal vessel function.
Methods: Middle aged to older individuals with pre-diabetes (n=15), type 2
diabetes (n=22) and healthy age matched controls (n=14) were recruited. Retinal
reactivity was measured using the Dynamic Vessel Analyzer (DVA) in the
dominant eye and coronary reactivity was measured using transthoracic
echocardiography ultrasound during a 5-minute hyperoxia (100% oxygen)
stimulus. We calculated the overall percent change in retinal vessel diameter and
coronary velocity from baseline.
Results: Although type 2 diabetics compared to controls had impaired retinal artery
responses to hyperoxia (-.04 .86% vs. -3.81 1.41%, P=.03) and smaller arteryvein ratios (AVR) (.83 .02 vs. .90 .02, P=.03), coronary velocity responses to
hyperoxia were not significantly different between the three groups. Individuals
with prediabetes compared to controls had non-significant impairments in coronary
and retinal reactivity. Coronary function was significantly correlated to AVR (r= .35, P=.01) and retinal artery diameter (r= -.50, P=.00).
Conclusions: Retinal but not coronary reactivity to a hyperoxic stimulus is
impaired with diabetes; however, smaller retinal artery diameters and AVR are
associated with impaired coronary function. Funded by Deans Feasibility
Grant/PA Tobacco Settlement Funds. DVA funded by PSIDO Equipment Grant,
PA Lions Club, PSHMC Research Grant, Departments of Heart and Vascular
Institute, Neurology, and Ophthalmology.
Commercial Relationships: Mary E. Lott, None; Bruce Smith, None; Julia E.
Slocomb, None; Vikram Shivkumar, None; Kerstin Bettermann, None
Support: Funded by Deans Feasibility Grant/PA Tobacco Settlement Funds.
DVA funded by PSIDO Equipment Grant, PA Lions Club, PSHMC Research
Grant, Departments of Heart and Vascular Institute, Neurology, and O
Changes Of Plasma Nitrate Level And Ocular Blood Flow In Patients With
Retinal Vein Occlusion After Treatments
Teruyo Kida, Hidehiro Oku, Tsunehiko Ikeda. Ophthalmology, Osaka Medical
College, Takatsuki, Japan.
Purpose: To investigate relationship between changes of ocular blood flow and
plasma nitrate, nitric oxide (NO) derivative, in fresh cases with retinal vein
occlusion (RVO) accompanied by macular edema after different treatments.
Methods: Prospective study was conducted. Thirty-four consecutive patients with
macular edema from RVO (15 male and 19 female, age 40-89 years old) referred to
Osaka Medical College from May to October 2011, were enrolled in this study.
Macular OCT, plasma nitrate level, and fundus blood flow were measured before
and 1- and 3-months after each treatment. Ocular blood flow was measured by laser
speckle flowgraphy (LSFG, Fukuoka, Japan). Of all 34 patients, 25 patients were
affected with branch retinal vein occlusion, 9 with central retinal vein occlusion,
and 3 patients have medical history of glaucoma. Each treatment was the following;
17 patients were treated with laser treatment for avascular lesion, 6 with intravitreal
injection of bevacizumab (IVB) and 2 with sub-Tenon triamcinolone acetonide
injection. The other cases were not treated and followed with observation.

Effect of Nitric Oxide Inhalation on Retinal Arteriolar Diameter in Minipigs
Ioannis K. Petropoulos1A, Anne-Laure Martin1B, Georgios Mangioris1A, Efstratios
Mendrinos1A, Peter C. Rimensberger1B, Constantin J. Pournaras1A. ALaboratory of
Neurobiology and Physiology of the Retinal Circulation, Department of
Ophthalmology, BDepartment of Pediatrics, 1Geneva University Hospitals, Geneva,
Switzerland.
Purpose: L-arginine retinal juxta-arteriolar microinjection exerts a vasodilatory
effect on retinal arterioles in minipigs, probably by stimulation of nitric oxide (NO)
production. The purpose of the present study was to investigate the effect of
inhaled NO on the retinal arteriolar diameter in minipigs.
Methods: Five minipigs were anesthetized, intubated and mechanically ventilated,
and a Swan-Ganz catheter was inserted for measuring cardiac output by the
thermodilution technique. Ocular fundus image (one eye per animal) was
monitored and recorded in a digital videotape under stable hemodynamic and
ventilatory conditions. After baseline recordings, inhaled NO at a concentration of
80 ppm was added to the breathing circuit for 30 minutes and was then weaned off.
Retinal arteriolar diameter was assessed in arbitrary units (AU) at baseline, 30
minutes after the initiation of inhaled NO, and again 30 minutes after interruption
of the inhaled NO, using a Retinal Vessel Analyzer (Imedos GmbH, Jena,
Germany). At the same time points, body temperature, arterial blood gases, heart
rate, arterial blood pressure, central venous pressure, pulmonary artery pressure,
pulmonary capillary wedge pressure, and cardiac output were measured, and
systemic and pulmonary vascular resistances were calculated.
Results: Mean baseline retinal arteriolar diameter was 161.4 18.4 AU. Thirty
minutes after the initiation of NO inhalation, mean retinal arteriolar diameter had
increased by 8.9 5.3 % compared to baseline (p<0.05), measuring 175.2 15.5
AU. Thirty minutes after the interruption of NO inhalation, mean retinal arteriolar

diameter had decreased to 171.1 21.0 AU, but was still bigger than at baseline by
5.5 2.6 % (p<0.05). None of the hemodynamic parameters recorded was altered
significantly. In particular, cardiac output, arterial blood pressure and systemic
vascular resistance were not modified.
Conclusions: Inhalation of 80 ppm NO induces vasodilation of the retinal
arterioles in minipigs, which is partially sustained over 30 minutes after weaning
the inhaled NO. Further studies are needed to evaluate a potential clinical
application of this treatment modality in vascular diseases of the retina.
Commercial Relationships: Ioannis K. Petropoulos, None; Anne-Laure
Martin, None; Georgios Mangioris, None; Efstratios Mendrinos, None; Peter
C. Rimensberger, None; Constantin J. Pournaras, None
Support: Swiss National Funding 320030-122190
Topical Latanoprost Increases Retinal Blood Flow In The Macular
Circulation
Naomi Niwa, Tomoaki Hattori, Miho Nozaki, Yuichiro Ogura. Department of
Ophthalmology, Nagoya City Univ Medical School, Nagoya, Japan.
Purpose: Retinal Function Imager (RFI, Optical Imaging Ltd.) is a novel device
offering a noninvasive diagnostic approach to retinal function assessment. The
purpose of this study was to evaluate the effect of topical latanoprost on the retinal
microcirculation using RFI in healthy volunteers.
Methods: Five healthy volunteers (mean age, 32.5 years) were recruited and
microcirculation velocities at perifovea were quantitatively analyzed by RFI.
Intraocular pressure (IOP), blood pressure (BP) were also measured. In a singleblind trial, all measurement was done before and after the instillation of latanoprost
once a day for 2 weeks.
Results: One week instillation of latanoprost decreased IOP from 13.0 4.3 mmHg
to 10.0 2.2 mmHg (p=0.05). Retinal arterial blood flow velocity significantly
increased at 2 weeks after instillation of latanoprost (from 1.93 2.0 mm/sec to
2.35 0.30mm/sec, p=0.04). Retinal venous blood flow velocity also increased
(from 1.850.30 mm/sec to 2.070.24 mm/sec, p=0.08). There was no significant
difference in BP and ocular perfusion pressure before and after instillation of
latanoprost.
Conclusions: Our data showed that topical latanoprost significantly increased
arterial blood flow velocity at perifovea in human eyes. These results suggested
that RFI might be useful to evaluate blood flow velocity in the glaucoma patients,
to assess the efficacy of topical glaucoma agent.
Commercial Relationships: Naomi Niwa, None; Tomoaki Hattori, None; Miho
Nozaki, None; Yuichiro Ogura, None
Support: None
Measurement of retinal blood flow using dual beam bi-directional Fourier
domain Doppler OCT - comparison with laser Doppler velocimetry
Rene M. Werkmeister1A, Nikolaus Dragostinoff1A, Stefan Palkovits1B, Reinhard
Told1B, Leopold Schmetterer1B. AMed Physics and Biomed Eng, BClinical
Pharmacology, 1Medical University of Vienna, Vienna, Austria.
Purpose: We have recently introduced a technique for the measurement of retinal
blood flow using bi-directional Fourier domain Doppler OCT. We have carefully
validated this system by showing that blood flow is preserved at vessel bifurcations
when measured with our system.
Methods: In the present study we set out to investigate the response of retinal
blood flow to 100% oxygen breathing. This was done in a group of 10 healthy
subjects. Retinal blood velocities were measured on one day using bi-directional
Laser Doppler Velocimetry (LDV). On another study day retinal blood velocities
were measured using bi-directional Fourier domain Doppler OCT. On both days
retinal vessel diameters were measured using the Imedos Retinal Vessel Analyzer.
Results: 100% oxygen breathing caused a highly significant decrease in blood
velocities, vessel diameters and blood flow (p < 0.01 each). The data obtained on
the 2 study days showed a high degree of correlation (p > 0.05 each), and the
amount of blood flow reduction was in good agreement between the two methods
employed.
Conclusions: Our data confirm our previous findings that bi-directional Fourier
domain Doppler OCT is a valid method for assessing retinal blood flow. The
results we obtained with this technique are in good agreement with previously
published data using other techniques.
Commercial Relationships: Rene M. Werkmeister, None; Nikolaus
Dragostinoff, None; Stefan Palkovits, None; Reinhard Told, None; Leopold
Schmetterer, None
Support: FWF P21570
Role of Endothelin-1 in Optic Nerve Head Blood Flow Regulation during

Isometric Exercise in Healthy Humans
Agnes Boltz1A,1B, Doreen Schmidl1A, Michael Lasta1A, Semira Kaya1A, Stefan
Palkovits1A, Reinhard Told1A, Gabriele Fuchsjger-Mayrl1A,1C, Gerhard
Garhfer1A, Leopold Schmetterer1A,1B. ADepartment of Clinical Pharmacology,
B
Center for Medical Physics and Biomedical Engineering, CDepartment of
Ophthalmology and Optometry, 1Medical University of Vienna, Vienna, Austria.
Purpose: We have previously shown that choroidal blood flow regulation during
isometric exercise in healthy subjects is modified by administration of an
endothelinA (ETA) receptor antagonist. The present study tested the hypothesis that
human optic nerve head blood flow (ONHBF) during isometric exercise is also
modified by administration of the same ETA receptor antagonist.
Methods: The study was performed in 16 healthy young subjects in a randomized,
placebo-controlled cross-over design. The effect of a six minutes squatting period
on ONHBF was investigated without drug administration as well as during infusion
of BQ-123 or placebo. ONHBF was measured using laser Doppler flowmetry.
Ocular perfusion pressure (OPP) was calculated as 2/3*mean arterial pressureintraocular pressure.
Results: During all squatting periods OPP as well as ONHBF increased (p < 0.001
each). The increase in ONHBF was, however, less pronounced than the increase in
OPP indicating for some degree of blood flow regulation. Placebo did not alter the
ONHBF response to isometric exercise. BQ-123 did not alter OPP or ONHBF at
baseline. The drug also did not alter the response of OPP during isometric exercise.
The increase in ONHBF during squatting was, however, more pronounced than
during placebo (p < 0.01). This is also evident by looking into the pressure/flow
relationship. During BQ-123 administration the increase in ONHBF started at
lower OPPs than during placebo administration.
Conclusions: Our data confirm previously published observations that ONHBF
under basal conditions is not altered by an ETA receptor antagonist. Our results do,
however, indicate that endothelin plays a role in the response of ONHBF during
isometric exercise, providing some of the vasoconstrictor tone that counteracts the
increase in OPP. Our data also show that the ONHBF response to an increase in
OPP can be modified by administration of an ETA receptor antagonist.
Commercial Relationships: Agnes Boltz, None; Doreen Schmidl,
None; Michael Lasta, None; Semira Kaya, None; Stefan Palkovits,
None; Reinhard Told, None; Gabriele Fuchsjger-Mayrl, None; Gerhard
Garhfer, None; Leopold Schmetterer, None
Support: FWF (Austrian Science Fund) Grant P21406
Evaluation of Ultrasound-Assisted Thrombolysis Using Nontargeted
Ultrasound Contrast Agents in a Model of Retinal Vein Occlusion
Walid F. Abdallah1,2, Hitenkumar Patel3, Edward Grant3, Gerald J. Chader1, Mark
S. Humayun1. 1Ophthalmology, Doheny Eye Institute, Los Angeles, CA;
2
Ophthalmology, Faculty of Medicine, Zagazig University, Zagazig, Egypt;
3
Radiology, Keck School of Medicine, University of Southern California, Los
Angeles, CA.
Purpose: To study the potential efficacy of ultrasound (US)-assisted microbubble
(MB) destruction as an innovative thrombolytic tool for the treatment of retinal
vein occlusion (RVO).
Methods: Experimental RVO in the right eyes of 40 rabbits was induced using
laser photothrombosis followed 48 hs later by the US experiment. Rabbits were
randomly divided into 4 equal groups: US+MB group, US+saline group, MB+sham
US group, and no treatment group. The last 3 groups acted as control. Fluorescein
fundus angiography (FFA) and pulse Doppler US were used to evaluate the retinal
blood flow. Pulse Doppler measurements were represented as mean SD and oneway ANOVA test was used to compare means. Statistical significance was defined
as P <0.05.
Results: MB-assisted US thrombolysis resulted in restoration of flow in 70% of
rabbits. (Fig.1) None of the control groups showed significant restoration of retinal
venous blood flow. The mean venous blood velocity for the US+MB group
improved from 0.050.1 cm/s before the experiment to 1.280.9 cm/s at the end of
the experiment with a statistically significant difference (p value = 0.002). On the
other hand, the final measurements of retinal venous blood velocity for the 3
control groups were not statistically different from the baseline i.e. postlaser
measurements. (Fig.2) Pulse Doppler US measurement of the retinal blood velocity
in retinal vessels correlated well with the FFA results.
Conclusions: US-assisted thrombolysis using non-targeted contrast agents is a
promising therapeutic tool for RVO and may serve as a new, noninvasive
intervention for RVO in
humans.


showed fluorescence in both somas and processes and co-localized with capillaries
at each stratification.
Conclusions: We have imaged for the first time retinal pericyte structure and
capillary blood flow simultaneously in the living animal. Large vessel vasomotion
in response to visual stimulation shows evidence of intact neurovascular response
in the anesthetized mouse. Additionally, the micron-level resolution provided by
FAOSLO provides the means for future investigation of the role of pericyte
contractility in regulating neurovascular coupling in individual capillaries in the
living animal.
Commercial Relationships: Jesse B. Schallek, None; Ying Geng, None; David
R. Williams, #6,199,986 (P), #6,338,559 (P), #6264,328 (P), Alcon (R),
GlaxoSmithKline (C, R), Polgenix (R), US#5,777,719 (P)
Support: CVS Core Grant EY001319, CVS training grant EY007125, BRP grant
EY014375
Commercial Relationships: Walid F. Abdallah, None; Hitenkumar Patel,

None; Edward Grant, None; Gerald J. Chader, 20080262512 (P); Mark S.
Humayun, 20080262512 (P)
Support: NEI core grant EY03040
In Vivo Adaptive Optics Imaging Of Retinal Pericytes And Capillary Blood
Velocity In Mice
Jesse B. Schallek1A, Ying Geng1A,1B, David R. Williams1A,1C. ACenter for Visual
Science, BThe Institute of Optics, CFlaum Eye Institute, 1University of Rochester,
Rochester, NY.
Purpose: The role of pericytes in actively regulating local capillary blood flow is
controversial, in part because of the challenges of imaging these small cells in vivo.
The development of adaptive optics retinal imaging combined with cell specific
fluorescent labeling in transgenic mice provides the necessary resolution to
simultaneously image pericytes and measure capillary blood flow noninvasively.
Methods: Adult mice, hemizygous for the NG2 DsRed transgene (Jackson Labs,
Bar Harbor, ME), were used to visualize fluorescent retinal pericytes in vivo. Mice
were anesthetized with inhaled isoflurane and fitted with a contact lens to prevent
corneal drying. Fluorescence adaptive optics scanning laser ophthalmoscopy
(FAOSLO) recorded two channels simultaneously. A reflectance channel imaged
the vascular network with 789 nm light, revealing individual blood cells moving
through the retinal circulation. A second channel captured 579+22 nm DsRed
fluorescence excited with a 514 nm laser. Modulation of the excitation light served
as a visual stimulus to examine the effects of neurovascular coupling in the mouse
retina. Light was flickered at frequencies spanning 5-25 Hz, within the temporal
tuning peak of mouse ganglion cells.
Results: We observed a biphasic dilation (~5%) and constriction (~9%) of the
central retinal artery when the retina was stimulated with flashing 514nm light.
This vascular response to visual stimulation in the mouse retina is consistent with
neurovascular coupling reported in other mammals. Adaptive optics imaging also
revealed hemostatic events in individual capillaries lasting tens of seconds.
Confocal z-stacks acquired with FAOSLO showed the mouse retinal circulation has
at least three distinct stratifications, consistent with histological reports. Pericytes

Changes in Choroidal and Optic Nerve Head Blood Flow Regulation During
an Experimental Increase in Ocular Perfusion Pressure
Doreen Schmidl1A, Agnes Boltz1A,1B, Semira Kaya1A, Rene M. Werkmeister1B,
Nikolaus Dragostinoff1B, Michael Lasta1A, Elzbieta Polska1A, Gerhard Garhofer1A,
Leopold Schmetterer1A,1B. ADepartment of Clinical Pharmacology, BCenter for
Medical Physics and Biomedical Engineering, 1Medical University of Vienna,
Vienna, Austria.
Purpose: The aim of the present study was to compare choroidal blood flow
(ChBF) and optic nerve head blood (ONHBF) in response to an increase in ocular
perfusion pressure (OPP) induced by isometric exercise.
Methods: 48 healthy subjects were included. Subjects performed 6 minutes of
squatting to increase OPP. Either ONHBF (n = 24) or ChBF (n = 24) was measured
continuously by the means of laser Doppler flowmetry. OPP was calculated as
2/3*mean arterial pressure-intraocular pressure.
Results: The response in blood flow was less pronounced than the response in OPP
indicating for some degree of blood flow regulation. ChBF and ONHBF increased
during isometric exercise (p < 0.001 each). However, ChBF was better
autoregulated in response to isometric exercise than ONHBF (p = 0.023). ChBF
remained constant until an OPP of about 60% above baseline, whereas ONHBF
started to increase when OPP rose about 40% over baseline. In 3 subjects, an
interesting phenomenon was observed: During squatting ONHBF decreased
considerably below baseline when OPP fluctuated due to exhaustion, although OPP
was still much higher than at baseline.
Conclusions: The results of the present study indicate that ChBF shows better
regulatory properties than ONHBF during isometric exercise, most likely due to the
rich neural innervation. In the ONHBF experiments, a slight decrease in OPP
during the squatting periods was accompanied by a pronounced decrease in ONH
blood flow. Whether this plays a role in ocular disease remains unclear.
Commercial Relationships: Doreen Schmidl, None; Agnes Boltz, None; Semira
Kaya, None; Rene M. Werkmeister, None; Nikolaus Dragostinoff,
None; Michael Lasta, None; Elzbieta Polska, None; Gerhard Garhofer,
None; Leopold Schmetterer, None
Support: Austrian Science Funds Project No. P15970 and P21406
Retinal Blood Flow In Healthy Young Subjects
Gerhard Garhofer1A, Rene M. Werkmeister1B, Nikolaus Dragostinoff1B, Leopold
Schmetterer1A,1B. ADepartment of Clinical Pharmacology, BBiomed Engineering &
Physics, 1Medical University of Vienna, Vienna, Austria.
Purpose: To characterize total retinal blood flow in a group of healthy subjects.
Methods: 64 healthy volunteers were included in this study. Retinal venous
diameters were measured using a Dynamic Vessel Analyzer (DVA). Retinal blood
velocities were measured using bi-directional laser Doppler velocimetry (LDV). All
vessels with a diameter of more than 60 m entering the optic nerve head were
measured. Total retinal blood flow was measured by summing up all data from the
individually measured vessels. In a subgroup of 10 healthy subjects measurements
were also taken from the arteries, and results obtained for total retinal blood flow as
measured from retinal veins as well as from retinal arteries were compared.
Results: Total retinal blood flow was 44.0 13.3 l/min. Retinal blood flow was
highest in the temporal inferior quadrant, followed by the temporal superior
quadrant, the nasal inferior quadrant and the nasal superior quadrant (p < 0.001
each). In all quadrants retinal blood velocities were linearly correlated to vessel
diameters. Retinal blood flow as measured in retinal veins (42.1 13.0 l/min) and
retinal arteries (42.1 12.1 l/min) was similar (p = 0.16).
Conclusions: The present study provides reference values for total retinal blood
flow in 64 healthy subjects. The inter-individual variability in retinal blood flow is
high, making it unlikely that individual diagnostics can be based on measurements

of retinal blood flow. Total retinal blood flow may, however, be important in risk
stratification, which needs to be proven in future studies.
Commercial Relationships: Gerhard Garhofer, None; Rene M. Werkmeister,
None; Nikolaus Dragostinoff, None; Leopold Schmetterer, None
Support: None
Hemorheologic and Hemodynamic Response of Conjunctival Microcirculation
to Acute Hypotension in Rabbits
Bruce I. Gaynes1, Pang-Yu Teng2, Justin M. Wanek2, Mahnaz Shahidi2.
1
Ophthalmology, Loyola University Chicago, Maywood, IL; 2Ophthalmology and
Visual Sciences, University of Illinois, Chicago, IL.
Purpose: Understanding microcirculatory pathophysiology as result of disease
and/or drug or surgical treatment is an important yet relatively underappreciated
facet of clinical patient management. Quantification of conjunctival microvascular
hemodynamics and hemorheologics may provide valuable insight into perturbation
of microcirculation perfusion as a consequence of vascular compromise. In this
study, the feasibility of quantifying the conjunctival hemodynamic response to
acute hypotension was evaluated.
Methods: Image sequences of the conjunctival microvasculature of anesthetized
rabbits were captured using a previously developed slit lamp biomicroscope
imaging system. Hemodynamic parameters consisting of venous diameter (D),
blood velocity (V), blood flow (Q), and wall shear stress (WSS) were derived by
image analysis. The reproducibility and validity of hemodynamic parameters were
established by obtaining repeated measurements in venules and measuring changes
after topical administration of phenylephrine. Conjunctival hemodynamic and
hemorheologic responses to acute hypotension were evaluated by intravenous
administration of esmolol.
Results: Venous D and V measurements ranged from 9 to 34 microns (19 7
microns, mean SD, N = 16) and 0.13 to 0.99 mm/s (0.37 0.27 mm/s),
respectively. Calculated values of Q and WSS ranged from 14 to 307 pL/s (102
80 pL/s) and 0.8 to 29.2 dynes/cm2 (5.5 7.6 dynes/cm2), respectively. The
coefficient of variations of D, V, Q, and WSS measurements were on average 7%,
21%, 25%, and 27%, respectively. With phenylephrine administration, venous
diameter was reduced on average by 21%. With esmolol administration, a marked
reduction in blood pressure was noted with a concomitant decrease in V, Q, and
WSS, with increased D. Venous V, Q, and WSS were positively correlated (R >
0.92; p < 0.002; N 7) and D was negatively correlated (R < -0.73; p < 0.035; N
7) with blood pressure.
Conclusions: Alterations in microvascular hemodynamics due to experimental
hypotension can be readily measured in the bulbar conjunctiva thereby providing
an accessible and potentially exploitable approach to effectively evaluate
microcirculatory hemorheologic and hemodynamic parameters related to
cardiovascular disease and its treatment.
Commercial Relationships: Bruce I. Gaynes, None; Pang-Yu Teng,
None; Justin M. Wanek, None; Mahnaz Shahidi, None
Support: NIH grants RO1 EY17918, P30EY01792, Research to Prevent
Blindness, Illinois Society for the Prevention of Blindness and the Richard A.
Perritt Charitable Foundation
Evaluation Of Retinal Vasomotor Reactivity During Changes In Arterial
Blood Oxygen Content
Helene Kergoat, Carl Dutrisac, John V. Lovasik. School of Optometry, University
Montreal, Montreal, QC, Canada.
Purpose: To compare vasomotor responses to flicker across retinal quadrants
during experimental changes in the arterial blood oxygen (O2) saturation level.
Methods: The diameter of paired retinal arteries and veins was quantified with a
Retinal Vessel Analyser in 15 healthy volunteers (20 to 30 years of age) before,
during, and after short-term hyperoxia and hypoxia. Hyperoxic vasoconstriction
was induced by inhaling 100% O2 while hypoxic vasodilation was elicited by
inhaling 88% O2 in a balance of nitrogen. Vasomotor responses to 30sec of flicker
were elicited during inhalation of normal room air and towards the end of inhaling
either test gas. The O2 saturation level (SaO2), end-tidal carbon dioxide (EtCO2),
pulse rate (PR) and respiratory rate (RR) were recorded throughout testing. The
blood pressure and intraocular pressure were measured to calculate the ocular
perfusion pressure (OPP). Statistics consisted of ANOVAs for alpha = 0.05.
Results: Hyperoxia increased arterial SaO2 by 1.3% (p= 0.0001), decreased EtCO2
by 12.0% (p= 0.0001), reduced PR by 3.6% (p= 0.0001), increased OPP by 6.0%
(p= 0.0001), had no effect on RR, but decreased the caliber of retinal arteries and
veins by 9.3% and 14.3% respectively (p= 0.0001). In contrast, hypoxia decreased
arterial SaO2 by 8.2% (p= 0.0001), decreased EtCO2 by 3.8% (p= 0.0042),
increased PR by 14.6% (p= 0.0001), decreased OPP by 10.8% (p= 0.0001) and
increased arterial and venous diameter by 5.7% and 5.1% (p= 0.0001) respectively.
The changes in the vessel caliber were the same across quadrants. The amplitude of
arterial/venous dilation to flicker at baseline increased by 1.5% and 1.8%

respectively in hyperoxia (p< 0.02) and decreased by 2.0% and 1.7% respectively
in hypoxia (p= 0.0001). The vasomotor changes were not uniform across all
quadrants.
Conclusions: While hyperoxia was associated with an increase in the vasomotor
response, hypoxia was associated with a reduction in the amplitude of flickerinduced vasodilation. The reduced dilation was attributed to a structural limit for
dilation beyond that caused by systemic hypoxia, or that the need for additional
nutrients due to flicker was already satisfied.
Commercial Relationships: Helene Kergoat, None; Carl Dutrisac, None; John
V. Lovasik, None
Support: Natural Sciences and Engineering Research Council of Canada;
Canadian Foundation for Innovation
Effect Of Breathing Pure Oxygen And A Mixture Of 92% O2 + 8% Co2 On
Flicker Induced Vasodilatation
Stefan Palkovits1A, Michael Lasta1A, Reinhard Told1A, Gerhard Garhfer1A,
Leopold Schmetterer1A,1B. AClinical Pharmacology, BCenter for Medical Physics
and Biomedical Engineering, 1Medical University of Vienna, Vienna, Austria.
Purpose: Flicker light stimulation was shown to increase retinal and optic nerve
head blood flow. It was suggested that higher oxygen demand, caused by increased
neural activity due to flicker light stimulation might be the reason for the vascular
flicker response. The aim of the present study was to investigate the effect of
flicker light on the diameters of the vessels while breathing either 100% oxygen or
a mixture of 92% oxygen plus 8% CO2 compared to ambient air breathing.
Methods: In total 34 subjects were included. 24 participants breathed ambient air
first followed by inhaling pure oxygen. 10 subjects breathed air first followed by a
mixture of 92% oxygen and 8% CO2. Among these three groups measurements of
the vessel diameters were done using the Dynamic Vessel Analyser. During the
first 30 seconds baseline measurement was performed, followed by a 30 second
flicker period (frequency 8 Hz). In addition, a blood gas sample was collected by
capillary blood draw from the earlobe.
Results: Flicker light stimulation significantly increased retinal venous diameters
while breathing ambient air compared to baseline (p<0.05). Oxygen partial pressure
(pO2) was significantly increased, from 855 mmHg to 39080 mmHg during
100% oxygen breathing. Inhaling of the gas mixture of 92% oxygen and 8% CO2
increased the value from 8818 mmHg to 27771 mmHg. No change in flicker
response compared to ambient air breathing could be observed in the 100% oxygen
group. In contrast, breathing 92% oxygen + 8% CO2 significantly increased flicker
induced vasodilatation in retinal veins compared to room air (p < 0.05).
Conclusions: Breathing pure oxygen does not alter the response of retinal vessels
to stimulation with flicker light, whereas breathing 92% O2 + 8% CO2 significantly
increases the flicker response.
Commercial Relationships: Stefan Palkovits, None; Michael Lasta,
None; Reinhard Told, None; Gerhard Garhfer, None; Leopold Schmetterer,
None
Support: None
Quantitative Choroidal Blood Flow Measurement Using Doppler Optical
Coherence Tomography With Pulse Synchronization
Masahiro Miura1, Shuichi Makita2, Takuya Iwasaki1, Yoshiaki Yasuno2. 1Dept of
Ophthalmology, Tokyo Med Univ, Ibaraki Med Ctr, Inashiki, Japan;
2
Computational Optics Group, University of Tsukuba, Tsukuba, Japan.
Purpose: To evaluate the absolute choroidal blood flow volume rate by using
synchronous measurement of Doppler optical coherence tomography (OCT) at
1020 nm and pulsation. To evaluate the accuracy and reproducibility of absolute
choroidal blood flow measurement.
Methods: Ten sets of four Doppler OCT volumes were obtained continuously in
the area of 2.0 mm by 2.0 mm (750 A-scans by 64 B-scans). Doppler frequency
shift of choroidal vessel at the same point was obtained from each volume of the
Doppler OCT. The Doppler frequency shift is then transformed to an absolute
choroidal blood flow velocity by using the three-dimensional structural of the blood
vessel obtained from structural OCT image. The volumetric flow rate was
calculated from an absolute choroidal blood flow velocity and a vessel diameter
measured in en face vasculature image. Plethysmograph was recorded by a pulse
oximeter with a finger probe as the Doppler OCT measurement was performed.
Doppler flow signals were classified as belonging to one of 7 phases of heartbeat
based on the plethysmograph data. We evaluated 3 eyes of 3 normal subjects.
Volumetric flow rates at total 7 choroidal vessels were measured. A reproducibility
and accuracy of the measurement were evaluated.
Results: Pulse curves of Doppler signals during single heartbeat period were
synthesized based on the classified heartbeat phase. From these curves, cyclic

change of the choroidal blood flow volume rate was quantitatively obtained.
Absolute choroidal blood flow volume rates at peak systole and end diastole were
12.0 6.2 l/min (3.9 - 20.3 l/min) and 5.2 2.4 l/min (2.1 - 8.2 l/min),
respectively. Difference of two measurements by same observer was 19.7 13.7 %
of volume flow rate. The volumetric flow rates of choroidal vessels before and after
a bifurcation were used to evaluate the accuracy of the measurement. The mean of
the total outgoing blood flow rate and the incoming blood flow rate were 11.3
l/min and 11.0 l/min, respectively.
Conclusions: Quantitative data of choroidal blood flow volume rate were obtained
by Doppler OCT with plethysmograph. This measurement could provide
fundamental data about regulation of choroidal blood flow in normal and
pathological condition.
Commercial Relationships: Masahiro Miura, None; Shuichi Makita, Topcon
(F); Takuya Iwasaki, None; Yoshiaki Yasuno, Topcon (F)
Support: None
Assessment of Oxygen Saturation in Retinal Vessels of Normal Subjects and
Diabetic Patients without Retinopathy using the Johns Hopkins Flow
Oximetry System
Rachel E. Annam1, Mohamed A. Ibrahim1, Long Luu2, Yasir J. Sepah1, Millena G.
Bittencourt1, Owhofasa Agbedia1, Hyun S. Jang1, Jithin Yohannan1, Jessica
Ramella-Roman2, Quan D. Nguyen3. 1Johns Hopkins University, Wilmer Eye
Institute, Baltimore, MD; 2Biomedical Engineering, Catholic University of
America, Washington, DC; 3Diseases of the Retina, and Uveitis, Johns Hopkins
Univ,Wilmer Eye Inst, Baltimore, MD.
Purpose: To determine oxygen saturation (SO2) in retinal vessels of normal
subjects and diabetic patients without retinopathy using the Johns Hopkins Flow
Oximetry System (JHFOS) with novel assessment software and to establish a range
of normal retinal oxygen saturation in the venous circulation and analyze the
differences across the study groups.
Methods: The novel assessment software was used to determine oxygen saturation
levels in veins located between 1 to 2 mm from the margin of the optic disc in
normal subjects and diabetic patients without retinopathy employing images
captured by JHFOS. The retina was divided into 4 quadrants (superior, inferior,
temporal, and nasal), although the majority of vessels were in the superior and
inferior quadrants. Only those vessels clearly identified as veins by the
ophthalmologists were included in the analysis.
Results: 21 normal subjects (12 females and 9 males, mean age = 56 years)
contributed 21 eyes, and 17 diabetic patients (8 females and 9 males, mean age =
61 years) contributed 17 eyes to the final analysis. The mean (+/-SD) SO2 in retinal
veins of normal subjects was 55.2 6.6% (95% CI 52.2 - 58.2) and the mean (+/SD) venous SO2 in diabetic patients without retinopathy was 59.6 10.4% (95%
CI 54.3 - 64.9).
Conclusions: A range of normal venous SO2 has been established using the
JHFOS.The mean venous SO2 was found to be higher in diabetic patients without
retinopathy compared with normal subjects. The results obtained from the JHFOS
are in agreement with previously published data. Further studies with additional
subjects are indicated to validate the use of JHFOS in detecting retinal oxygenation
as well as to assess levels of retinal oxygenation among normal subjects and
diabetics with different stages of retinopathy.
Commercial Relationships: Rachel E. Annam, None; Mohamed A. Ibrahim,
None; Long Luu, None; Yasir J. Sepah, None; Millena G. Bittencourt,
None; Owhofasa Agbedia, None; Hyun S. Jang, None; Jithin Yohannan,
None; Jessica Ramella-Roman, None; Quan D. Nguyen, None
Support: National Eye Institute RO1 Award (EY17577)
Bloodflow Regulation In The Optic Nerve Head During Prolonged Elevation
Of The Intraocular Pressure
John V. Lovasik1, Helene Kergoat1, Mireille Parent1, Michael G. Quigley2. 1School
of Optometry, University of Montreal, Montreal, QC, Canada; 2Department of
Ophthalmology, McGill Univ/Univ of Montreal, Montreal, QC, Canada.
Purpose: Vascular autoregulation (AR) for the eye refers to the ability of a
vascular bed to maintain constant blood flow (BF) despite changes in the ocular
perfusion pressure (OPP) that arise from variations in the BP or IOP. AR in the
large retinal vessels occurs by modulation of vessel caliber. Perfusion of the optic
nerve head (ONH) is entirely by a capillary network. Laser Doppler flowmetry
(LDF) was introduced by Riva et al. for quantifying capillary blood flow, volume,
and velocity in the ONH, but its sophistication, limited availability and
measurements limited to short intervals have restricted its use to laboratory
research. Consequently there is a remarkable paucity of needed clinical studies on
AR in the ONH even though subnormal perfusion is implicated in low-tension
glaucoma and standard therapy is directed at decreasing IOP for improving BF. The
objective of this study was to determine if the effects of an acute reduction in the
OPP could be measured continuously over extended periods using computer-based

imaging.
Methods: Five healthy experienced adult volunteers (54.6 8.8 yrs of age)
participated in this study. The pupil of the test eye was dilated. A suction cup, to
increase the IOP, was placed on the temporal sclera ~2mm from the cornea after
instillation of 2 drops of a topical anesthetic and 2 drops of a topical lubricant.
Testing consisted of a 2-min baseline, 4-min increased IOP, and a 2-min recovery
during which the ONH was imaged at 25fps through a broadband green filter
(Imedos DVA) for better contrast of blood against tissue. Statistical analysis
consisted of ANOVAs (alpha of 5%). Changes in ONH density during an increase
in IOP were normalized to baseline. The IOP was increased to reduce the OPP
between 10% to 40%. Subjects were prompted to blink fully to maintain clear
corneas. Room lighting was minimal to eliminate scattering of ambient light into
the test eye.
Results: The abrupt increase in IOP caused a reduction of ~15% (p < 0.04) in the
group-averaged normalized BF that peaked at ~35 sec. Thereafter, BF manifested
low frequency undulations that slowly restored baseline BF in 135 sec.
Surprisingly, BF continued to exceed baseline and was ~18% above baseline in
~110 sec that coincided with the end of the 4-min OPP reduction phase. When
scleral suction was stopped abruptly, BF decreased linearly to be within 5% of
baseline by the end of recovery.
Conclusions: The physiological stress localized to the eye, i.e. an abrupt increase
in the IOP, elicited significant changes in the capillary BF in the ONH. None of our
subjects demonstrated a rapid AR response. The hyper-perfusion trend midway
through provocation may represent a regulatory-type response with diagnostic
value.
Commercial Relationships: John V. Lovasik, None; Helene Kergoat,
None; Mireille Parent, None; Michael G. Quigley, None
Support: Natural sciences and engineering research council of Canada; Canada
Foundation for Innovation
Sphingosine 1-Phosphate (S1P) Elicits Constriction of Isolated Porcine Retinal
Arterioles by Activation of the Rho/ROCK Pathway
Takayuki Kamiya, Taiji Nagaoka, Tsuneaki Omae, Ichiro Tanano, Shinji Ono,
Akitoshi Yoshida. Ophthalmology, Asahikawa Medical University, Asahikawa,
Japan.
Purpose: Sphingosine 1-phosphate (S1P) has been shown to play a role in
regulating immune responses, inflammatory processes, and vascular tone, and S1P
might be involved in diabetic retinopathy. The purpose of this study was to
investigate the effect of S1P on the retinal vessels and whether the Rho/ROCK
pathway is involved in S1P-mediated vasoconstriction.
Methods: The porcine retinal arterioles (70-102 m internal diameter) were
isolated, cannulated, and pressurized (55 cm H2O) without flow for in vitro study.
Videomicroscopic techniques were used to record diameter changes in response to
S1P with or without a novel ROCK inhibitor, K115 (Kowa Tokyo, Japan).
Results: Retinal arterioles constricted in response to S1P (0.1 nM-10 M). The
S1P-induced constriction was significantly (P<0.05) reversed by administration of
K-115. In addition, pretreatment with K-115 stopped the S1P-induced
vasoconstriction.
Conclusions: This study suggested that S1P may elicit constriction of the retinal
arterioles via the Rho/ROCK pathway in porcine retinal arterioles.
Commercial Relationships: Takayuki Kamiya, None; Taiji Nagaoka,
None; Tsuneaki Omae, None; Ichiro Tanano, None; Shinji Ono, None; Akitoshi
Yoshida, None
Support: None
Homocysteine Inhibits Endothelial-dependent Nitric Oxide-mediated Dilation
Of Retinal Arterioles Via Enhanced Superoxide Production
Taiji Nagaoka, Tsuneaki Omae, Ichiro Tanano, Akitoshi Yoshida. Ophthalmology,
Asahikawa Medical University, Asahikawa, Japan.
Purpose: Homocysteine, a sulfur-containing amino acid, is an emerging risk factor
for cardiovascular diseases. Furthermore, recent epidemiological studies confirm
that elevated homocysteine levels are associated with ocular vascular diseases.
However, the direct effect of homocysteine on ocular microvascular reactivity
remains unknown. Herein, we examined whether homocysteine can affect
endothelium-dependent nitric oxide (NO)-mediated dilation of retinal arterioles and
whether oxidative stress and distinct protein kinase signaling pathways are
involved in the homocysteine-mediated effect.
Methods: Porcine retinal arterioles were isolated, cannulated, and pressurized
without flow in vitro. Diameter changes were recorded using videomicroscopic
techniques.
Results: Intraluminal treatment with homocysteine (1 mM, 180 minutes)
significantly attenuated arteriolar dilation to endothelium-dependent NO-mediated

agonists bradykinin and A23187 but not to endothelium- independent NO donor
sodium nitroprusside. In the presence of superoxide scavenger TEMPOL,
NAD(P)H oxidase inhibitor apocynin, p38 kinase inhibitor SB203580, or
pioglitazone, but not xanthine oxidase inhibitor allopurinol, JNK inhibitor
SP600125 and pioglitazone with GW9662, peroxisome proliferator-activated
receptor- inhibitor (PPAR-), the detrimental effect of homocysteine on
bradykinin-induced dilation was prevented.
Conclusions: Homocysteine inhibits endothelium-dependent NO-mediated dilation
in retinal arterioles by producing superoxide from NAD(P)H oxidase, which
appears to be linked with p38 kinase. By impairing endothelium- dependent NOmediated vasoreactivity, homocysteine can potentially facilitate the development of
retinal vascular diseases. In addition, pioglitazone are beneficial by preserving
endothelial function, possibly through activation of PPAR-.
Commercial Relationships: Taiji Nagaoka, None; Tsuneaki Omae,
None; Ichiro Tanano, None; Akitoshi Yoshida, None
Support: None
Basal Blood Flow And Autoregulation Changes Within the Optic Nerve Head
Of Rhesus Monkey With Idiopathic Bilateral Optic Atrophy
Chelsea Piper1, Brad Fortune2, Grant Cull1, Claude F. Burgoyne1A, George A.
Cioffi1B, Lin Wang3. AOptic Nerve Head Research Lab, BOphthal-Discoveries in
Sight, 1Devers Eye Institute, Portland, OR; 2Devers Eye Institute, Legacy Health,
Portland, OR; 3Devers Eye Institute, Legacy Research Institute, Portland, OR.
Purpose: Idiopathic bilateral optic atrophy (BOA) of rhesus macaques is a disease
affecting primarily macular ganglion cells, causing thinning of the retinal nerve
fiber layer (RNFL) of papillomacular bundles and temporal optic disk pallor.
Histopathology revealed axon loss and gliotic changes within the temporal
quadrant of the anterior orbital optic nerve (ON). The purpose of the study was to
assess basal blood flow (BF) and autoregulation (AR) within the optic nerve head
(ONH) in eyes with BOA.
Methods: In six BOA and ten normal control (CTL) animals, two needles were
inserted into the anterior chamber of both eyes. One needle was to manometrically
control the intraocular pressure (IOP); the other was connected to a pressure
transducer to measure IOP. A laser speckle flowgraph (Softcare, Japan) was used to
measure BF in the ONH. Under stable systemic blood pressure, basal BF was
measured while the IOP was set at 10mmHg; AR capacity was assessed by
comparing the BF changes after the IOP was raised from 10mmHg to 30mmHg for
3min. Spectral Domain OCT (Spectralis Heidelberg GmbH) was used to measure
RNFL thickness (RNFLT). The averaged BF difference was analyzed using
ANOVA and the Mann-Whitney test was used to compare the mean RNFLT in
each quadrant between BOA and CTL eyes.
Results: The average RNFLT was 69.97.4m in BOA eyes, which was
significantly thinner than in CTL eyes (101.712.5m, P<0.05). The average basal
ONH BF was 8.241.24 in BOA eyes, which was significantly lower compared
with 10.691.33 of CTL eyes (P<0.001; see graph). The magnitude of basal BF
decrease was positively correlated with RNFLT (P<0.05). After IOP was raised to
30mmHg, BF decreased to 6.721.33 (22%, P<0.01) in BOA, but did not change in
CTL eyes (10.941.38, P=0.18). Blood flow was uniformly reduced in all four
quadrants of the ONH despite dominate structural damage in the temporal quadrant
(see graph).
Conclusions: The basal BF and autoregulation capacity in the ONH were
significantly reduced. The magnitude of the hemodynamic changes was correlated
with RNFLT, and blood flow reduction in BOA eyes were equally decreased in all
quadrants.
Commercial Relationships: Chelsea Piper, None; Brad Fortune, equipment

from Heidelberg Engineering, GmbH (F); Grant Cull, None; Claude F.
Burgoyne, equipment and unrestricted research support from Heidelberg
Engineering, GmbH (F); George A. Cioffi, None; Lin Wang, None
Support: NIH EY19939; Good Samaritan Foundation; Heidelberg Engineering,
GmbH, Heidelberg, Germany (equipment)
Innervation Pattern Of NG2 Positive Pericytes In The Rat Choroid
Herbert A. Reitsamer1A, Andrea Trost1A, Barbara Bogner1A, Clemens Strohmaier1A,
Christian Runge1A, Guenther Grabner1A, Ludwig Aigner1B, Falk Schroedl1A,1C.
Ophthalmology, BMolecular Regenerative Medicine, CAnatomy, 1Paracelsus

University Salzburg, Salzburg, Austria.
Purpose: Pericytes are contractile cells surrounding blood vessels. They might be
involved in the vessels caliber regulation and therefore in blood flow homeostasis.
In this study, we address the question whether pericytes in the choroid receive input
from the autonomic nervous system and therefore might be involved in ocular
blood flow regulation.
Methods: Rat choroidal wholemounts and sections were prepared for
immunohistochemistry of the pericyte marker chondroitin sulfate proteoglycan
(NG2) and the pan-neuronal marker PGP9.5, or tyrosine hydroxilase (TH),
vasoactive intestinal polypeptide (VIP), choline acetyl transferase (ChAT) and
calcitonin-gene related peptide (CGRP). Additionally, PGP9.5 and TH were
analyzed in the DCX-dsRed transgenic rat choroid, a putative model to study
pericytes function in-vivo.
Results: In the rat choroid, NG2 immunoreactivity was detected in cells and
processes surrounding blood vessels. These NG2 positive cells were not colocalized with PGP9.5, but received close appositions of PGP9.5, TH, VIP and
ChAT immunoreactive boutons. Blood vessels were densely surrounded by NG2
positive processes, in close vicinity to TH, VIP, ChAT and CGRP positive fibres
and boutons. In the DCX-dsRed transgenic rat, PGP9.5 and TH were screened and
revealed dense appositions of the respective markers on the dsRed positive cells, a
subpopulation of which is also positive for NG2.
Conclusions: Besides the innervations of vascular smooth muscle cells, the close
relationship of sympathetic (TH) and parasympathetic (VIP, ChAT) nerve fibres on
NG2 positive processes might indicate an additional target of the autonomic
nervous system for choroidal blood flow regulation, while the role of primary
afferent fibres (CGRP) remains unclear. Although additional experiments are
needed, regarding the autonomic innervation, similar findings in the DCX-dsRed
transgenic rat indicate this model potentially suitable for in-vivo experiments
unveiling the role of pericytes in blood flow regulation.
Commercial Relationships: Herbert A. Reitsamer, None; Andrea Trost,
None; Barbara Bogner, None; Clemens Strohmaier, None; Christian Runge,
None; Guenther Grabner, None; Ludwig Aigner, None; Falk Schroedl, None
Support: None
Retinal Arteriolar Reactivity Response Characteristics Assessed Using a
Sinusoidal Hyperoxic Provocation
Richard W. Cheng1A, Joseph A. Fisher1A, James Duffin1A, John G. Flanagan2, Tien
Wong3, Monica Jong1B, Sunni R. Patel3, Alanna Adleman1C, Christopher Hudson4.
A
Physiology, BOphthalmology and Vision Sciences, CMedical Science, 1University
of Toronto, Toronto, ON, Canada; 2Dept of Ophthal & Vision Sci, Univ of
Toronto,Toronto Western Hosp, Toronto, ON, Canada; 3Vision Science Division,
University Health Network, Toronto Western Research Institute, Toronto, ON,
Canada; 4School of Optometry, University of Waterloo, Waterloo, ON, Canada.
Purpose: To investigate the vascular reactivity response characteristics of the
retinal arterioles to hyperoxic provocation.
Methods: Four healthy volunteers were subjected to steady baseline (PETO2
100mmHg) and then sinusoidal hyperoxic provocation (PETO2 varied according to
a sinusoid waveform between 100 and 500mmHg) using the RespirActTM gas
sequencer. The Canon Laser Blood Flowmeter (CLBF-100) was used to repeatedly
measure retinal arteriole reactivity. Sinusoidal end-tidal O2 and retinal arteriole
reactivity data were fitted with a sine wave curve. The fitting procedure was
assisted by specially-written analysis programs (National Instruments Inc,
LabVIEW) that used the Levenberg-Marquardt algorithm. The phase lag, time
constant, response time, gain, and "cut-off period" of the arteriole reactivity
measurements to the sinusoidal hyperoxic provocation was computed.
Results: The average correlation coefficient for the sinusoidal hyperoxic and
diameter fits were r=0.9990.0009 and r=0.7300.1735, respectively, while for
velocity and blood flow the fit values were r=0.4520.1687 and r=0.5300.1621,
respectively. For diameter, the mean phase lag, time constant and response time
were 1.300.59 min, 1.300.59 min, and 3.891.78 min, respectively. The mean
diameter cutoff period, the sinusoidal period that gives the corresponding
vascular response of the time constant during a 500 mmHg step change, was
8.153.72 min. The average gain defined by the mean maximal diameter/ 100
mmHg O2 was -1.920.37. Based on the sinusoidal diameter fit, the average
maximal change in diameter was -7.691.50 m.
Conclusions: Assuming first order linear dynamics, we demonstrate a novel
method of calculating the response time of the retinal arterioles to hyperoxic
provocation.
Commercial Relationships: Richard W. Cheng, None; Joseph A. Fisher,
Thornhill Research Inc. (I); James Duffin, Thornhill Research Inc. (I); John G.
Flanagan, None; Tien Wong, None; Monica Jong, None; Sunni R. Patel,
None; Alanna Adleman, None; Christopher Hudson, Thornhill Research Inc. (I)
Support: ORF-RE

Signaling Pathway for Porcine Retinal Arteriolar Constriction to PKC
Activation: Roles of L-type Voltage-operated Calcium Channels, Myosin Light
Chain Kinase and Myosin Light Chain Phosphatase
Luke B. Potts1, Lih Kuo1,2, Wenjuan Xu2, Travis W. Hein2. 1SBTM, Texas A&M
Health Science Ctr, Temple, TX; 2Surgery, Scott & White Memorial Hospital,
Temple, TX.
Purpose: Increased protein kinase C (PKC) activity has been implicated in several
ischemic retinal diseases, possibly due to increased retinal arteriolar constriction.
Opening of plasmalemmal calcium channels and subsequent calcium/calmodulinmediated activation of myosin light chain (MLC) kinase, as well as inhibition of
myosin light chain phosphatase (MLCP), contribute to smooth muscle contraction
by regulating MLC phosphorylation. However, involvement of these signaling
entities in PKC-elicited retinal vasoconstriction has not been ascertained. Hence,
the purpose of this study was to determine the roles of L-type voltage-operated
calcium channels (L-VOCCs) and MLC kinase in PKC activation-induced
vasoconstriction. Whether the activation of PKC or L-VOCCs leads to MLCP
phosphorylation in retinal arterioles was also assessed.
Methods: We utilized an isolated vessel approach and pharmacological tools to
assess porcine retinal arteriolar constriction to activators of PKC (PDBu, 0.1 M)
and L-VOCC (Bay K 8644, 1 M). Immunoblotting was used to determine
phospho-MLCP levels in retinal arterioles.
Results: Isolated and pressurized (55 cmH2O) porcine retinal arterioles (~80 m
maximum diameter) developed stable basal tone (~42% of maximum diameter),
and constricted by 226% and 165% in response to PDBu and Bay K 8644,
respectively. The broad-spectrum PKC inhibitor G-6983 (3 M) and L-VOCC
blocker nifedipine (1 M) prevented, as well as reversed, the vasoconstriction
elicited by PDBu. Inhibition of MLC kinase with ML-9 did not prevent the
development of vasoconstrictions to PDBu or Bay K 8644, but reversed
vasoconstrictions to both agonists. Both PDBu and Bay K 8644 elicited an increase
in phospho-MLCP levels relative to control.
Conclusions: PKC activation stimulates calcium influx through L-VOCCs, which
leads to retinal arteriolar constriction. MLC kinase is activated by calcium from LVOCCs for maintenance, but not for development, of PDBu-induced retinal
vasoconstriction. Increased inhibitory phosphorylation of MLCP with PDBu and
Bay K 8644 treatments suggests that this signaling mechanism may enhance MLC
phosphorylation and subsequently contribute to retinal vasoconstriction in response
to PKC activation.
Commercial Relationships: Luke B. Potts, None; Lih Kuo, None; Wenjuan
Xu, None; Travis W. Hein, None
Support: NIH EY018420 and Retina Research Foundation
Correlation Of Retinitis Pigmentosa Disease Stage With Orbital Color
Doppler Imaging
Amani S. Albakri, Eman Al-Shahwan, Sawsan R. Nowilaty. Vitreoretinal Division,
King Khaled Eye Specialist Hospital, P.O Box 7191, Riyadh 11462, Saudi Arabia.
Purpose: To prospectively explore if disease stage in retinitis pigmentosa (RP)
correlates with retinal and/or choroidal blood flow velocities measured by orbital
color Doppler imaging.
Methods: Twenty nine patients with RP (age 11-67years, mean 34) were
prospectively evaluated with 1) standardized clinical staging of the fundus guided
by photographic templates of 4 parameters (degree of optic disc pallor, degree of
retinal vascular attenuation, site of intraretinal pigmentation and amount of
intraretinal pigmentation); 2) standardized orbital color Doppler imaging (CDI) of
the ophthalmic artery, central retinal artery, posterior ciliary arteries and central
retinal vein; 3) standardized Goldmann visual fields and 4) standardized full-field
electroretinography (ERG). Ten age-matched controls without systemic vascular or
ocular disease underwent standardized CDI for comparison.
Results: Peak-systolic and end-diastolic velocities in the central retinal artery,
posterior ciliary arteries and maximal and minimal velocities in the central retinal
vein were consistently and significantly lower in RP patients than in controls
(p<0.001). Furthermore, the central retinal artery peak-systolic velocity correlated
inversely with advancing clinical stage of RP (r -.367, p 0.005), greater disc pallor
(r -.032, p 0.015), greater retinal vascular attenuation (r-0.47,p<0.001), more
widespread pigmentation than the midperipheral retina alone (r -0.32, p 0.016),
smaller remaining central visual field (r -0.53, p < 0.001) and lower ERG
amplitudes (r .354, p 0.006). The central retinal artery was the vessel which
displayed the most significant changes in velocity as RP progressed, followed by
the central retinal vein. The ophthalmic and ciliary arteries velocities showed no
significant correlation with the stage of RP.
Conclusions: Retinal arterial velocities are decreased in eyes with retinitis
pigmentosa. With disease progression, central retinal arterial velocities further
decrease. These findings may add to the characterization of retinitis pigmentosa and
to a better understanding of the pathogenesis of the disease and its progression.
Commercial Relationships: Amani S. Albakri, None; Eman Al-Shahwan,

None; Sawsan R. Nowilaty, None
Support: None
Theoretical Analysis Of Myogenic And Metabolic Responses In Retinal Blood
Flow Autoregulation
Julia Arciero1, Aaron Pickrell2, Brent Siesky3, Alon Harris3. 1Mathematics, Indiana
University-Purdue University Indianapolis, Indianapolis, IN; 2St. George's
University School of Medicine Grenada West Indies, Great River, NY;
3
Ophthalmology, Indiana University School of Medicine, Indianapolis, IN.
Purpose: Retinal blood flow autoregulation is achieved by altering the tone of
arteriolar smooth muscle cells and capillary pericytes according to myogenic,
shear-dependent, and metabolic mechanisms. A failure in retinal autoregulation
may be a risk factor for glaucoma, suggesting that glaucoma therapies should target
vascular regulatory mechanisms. Here, a mathematical model is used to predict the
relative importance of regulatory mechanisms in achieving retinal autoregulation.
Methods: Resistance vessels are assumed to respond to local changes in pressure,
shear stress, and lactate production and to the downstream metabolic state
communicated via conducted responses. Arteriolar diameters are calculated based
on vascular smooth muscle mechanics, and capillary diameters remain fixed unless
lactate production exceeds a certain threshold. Model parameters governing wall
tension are fit to data from porcine retinal arterioles and differ for each vessel type.
The response to lactate production is assumed to differ in arterioles and capillaries
and under conditions of normoxia or hypoxia, as has been seen experimentally.
Results: The factor by which flow changes as the blood pressure exiting the central
retinal artery is varied between 32 and 44 mmHg is used to indicate the degree of
autoregulation. A factor of 1 indicates perfect autoregulation. In the presence of
only myogenic and shear-dependent mechanisms, the model predicts a poor degree
of autoregulation (factor of 1.74). However, including the effects of both the
conducted and lactate responses in arterioles significantly improves the degree of
autoregulation (factor of 1.14).
Conclusions: The model results indicate that metabolic responses are crucial in
achieving autoregulation of blood flow. This model can also be used to predict the
relative roles of lactate and conducted responses in both autoregulation and
metabolic flow
regulation.
Commercial Relationships: Julia Arciero, None; Aaron Pickrell, None; Brent

Siesky, None; Alon Harris, None
Support: None
The Dcx-dsRed Transgenic Rat As A Model To Study Pericyte Function?
Andrea Trost1A, Falk Schroedl1A,1B, Barbara Bogner1A, Clemens Strohmair1A,
Christian Runge1A, Guenther Grabner1A, Ludwig Aigner1C, Herbert A. Reitsamer1A.
A
Ophthalmology/Optometry, BAnatomy, CMolecular Regenerative Medicine,
1
Paracelsus Medical University, Salzburg, Austria.
Purpose: The function of pericyte contraction and its role in blood flow regulation
is based on contraction experiments in cell culture and on isolated microvessels. In
vivo, due to a lack of appropriate models and technical limitations, the contribution
of pericytes to local blood flow regulation has not been proven yet.
Retinal and choroidal flat mount preparations of the doublecortin-promoter-dsRed
transgenic rat (DCX-dsRed) unveiled dsRed positive cells with perivascular
localization. The aim of the present study was to characterize these cells and to
analyse whether the DCX-dsRed rat model will be suitable to study pericyte

function in vivo.
Methods: Eyes from DCX-dsRed rats were analyzed for pericyte, endothelial and
neuronal markers using immunohistochemistry and confocal laserscanning
microscopy. Whole mounts and sections were tested for the potential pericyte
markers NG2, PDGFRb, CD146, as well as CD31 (endothelial marker) and
compared with cells positive for DCX-dsRed.
Results: In the retina, perivascular dsRed positive cells did not co-localize with
NG2, PDGFRb, CD146, and CD31. In the choroid dsRed positive perivascular
cells protrude their processes around the vessels and co-localize with the
aforementioned pericyte markers.
These dsRed positive cells, however, represent a subgroup of the NG2, PDGFRb
and CD146 immunreactive cells since perivascular structures were detectable with
the pericyte markers lacking the dsRed positive signal.
Conclusions: While retinal dsRed positive perivascular cells in the DCX-dsRed rat
do not represent pericytes, this is the case in the choroid, since here a colocalization with the pericyte markers NG2, PDGFRb and CD146 was obvious.
Although these choroidal dsRed positive cells do not represent the entity of
choroidal pericytes, they may be used for further studies on pericyte function and
blood flow in vivo.
Commercial Relationships: Andrea Trost, None; Falk Schroedl,
None; Barbara Bogner, None; Clemens Strohmair, None; Christian Runge,
None; Guenther Grabner, None; Ludwig Aigner, None; Herbert A. Reitsamer,
None
Support: None
Caffeine Affects Ocular Microcirculation In Young Healthy Subjects
Naim Terai1A, Eberhard Spoerl1A, Richard P. Stodtmeister2, Lutz E. Pillunat1B.
A
Ophthalmology, BDept of Ophthalmology, 1University of Dresden, Dresden,
Germany; 2Ophthalmology, University Hospital Carl Gustav Carus, Rodalben,
Germany.
Purpose: To investigate the effect of caffeine on ocular microcirculation in young
healthy subjects
Methods: Seventeen healthy subjects were included in this study.The diameter of
retinal vessels was measured continuously with the dynamic vessel analyzer (DVA)
before and 1 hour after 200 mg caffeine intake. After baseline assessment a
monochromatic luminance flicker was applied to evoke retinal vasodilatation.
Flicker response was then analyzed 50, 150 and 250 sec after baseline
measurement. Additionally, blood pressure parameters and intraocular pressure
(IOP) were obtained before and after caffeine intake.
Results: The mean diameter of the arterioles at baseline before caffeine intake was
123.30 14.0 m and after caffeine 117.30 13.0 m which was significantly
different (P=.004) before and after treatment. The mean diameter of the venules at
baseline before caffeine intake was 147.60 19.5 m and after caffeine 137.73
19.9 m which was significantly different (P=.005.). The flicker-induced increase
in vasodilatation from baseline measurement in the arterioles was 3.4 2.3 m
before and 4.3 2.5 after caffeine consumption (P=.020). The flicker-induced
increase in vasodilatation in the venules was 4.3 3.4 m before and 7.4 2.6 after
caffeine consumption from baseline measurement (P=.0001). The flicker response
of the arterioles (defined as FR=100 x (flicker-baseline)/flicker %) increased from
2.8 % before caffeine to 3.8 % after caffeine intake (P=.010). The flicker response
of the venules increased from 3.4 % before caffeine to 5.5 % after caffeine intake
(P=.0001). Systolic blood pressure changed from 126.5 11.0 mmHg to 135.3
13.2 mmHg after caffeine (P=.001). Diastolic blood pressure increased from 83.3
8.4 mmHg to 88.2 10.5 mmHg after caffeine intake (P=.010). Pulse rate
decreased from 77.2 10.5 bpm to 68.4 12.3 bpm (P=.010). IOP changed from
12.5 2.8 mmHg to 12.9 2.5 mmHg after caffeine which was without statistical
significance (P=.387)
Conclusions: The present study showed a significant vasoconstrictory response of
the retinal arterioles and venules 1 hour after caffeine intake in young healthy
subjects measured by the DVA. Also the reacting capacity of the arterioles and the
venules showed a significant improvement before and after caffeine consumption.
These effects might be elicited by the inhibitory effect of caffeine on adenosine, a
potent vasodilatator of the retinal vasculature.
Commercial Relationships: Naim Terai, None; Eberhard Spoerl,
None; Richard P. Stodtmeister, None; Lutz E. Pillunat, None
Support: None
The Effect Of Adenosine On The Optic Nerve Head Blood Flow In Rabbits
Clemens Strohmaier, Christian Runge, Barbara Bogner, Andrea Trost, Falk
Schrdl, Gnther Grabner, Herbert A. Reitsamer. Ophthalmology & Optometry,
Paracelsus University, Salzburg, Austria.
Purpose: To investigate the effect of different doses of adenosine on optic nerve
head blood flow in New Zealand rabbits, using a novel, non-invasive Laser
Doppler Flowmetry instrument.

Methods: White New Zealand rabbits (2-3 kg, n = 12) were anesthetized with
Pentobarbital Sodium (50 mg/kg i.v.) and respired with room air. The mean arterial
pressure (MAP) and the intraocular pressure (IOP) were measured through
cannulation of the central ear artery and the vitreous body, respectively. Noninvasive Laser Doppler Flowmetry was used to measure optic nerve head blood
flow continuously. In group 1, the dose-response characteristics at baseline MAP
was investigated, adenosine was applied intravenously at different rates of
2,5mg/kg/h, 5mg/kg/h, 7,5mg/kg/h und 10mg/kg/h. In group 2, MAP was
manipulated mechanically with occluders placed around the aorta and vena cava
and thus, ocular perfusion pressure was changed over a wide range. Pressure-flow
relationships were measured at control and in response to i.v. administration of
adenosine at 5 mg/kg/h.
Results: Adenosine caused a statistically significant increase in blood flow by 36,1
3,9 % at a rate of 5 mg/kg/h. At the higher doses of 7,5 and 10 mg/kg/h blood
flow increased further by 79,2 8,7 % and 95,8 6,9 % from baseline. The dose of
2,5 mg/kg/h had no significant effect on any measured parameter. The high dose
(10 mg/kg/h) caused a significant reduction of the mean blood pressure by 11,3
3,2 %. In addition, the doses of 5, 7,5 and 10 mg/kg/h increased the IOP
significantly by 22,1 2,3 % , 34,6 7,5 % and 33,2 9,1 %. Adenosine at 5
mg/kg/h caused a significant upward shift of the pressure-flow relationship.
Conclusions: A concentration dependent vasodilation through adenosine in the
optic nerve head was found. These results are in good accordance with the literature
and previously published data from our group with other measurement techniques.
Commercial Relationships: Clemens Strohmaier, None; Christian Runge,
None; Barbara Bogner, None; Andrea Trost, None; Falk Schrdl,
None; Gnther Grabner, None; Herbert A. Reitsamer, None
Support: PMU FFF, Austrian Academy of Sciences, Fuchs Foundation, Adele
Rabensteiner Foundation
Lower Limit of Blood Flow Autoregulation in Optic Nerve Head
Lin Wang, Grant A. Cull, Chelsea Piper, Claude F. Burgoyne, Brad Fortune.
Devers Eye Institute, Legacy Research Institute, Portland, OR.
Purpose: The lower limit of blood flow autoregulation (LLA) refers to a critical
ocular perfusion pressure (OPP) below which the autoregulation capacity fails and
blood flow (BF) can no longer be maintained at a normal level. The LLA is
therefore defined as the minimal OPP that ensures sufficient blood perfusion to a
tissue. The purpose of this study was to determine the LLA within the optic nerve
head (ONH) of normal monkey eyes.
Methods: In 12 rhesus monkeys anesthetized with pentobarbital (8-10 mg/kg/h, IV
infusion), with mean arterial blood pressure (BP) ranging from 63 to 116 mmHg
and intraocular pressure (IOP) set at 10 mmHg manometrically, the BF in the ONH
was measured (arbitrary unit) with a laser speckle flowgraph device and plotted
against corresponding OPP (calculated as BP - IOP) (Fig A). The OPP was then
reduced by increasing IOP from 10 mmHg to 30, 40 or 50 mmHg. Three minutes
after IOP elevation, BF was measured again. The percentage BF change in response
to each IOP increase was plotted against corresponding OPP after IOP was elevated
(Fig B).
Results: At IOP 10 mmHg and within a range of OPP from 90 to 47 mmHg, ONH
BF was essentially constant with no or little association with OPP (Fig A, R 2 =
0.01, linear regression). After acute IOP elevation, the relationship between percent
BF changes and OPP (from 18 to 76 mmHg) proved to be best fit by a two segment
linear model (vs. a single linear function; F [2, 269 df] = 26.2, P<0.0001). The
resultant function had a break point at 35.7 mmHg (95% CI: 33-38). BF above 35.7
mmHg remained stable and was independent of OPP (Fig B blue, y=0.0001*OPP0.0016, P<0.001); however, BF below 35.7 mmHg declined linearly with
decreasing OPP (Fig B red, y=0.012*OPP-0.43 , R2=0.31).
Conclusions: The normal monkey ONH has strong capacity of BF autoregulation.
The LLA of BF autoregulation in normal monkey ONH is approximately 36 mmHg
(OPP). This value is within the range of most previous studies in both monkeys and
humans.
Commercial Relationships: Lin Wang, None; Grant A. Cull, None; Chelsea

Piper, None; Claude F. Burgoyne, None; Brad Fortune, None
Support: NIH Grant EY019939, Good Samaritan Hospital/Devers Eye Institute
Foundation

Effect of Aging on the Retinal Arterial Stiffness in Healthy Subjects
Eiichi Sato, Taiji Nagaoka, Atsushi Takahashi, Kenji Sogawa, Tsuneaki Omae,
Seigo Nakabayashi, Akitoshi Yoshida. Ophthalmology, Asahikawa Medical
University, Asahikawa, Japan.
Purpose: The pulsatility ratio (PR) is an index of vessel rigidity. Although arterial
stiffness is thought to increase with age, it is unclear how the PR in the retinal
artery changes with age. The purpose of this study was to evaluate the effect of
aging on the PR in the retinal artery in healthy individuals.
Methods: One hundred eight healthy subjects (57 men; 51 women; age range,
20~77 years) were included. Subjects were divided into the following age groups
(in years): 20s, 30s, 40s, 50s, 60s, and 70s. Using a retinal laser Doppler system
(Canon Laser Blood Flowmeter, Model CLBF 100, Canon Inc, Tokyo, Japan), the
vessel diameter and blood velocity were measured and the retinal blood flow (RBF)
was calculated in the superior temporal retinal artery. The PR was calculated from
the blood velocity profile. The PR is expressed as Vmax/Vmin, where Vmax is the
peak systolic velocity during systole and Vmin is the end diastolic velocity.
Results: No significant differences were seen between the groups in blood
pressure, vessel diameter, blood velocity, or RBF. The PRs of individuals in their
60s (3.79 0.88, mean standard deviation) and 70s (5.14 1.48) increased
markedly and were significantly (P < 0.001) higher than that of those in their 20s
(2.67 0.35).
Conclusions: Aging did not significantly affect diameter, velocity, or RBF in
healthy individuals. However, the PR markedly increased in individuals over 60
years of age, possibly indicating that the retinal arterial stiffness might progress
after 60 years of age.
Commercial Relationships: Eiichi Sato, None; Taiji Nagaoka, None; Atsushi
Takahashi, None; Kenji Sogawa, None; Tsuneaki Omae, None; Seigo
Nakabayashi, None; Akitoshi Yoshida, None
Support: None
Changes In The Blood Volume Of The Optic Nerve Head By Epinephrine In
Intravitreal Infusion Solution During Vitrectomy
Makoto Ubuka, Yasutaka Onoda, Koichiro Hitani, Tomoaki Shiba, Yuichi Hori,
Takatoshi Maeno. Ophthalmology, Toho Univ Sakura Med Ctr, Sakura, Japan.
Purpose: To examine whether epinephrine in addition to intravitreal infusion
solution affects the blood flow volume in eyes during vitrectomy, based on
measurements by laser speckle flowgraphy (LSFG).
Methods: In this study we analyzed 11 eyes of 11 patients treated by vitrectomy, 7
of 11 with epimacular membrane and 4 of 11 with idiopathic macular hole. All
procedures were in full compliance with the guidelines of the Declaration of
Helsinki and were approved by the Institutional Review Board/Ethics Committee.
All patients underwent micro incision vitreous surgery with a 23 gauge instrument
using the Accurus (Alcon) Vented Gas Forced Infusion (VGFI) system. After
performing the core vitrectomy and posterior vitreous detachment under an
epinephrine-free BSS PLUS500 intraocular irrigating solution, the blood flow
volume was measured under a stabilized infusion pressure of 10 mmHg using the
VGFI system. The blood flow volume in the optic nerve head was measured by
LSFG with the patient in the supine position. Next, 50 ml of 1 mg epinephrinefortified BSS PLUS500 intraocular irrigating solution was infused at 10 mmHg
with the same VGFI system, and the blood flow volume was measured again by the
same method. During the LSFG measurement, there were no significant changes
noted in simultaneous measurements of the intraocular pressure, pulse rate, or
systolic and diastolic blood pressure. The relative blood flow volume in the LSFG
was expressed as the mean blur rate (MBR).
Results: The MBR, the value representing the blood flow volume of the optic
nerve head, significantly decreased after the switchover to the epinephrine-fortified
infusion solution from the epinephrine-free solution (average decrease of
9.88.9%; P=0.004; paired t-test) . There were no significant differences in the
blood pressure, pulse rate, or intraocular pressure between the two measurements in
any patient.
Conclusions: The results of this study suggest that the blood flow volume of the
optic nerve head is significantly affected by epinephrine in addition to intravitreal
infusion solution during vitrectomy.
Commercial Relationships: Makoto Ubuka, None; Yasutaka Onoda,
None; Koichiro Hitani, None; Tomoaki Shiba, None; Yuichi Hori,
None; Takatoshi Maeno, None
Support: None
Dilation of Porcine Retinal Arterioles via a cAMP/Protein Kinase A and AMPActivated Protein Kinase-Dependent Mechanism with Cilostazol
Ichiro Tanano, Taiji Nagaoka, Tsuneaki Omae, Takayuki Kamiya, Shinji Ono,
Akitoshi Yoshida. Ophthalmology, Asahikawa Medical University, Asahikawa,
Japan.
Purpose: Cilostazol, a selective inhibitor of phosphodiesterase 3, has antiplatelet
aggregation and peripheral vasodilation effects. We examined the effects of
cilostazol on the retinal microvascular diameter to determine if they depend on the
endothelium and/or potassium (K) channels in smooth muscle to reveal the
signaling mechanisms involved in this vasomotor activity.
Methods: Porcine retinal arterioles were isolated, cannulated, and pressurized
without flow in vitro. Video microscopic techniques recorded diametric responses
to cilostazol.
Results: The retinal arterioles dilated in a cilostazol concentration-dependent (0.1
nM-10 M) manner and decreased by 60% after endothelial removal. The nitric
oxide (NO) synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME),
inhibited cilostazol-induced vasodilation comparable to denudation. Inhibition of
soluble guanylyl cyclase and blockade of protein kinase A (PKA) were comparable
to L-NAME. Compound C, an AMP-activated protein kinase (AMPK) inhibitor,
partially inhibited cilostazol-induced vasodilation, which exhibited a weaker
inhibitory effect on cilostazol-induced vasodilation than blockade of PKA. The
large-conductance Ca2+-activated K channel (BK channel) blocker, iberiotoxin,
also inhibited cilostazol-induced vasodilation.
Conclusions: Cilostazol elicits endothelium-dependent and -independent dilation
of retinal arterioles mediated by NO release and BK channel activation,
respectively.
Commercial Relationships: Ichiro Tanano, None; Taiji Nagaoka,
None; Tsuneaki Omae, None; Takayuki Kamiya, None; Shinji Ono,
None; Akitoshi Yoshida, None
Support: None
Effect of Intravitreal Rho Kinase Inhibitors on Retinal Microcirculation in
Cats
Takafumi yoshioka, Seigo Nakabayashi, Taiji Nagaoka, Tomofumi Tani, Akitoshi
Yoshida. Ophthalmology, Asahikawa Medical University, Asahikawa, Japan.
Purpose: To investigate the effects of Rho kinase inhibitors, fasudil and K115, on
retinal microcirculation in cats.
Methods: Same concentration of fasudil (n=5), a novel Rho kinase inhibitor K-115
(n=5), or vehicle (PBS; n=5) was injected intravitreously. The vessel diameter and
blood velocity were measured simultaneously in the first-order retinal arterioles by
laser Doppler velocimetry (CLBF model 100, Canon); the retinal blood flow (RBF)
was calculated. The measurements started 15 minutes before the injection, and
were performed every 10 minutes for 2 hours after the injection.
Results: In the PBS group, vessel diameter, blood velocity, and RBF did not differ
significantly from the pre-injection level. In the fasudil group, vessel diameter and
blood velocity did not change significantly. RBF tended to increase (P=0.09) but
there was no statistically significance. At 120 minutes in the K-115 group, vessel
diameter did not change significantly, but blood velocity (33.38.0%, P<0.001) and
RBF (32.57.9%, P<0.001) significantly increased from the pre-injection levels.
The blood velocity (P<0.01) and RBF (P=0.02) were significantly increased in the
K-115 group compared with those in other groups.
Conclusions: Our data showed that intravitreal administration of a novel Rho
kinase inhibitor K-115 increased blood velocity and RBF in cats. These finding
indicate that the increase in blood velocity in the K-115 reflect a dilation of the
downstream from the measured vessels.
Commercial Relationships: Takafumi yoshioka, None; Seigo Nakabayashi,
None; Taiji Nagaoka, None; Tomofumi Tani, None; Akitoshi Yoshida, None
Support: None
Interocular Vascular Communication Through Collateral Vessels In An
Experimental Pig Model
Hakan Moren1, Bodil Gesslein1, Per Undren2, Sten Andreasson1, Malin Malmsj1.
1
Ophthalmology, Retinal Vascular Research, Lund University, Lund, Sweden;
2
Department of Neuroradiology, Skne University Hospital, Lund, Sweden.
Purpose: The authors recently presented an endovascular coiling model of retinal
ischemia. In order to elaborate this model, the aim of this study was to examine if
there is collateral blood supply with direct communication between the right and
left eye. Also, the aim was to examine if the extent of ischemia following vascular
occlusion is dependent on this collateral blood supply.
Methods: The external carotid system of 8 pigs (mean weight 70kg) was
catheterized using a fluoroscopy monitored, transfemoral, endovascular approach.
Vascular occlusion was performed in the ophthalmic artery using coils. Retinal
function was evaluated after occlusion using multifocal electroretinography
(mfERG).
Results: Unilateral angiograms of the ophthalmic artery showed bilateral retinal

contrast filling almost simultaneously. There were angiographic signs of net
collateral flow from the right to the left eye or vice versa. Occlusion of the
ophthalmic artery in eyes where the net collateral flow originated resulted in
attenuated mfERG b-wave amplitudes on day 1 in both eyes indicating retinal
ischemia. By contrast, occlusion in eyes with both direct and collateral blood
supply resulted in less attenuated amplitudes in both eyes indicating less ischemia.
Conclusions: The present study shows evidence of direct vascular communication
through collateral blood vessels between the two eyes. This has not been shown in
pigs before. An interocular collateral net flow could be present in either direction.
The degree of ischemia following occlusion of the ophthalmic artery seems to be
influenced by whether the occluded eye has only direct or also additional collateral
supply. This collateral blood circulation may have clinical importance, i.e. to
modulate the level of ischemic damage to the retina in the event of vascular
occlusion. It may also be of importance to further development of experimental
models of retinal
ischemia.
Commercial Relationships: Hakan Moren, None; Bodil Gesslein, None; Per

Undren, None; Sten Andreasson, None; Malin Malmsj, None
Support: None
Dorzolamide-induced Vasorelaxation of Porcine Ciliary Arteries is Mediated
by Nitric Oxide
Sidse Kringelholt1, Ulf Simonsen2, Toke Bek1. 1Department of Ophthalmology,
Aarhus University Hospital, Aarhus C, Denmark; 2Department of Biomedicine,
Aarhus University, Aarhus C, Denmark.
Purpose: Carbonic anhydrase inhibitors (CAIs) are known to lower the intraocular
pressure in glaucoma by reducing aqueous humor production. However, it is
possible that other mechanisms, such as the regulation of blood flow to the ciliary
body, are involved in the pressure lowering effect. This effect might be mediated
by different transmitters including nitrogen oxide (NO).
Methods: The intraocular part of porcine ciliary arteries was isolated and mounted
in a wire myograph system for isometric tension recordings.
The effect of the CAI dorzolamide was tested on the tone of uveal arteries with and
without endothelium, and in the presence or absence of the NO synthesis inhibitor
L-NAME.
Results: Dorzolamide induced a significant concentration-dependent relaxation of
ciliary arteries. Removal of the endothelium and NO-inhibition significantly
reduced dorzolamide-induced vasorelaxation (p<0.05, Students unpaired t-test).
Conclusions: Dorzolamide-induced vasorelaxation of ciliary arteries is mediated
by endothelial nitric oxide. Several mechanisms may be responsible for the drugs
beneficial effects in glaucoma, both including regulation of aqueous humor
production and modulation of vascular tone.
Commercial Relationships: Sidse Kringelholt, None; Ulf Simonsen,
None; Toke Bek, None
Support: None
The Effect Of Galanin On Choroidal Blood Flow In Rabbits And Rats
Christian Runge, Barbara Bogner, Clemens Strohmaier, Falk Schrdl, Andrea
Trost, Gnther Grabner, Herbert A. Reitsamer. Ophthalmology and Optometry,
Paracelsus University, Salzburg, Austria.
Purpose: To study the effect of galanin on choroidal blood flow (ChorBF) in
anesthetized rabbits and rats with Laser Doppler flowmetry.
Methods: In anesthetized New Zealand white rabbits (n=8) mean arterial pressure
(MAP) and intraocular pressure (IOP) were measured by direct cannulation of the
central ear artery and the vitreous, respectively. Laser Doppler flowmetry (LDF)
was used to measure ChorBF. Galanin was administrated intravenously with a
concentration of 1mg/kg/h.
In Brown Norway rats mean arterial pressure (MAP) was measured by cannulation
of the femoral artery. ChorBF measurements (n=6) were performed with non
contact LDF. Galanin was infused at a dose of 1mg/kg/h and 3mg/kg/h.
Control experiments in rat and rabbit forehead skin were performed to reproduce
the known galanin effect on skin blood flow. Since the rabbit specific galanin
sequence has not been identified up to now, rat specific galanin was used in both
species.
Results: In both species no significant effect on ChorBF was detectable. However
in 3/8 experiments choroidal vasodilation was measured in rabbit but not in rat.
Nevertheless, control experiments in rat forehead skin revealed an increase in blood
flow (36%) after 1mg/kg/h galanin application. The increase of galanin
concentration to 3mg/kg/h still had no effect on choroidal blood flow. Control
experiments in rabbit forehead skin showed no effect.
Conclusions: In the present study the administration of galanin revealed no
significant effect on choroidal blood flow in the species investigated. The
discrepancy in choroidal blood flow in the rabbit group needs to be further
explored. Differences in the amino acid sequence or receptor binding properties
between the used rat specific galanin and rabbit galanin might be responsible for
the effect on rat skin blood flow, which was not observed in rabbits.
Commercial Relationships: Christian Runge, None; Barbara Bogner,
None; Clemens Strohmaier, None; Falk Schrdl, None; Andrea Trost,
None; Gnther Grabner, None; Herbert A. Reitsamer, None
Support: None
Relationship between Subfoveal Choroidal Thickness and Choroidal
Circulation in Response to Increased Systemic Blood Pressure Induced by
Cold Pressure Test
Kenji Sogawa1, Taiji Nagaoka1, Tomofumi Tani1, Ichiro Tanano2, Tsuneaki Omae2,
Akitoshi Yoshida1. 1Ophthalmology, Asahikawa Medical University, Asahikawa,
Japan; 2Ophthalmology, Asahikawa Medical College, Asahikawa, Japan.
Purpose: To investigate changes in the choroidal thickness during changes in the
choroidal blood flow resulting from the increased systemic blood pressure induced
by cold pressure test in healthy young subjects.
Methods: We examined 7 eyes of 7 healthy young Japanese subjects. The
increased systemic blood pressure was induced by cold pressure test by submerging
the subjects right hands in 4-5C cold water for 5 minutes. Once each minute
during the cold pressure test, we measured the subfoveal choroidal thickness using
enhanced depth imaging optical coherence tomography and the total choroidal
blood flow by measuring the pulsatile ocular blood flow with Langham OBF
computerized tonometry. We also measured the systolic and diastolic blood
pressures.
Results: One minute after the cold pressure test, the mean blood pressure increased
(11.1% 3.2%) compared with the baseline. Two minutes after the clod pressure
test, the choroidal blood flow increased by 7.9% 2.6% of the baseline. In contrast,
there was no significant change in the subfoveal choroidal thickness flow during
the cold pressure test.
Conclusions: Our results suggested that the increased mean blood pressure may
cause increased choroidal blood flow. In addition, the increased choroidal blood
flow did not attenuate the subfoveal choroidal thickness in healthy young subjects.
Commercial Relationships: Kenji Sogawa, None; Taiji Nagaoka,
None; Tomofumi Tani, None; Ichiro Tanano, None; Tsuneaki Omae,
None; Akitoshi Yoshida, None
Support: None
Retinal Blood Flow Velocity in Patients with Active Uveitis Using the RFI
Sanjay R. Kedhar1, Xing Feng2, Richard B. Rosen1, C. Michael Samson1.
1
Ophthalmology, New York Eye & Ear Infirmary, New York, NY;
2
Ophthalmology, Beijing Tongren Eye Center, Beijing, China.
Purpose: To evaluate differences in the retinal blood flow velocities of patients
with active uveitis and healthy controls using the Retinal Function Imager (RFI,
Optical Imaging, Israel) and to determine the correlation between the retinal blood
flow velocity and central macular thickness in uveitis patients.
Methods: 16 eyes of 14 patients with active uveitis and 51 eyes of 51 normal
control subjects were enrolled. Five eyes had uveitic cystoid macular edema (CME)
by optical coherence tomography (SLO-OCT, OTI, Canada). Eyes with diabetic
retinopathy, posterior uveitis and glaucoma were excluded. Retinal blood flow
velocities by RFI and central macular thickness by SLO-OCT were obtained.
Differences among the groups were assessed.
Results: Median (first quartile, third quartile) venous velocity for uveitic eyes with

CME, uveitic eyes without CME and controls were 2.18(1.86, 2.53), 2.57(2.26,
2.91) and 2.82(2.39, 3.53) mm/s. Median (first quartile, third quartile) arterial
velocity for uveitic eyes with CME, uveitic eyes without CME and controls were
3.83(3.68, 4.15), 3.42(2.99, 4.17) and 3.93(3.35, 4.65) in mm/s. Uveitic eyes with
CME had significantly lower venous velocity than controls (P=0.044). There was a
similar trend for uveitic eyes without CME, but it did not reach statistical
significance (P=0.239). Correlation between RBF and central retinal thickness (55
mm grid pattern) showed a strong linear relationship between venous velocity and
central retinal thickness [coefficient -0.001572 (P = 0.007)]. Arterial velocity did
not correlate with central thickness [coefficient 0.000573 (P = 0.697)].
Conclusions: The RFI detected significantly decreased retinal venous velocities in
eyes with uveitic CME relative to healthy controls, suggesting abnormal vessel
function in eyes with uveitic CME. A similar trend was seen in uveitic eyes as a
whole, although the difference was not statistically significant. In eyes with uveitis,
a decrease in venous velocity was correlated with an increase in central retinal
thickness.
Commercial Relationships: Sanjay R. Kedhar, None; Xing Feng,
None; Richard B. Rosen, None; C. Michael Samson, None
Support: None
Time of Collapse of Spontaneous Venous Pulsation
Fabrice Moret1A, Wolf A. Lagrze1B, Charlotte M. Poloschek1B, Michael Bach1A.
A
Sect. Visual Function and Electrophysiology, BSect. Neuroophthalmology, 1Eye
Hospital, University of Freiburg, Freiburg, Germany.
Purpose: The spontaneous venous pulsation is a negative marker for elevated
intraocular pressure and papilledema. Its relationship to the venous outflow
resistance is also of interest in the context of glaucoma. At this time, pulsation's
etiology remains unclear. A key element to elucidate the pulsation mechanism is
the time at which collapse occurs with respect to the heart cycle, but previous
reports are contradictory. We seek here to assess this question using quantitative
measurements of both vein diameters and arteries's lateral displacements; the later
being used as marker of ocular systole time.
Methods: We recorded 4-s fundus sequences with a near-infrared SLO in 12
subjects of age 22 to 31 years and with normal ocular status. Image sequences were
co-registered, cleaned from microsaccades and filtered via a principal component
analysis (PCA) to remove non-pulsatile dynamic features. Time courses of
maximal arterial lateral displacements and of venous diameters at sites of
spontaneous venous pulsation or proximal to the disk were derived. We analyzed
time courses from both raw and PCA-filtered data.
Results: Signals in seven subjects offered recognizable arterial pulsatile waveforms
and were analyzed. Signal waveforms presented the strongest interest. Veins
diameter is not just binary: either open or collapsed; No single collapse is observed
over time, instead the venous signal appears as a damped and delayed replica of the
arterial signal. The collapse is thus a normal trough as observed in arterial
pressure waveforms. Waveforms and delays vary significantly between subjects but
delays between waveform up-slope feet are always positive and never exceed
arterial systole duration.
Conclusions: On this young cohort, the results support the traditional view: venous
collapse occurs near ocular arterial systole. Diameter's waveforms show no
collapse as such but rather a delayed arterial pressure waveform. Waveforms and
limited positive delays suggest that the arterial pressure pulsation may be carried
over the retinal microcirculation and that intraocular pressure pulse - if involved at
all - may not be a primary actor in venous pulsation generation.
Commercial Relationships: Fabrice Moret, None; Wolf A. Lagrze,
None; Charlotte M. Poloschek, None; Michael Bach, None
Support: Deutsche Forschungsgemeinschaft BA877/19-2
Manometric Investigation Of The Relationship Between Pulsatile Ocular
Blood Flow And Intraocular Pressure In Living Human Eyes
Nikolaos Karyotakis1, Harilaos S. Ginis2A, Anna I. Dastiridou3, Miltiadis K.
Tsilimbaris2B, Ioannis G. Pallikaris4. 1Medicine School, University Of Crete,
Heraklion, Greece; AInstitute of Vision & Optics, BOphthalmology-Research Acct,
2
University of Crete, Heraklion, Greece; 3Medicine School, University Of Larisa,
Larisa, Greece; 4School of Medicine, University of Crete, Heraklion - Crete,
Greece.
Purpose: The hemodynamic impact of elevated intraocular pressure has been
implicated in ischemic or hypoxic models of ocular disease. The purpose of this
study was to employ a manometric method that involves cannulation of the anterior
chamber to investigate the relationship between intraocular pressure (IOP), ocular
pulse amplitude (OPA) and pulsatile ocular blood flow (POBF).
Methods: Nine patients (3 men and 6 women, aged 64 years, sd 14) were measured
during cataract surgery. The study was approved by the Institutional Review Board.
The anterior chamber of each eye was cannulated and connected to a device
featuring a pressure sensor and a dosimetric pump. The transducer output can be
continuously recorded while the dosimetric pump can be used to control IOP. The
anterior chamber of each eye was infused in steps of known volume of saline until
the IOP was increased to 40 mmHg. At 40 mmHg the infusion stopped and the
sensor recorded the IOP decay curve over time for 3 minutes. The POBF was
estimated based on the ocular volume and pressure changes. A purposely
developed Matlab (The Mathworks, Inc, MA, USA) script was applied in order to
identify, filter and average series of ocular pulses. The POBF was estimated in
range of pressures from 40 mmHg to 20 mmHg depending on the IOP decay curve
over time of each subject. Ocular rigidity of each eye was calculated during the
infusion process and the rigidity coefficient was used to convert the measured
pressure changes to corresponding volume changes.
Results: The average POBF at maximum IOP 40 mmHg was 707.5 (sd 249.1)
L/min, and the POBF at minimum IOPs was 1019.5 (sd 297) L/min. The POBF
was increasing inversely with IOP for all subjects, with mean difference between
the maximum and minimum values at 304.5 (sd 102.8) L/min.
Conclusions: The POBF is decreased at elevated IOP levels suggesting that the
pulsatile component of blood flow is determined by the value of IOP. Also other
parameters like ocular rigidity systemic and baseline IOP are correlated to POBF.
Commercial Relationships: Nikolaos Karyotakis, None; Harilaos S. Ginis,
None; Anna I. Dastiridou, None; Miltiadis K. Tsilimbaris, None; Ioannis G.
Pallikaris, None
Support: None
Mechanisms of Autoregulation of Retinal blood flow in Response to Decreased
Ocular Perfusion Pressure in Cats; Comparison of Increased Intraocular
Pressure and Decreased Systemic Blood Pressure
Tomofumi Tani, Taiji Nagaoka, Seigo Nakabayashi, Kenji Sogawa, Akitoshi
Yoshida. Ophthalmology, Asahikawa Medical University, Asahikawa, Japan.
Purpose: To investigate the regulatory mechanism of retinal circulation during
decreased ocular perfusion pressure (OPP) in cats.
Methods: The effect of acute decreased OPP on retinal arteriolar diameter (D),
flow velocity (V) and blood flow (RBF) was assessed with laser Doppler
velocimetry. We manipulated the OPP by either elevated intraocular pressure (IOP)
or systemic hypotension. The involvements of nitric oxide (NO), adenosine and/or
N-methyl-D-aspartic acid (NMDA) in the regulation of retinal arteriolar
hemodynamics during decreased OPP were determined at two hours after
intravitreal injections of respective inhibitors.
Results: The OPP was gradually decreased from 90 to 40 mmHg. In the PBS
group, the V decreased in proportion to the decreased OPP whereas the D gradually
increased. As a result, RBF was maintained at more than 70 mmHg of OPP but
significantly decreased less than 60 mmHg of OPP during decreased OPP by both
elevated IOP and systemic hypotension. 8-(p-Sulfophenyl)theophylline hydrate
(8SPT; an adenosine receptor blocker) also enhanced the reduced RBF in response
to both elevated IOP and systemic hypotension whereas N-nitro-L-arginine
methylester (L-NAME; NO synthase inhibitor) and DL-2-Amino-5phosphonopentanoic acid (DL-APV; an NMDA receptor antagonist) enhanced the
decreased RBF in response to only elevated IOP.
Conclusions: Our data suggest that the RBF may be maintained at more than 70
mmHg of OPP. In addition, NO, adenosine and NMDA may be responsible for the
RBF preservation in response to the decreased OPP by elevated IOP whereas
adenosine may be responsible in response to that by systemic hypotension,
suggesting that different vasoregulatory factors may regulate the retinal
microcirculation during the decreased OPP between elevated IOP and systemic
hypotension.
Commercial Relationships: Tomofumi Tani, None; Taiji Nagaoka,
None; Seigo Nakabayashi, None; Kenji Sogawa, None; Akitoshi Yoshida, None
Support: None
Optic Nerve Head Capillaries Blood Oxygenation Following Dynamic Exercise
in Human
Vasile Diaconu, Patrick Sauvageau, Valentina Vucea. Ecole D'optometrie,
University of Montreal, Montreal, QC, Canada.
Purpose: It is generally suggested that the blood flow in the human eye retinal
vessels can be regulated to satisfy the metabolic requirements. This physiological
process called auto-regulation is engaged to maintain a relatively constant ocular
blood flow (OBF) to compensate for changes in the ocular perfusion pressure
(OPP). The goal of the present study was to investigate the blood oxygenation of
the optical nerve capillaries, in function of the OPP changes, after dynamic
exercise.
Methods: Six healthy non-smoking men (mean age: 22; range: 20-25 years) have
participated in the study. A high level physical effort has been generated by using

the computerized stationary bicycle. The pulse rate (PR) of 160 beats per minute
has been always reached after a session of 15 minutes of exercise for each subject.
The blood oxygenation of the optic nerves capillaries (BOONH) has been derived
by the multichannel spectroscopy technique (Vucea et al., 2011). The BOONH,
brachial blood pressure (Bp), intraocular pressure (IOP) and systemic arterial blood
oxygenation have been evaluated in each subject before and after the exercise
session.
Results: The results demonstrate the correlations between the IOP and BOONH
variations for each subject, R2=0,98. These correlations indicate that following an
intense physical exercise the subjects who have demonstrated an important
reduction in IOP also have experienced an important reduction in BOONH (i.e. blood
flow).
Conclusions: The results of the present study are in agreement with previous
studies (Stefansson et al., 2005) which explain that the optic nerves blood
oxygenation is regulated by the IOP, the mean arterial blood pressure and the
retinal blood vessels resistance.
Commercial Relationships: Vasile Diaconu, None; Patrick Sauvageau,
None; Valentina Vucea, None
Support: CRSNG to Vasile Diaconu
Age Effects on Retinal Blood Flow Assessed Using Spectral-Domain Optical
Coherence Tomography Doppler
Firdaus Yusof1,2, Faryan Tayyari1, John G. Flanagan1,3, Christopher Hudson1,3.
1
School of Optometry and Vision Sciences, University of Waterloo, Waterloo, ON,
Canada; 2Department of Optometry and Visual Science, International Islamic
University of Malaysia, Bandar Indera Mahkota, Kuantan, Malaysia; 3Department
of Ophthalmology and Vision Sciences, University of Toronto, Toronto, ON,
Canada.
Purpose: To determine the impact of healthy aging upon total retinal blood flow as
derived by Spectral-Domain, Optical Coherence Tomography Doppler SD-OCT.
Methods: SD-OCT Doppler blood flow was non-invasively measured using the
RTVue system (Optovue Inc., USA). One eye of each of 6 healthy young (mean
age 25.7; SD 3.5 years) and 6 healthy elderly (mean age 63.5; SD 1.8 years)
subjects was randomly selected for the study and dilated using Mydriacyl 1%. A
double circular scanning pattern was employed. A minimum of six separate SDOCT Doppler measurements (i.e. each separate measurement comprising a superior
nasal pupil scan and an inferior nasal pupil scan) were acquired. Total retinal blood
flow was calculated, using data from valid scans only, by summing flow from all
detectable venules.
Results: 117 of 170 images (68.82%) were determined to be valid using the
DOCTORC software (Centre for Ophthalmic Optics and Lasers, CA, USA). Mean
total retinal blood flow for the young group was 44.9615.59 l/min, while for the
elderly group it was 39.4513.20 l/min. Two-sided t-test showed no significant
difference between both groups (p=0.52). Linear regression analysis showed no
significant correlation between total retinal blood flow and age (r=-0.31, p=0.33).
Conclusions: Preliminary data acquired using SD-OCT Doppler shows no
significant difference in retinal blood flow between a young and elderly group.
Commercial Relationships: Firdaus Yusof, None; Faryan Tayyari, None; John
G. Flanagan, Carl Zeiss Meditec (F), Heidelberg Engineering (F, C); Christopher
Hudson, Optovue Inc. (F)
Support: Ontario Research Fund
Effect of Slow Releasing Hydrogen Sulfide Donor GYY4137 on Isolated
Bovine Ciliary Artery
Madhura S. Kulkarni1A, Ya Fatou Njie-Mbye1A, Catherine A. Opere2, Matt
Whiteman3, Sunny E. Ohia1B. APharmaceutical Sciences, BProvost/Academic
Affairs, 1Texas Southern University, Houston, TX; 2Pharmacy Sciences, Creighton
University, Omaha, NE; 3University of Exeter, Peninsula Medical School, Exeter,
United Kingdom.
Purpose: Hydrogen sulfide (H2S), a colorless gas characterized by its pungent odor
of rotten eggs has been reported to elicit relaxation effects on non-ocular smooth
muscles of several mammalian species. The purpose of the present study is to
investigate whether the slow releasing H2S donor, GYY4137 can induce
pharmacological actions on isolated bovine ciliary artery.
Methods: Isolated bovine posterior ciliary arteries were set up in 25 mL organ
baths containing oxygenated Krebs solution (pH 7.4) at 37 0 C. Changes in
longitudinal isometric tension were recorded via a grass FT03 Force-displacement
Transducers and analyzed using the Grass PolyView computer software. The
relaxant action of GYY 4137 on phenylephrine induced tone was studied in the
absence or presence of inhibitors of enzymes for the biosynthetic pathways of H2S
and prostanoids. In addition, we examined the effects of ATP-sensitive K+ (KATP)
channel antagonist, glibenclamide on GYY4137 induced response.
Results: The slow releasing H2S donor, GYY4137 (10nM-0.1M) elicited a
concentration-dependent relaxation of phenylephrine induced tone in bovine ciliary

artery. This response was significantly (P<0.05) enhanced in the presence of the
cyclo-oxygenase inhibitor, flurbiprofen (3M). Other H2S donors (MC05, MC06
and sodium hydrosulfide (NaHS) also exerted similar response in the following
order of magnitude; MC06>NaHS>GYY4137>MC05. Interestingly, the inhibitor
of cystathionine -synthase, AOA (30M) caused a rightward shift in the
concentration-response curve to GYY4137 whilst, the inhibitor of cystathionine lyase, PAG (1mM) only attenuated relaxations caused by high concentrations of
GYY4137 (>1M). The KATP channel antagonist, glibenclamide (100M) did not
affect the relaxation action induced by GYY4137 on ciliary artery.
Conclusions: The slow releasing H2S donor, GYY4137 can relax isolated bovine
ciliary artery, an effect dependant on endogenous production of H2S. Prostanoids
are involved in the inhibitory action of GYY4137. The observed vascular
relaxation induced by GYY 4137 suggest that slow releasing H2S molecules might
serve as potential therapeutic agents to increase ocular blood flow, consequently
preserving the optic nerve and alleviating vision loss.
Commercial Relationships: Madhura S. Kulkarni, None; Ya Fatou NjieMbye, None; Catherine A. Opere, None; Matt Whiteman, None; Sunny E.
Ohia, None
Support: None
558 Tumors: New Drugs, Delivery Systems and Mechanisms of Action
Thursday, May 10, 2012, 11:15 AM - 1:00 PM
Intra-arterial Chemotherapy for the Management of Retinoblastoma in Eyes
with Extensive (>50%) Retinal Detachment
Sotiria Palioura1,2, Y. Pierre Gobin3, Scott E. Brodie1,4, Ira J. Dunkel5, Brian P.
Marr1, David H. Abramson1. 1Ophthalmic Oncology Service, Memorial SloanKettering Cancer Center, New York, NY; 2Currently, Department of
Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School,
Boston, MA; 3Division of Interventional Neuroradiology, Departments of
Radiology, Neurosurgery and Neurology, Weill Cornell Medical College, New
York Presbyterian Hospital, New York, NY; 4Department of Ophthalmology,
Mount Sinai School of Medicine, New York, NY; 5Department of Pediatrics,
Memorial Sloan-Kettering Cancer Center, New York, NY.
Purpose: Superselective delivery of chemotherapy through the ophthalmic artery
was introduced by our group 5 years ago for the treatment of eyes with advanced
intraocular retinoblastoma otherwise scheduled for enucleation. We now aim to
evaluate the efficacy and toxicity of intra-arterial chemotherapy for advanced
intraocular retinoblastoma associated with > 50% retinal detachment.
Methods: Retrospective review of 37 eyes of 34 retinoblastoma patients who had
extensive (>50%) retinal detachments and were treated (September 2006-December
2010) with intra-arterial chemotherapy either as primary treatment or as salvage
treatment after failed systemic chemoreduction and/or external beam radiation
(mean follow up, 21 months). Data on patient survival, ocular survival, systemic
and ocular complications of intra-arterial chemotherapy treatments, retinal
reattachment rates and time course, and serial electroretinograms (ERG) were
collected.
Results: A total of 134 chemotherapy injections (median, 3 per eye) were
performed. All children survived. Five eyes (5/37; 14%) were enucleated for
progression of disease. No eye was enucleated for complications from treatment.
The Kaplan-Meier enucleation-free survival rate at 2 years was 87.9% (95%
confidence interval, 76.5%-99.3%). The retina reattached in 28 eyes (28/37; 76%)
and the 30-Hz flicker ERGs improved by greater than 25 V in 7 eyes (7/37; 19%),
remained stable (change < 25 V) in 26 eyes (26/37; 70%), and decreased by
greater than 25 V in 4 eyes (4/37; 11%). The Kaplan-Meier retinal reattachment
rate was 58% after 3 intra-arterial chemotherapy infusions (95% confidence
interval, 41.9%-74.1%).
Conclusions: Intra-arterial chemotherapy is effective in preventing enucleation,
promoting retinal reattachment and preserving or improving retinal function in the
majority of eyes with advanced intraocular retinoblastoma and >50% retinal
detachment that would otherwise be considered for enucleation.
Commercial Relationships: Sotiria Palioura, None; Y. Pierre Gobin,
None; Scott E. Brodie, None; Ira J. Dunkel, None; Brian P. Marr, None; David
H. Abramson, None
Support: None
Effects Of Zeaxanthin On Cell Viability Of Cultured Human Uveal Melanoma
Cells And Normal Ocular Cells In Vitro

Dan-Ning Hu1A, Richard B. Rosen1B, Min Chen1C, Steven A. McCormick1C.
A
Pathology & Ophthalmology, BOphthalmology, CPathology, 1New York Eye &
Ear Infirmary, New York, NY.
Purpose: Higher intakes and blood levels of zeaxanthin have been repeatedly
associated with a lower incidence of a variety of malignant tumors. Zeaxanthin has
also been shown to inhibit proliferation and induce apoptosis in various tumor cells.
However, the effect of zeaxanthin on uveal melanoma cells has not been reported.
The present study investigated the influence of zeaxanthin on viability of cultured
human uveal melanoma cells compared to normal ocular cells.
Methods: Cell viability of three immortal cell lines of uveal melanoma (SP6.5,
M17 and C918) in the presence of zeaxanthin was studied using the MTT test.
Normal uveal melanocytes and retinal pigment epithelial cells (ARPE19) were used
as controls. Cell apoptosis was determined by annexin V-FITC staining. All studies
were conducted in triplicate.
Results: Zeaxanthin at 10-100 M did not change the cell viability in normal uveal
melanocytes or retinal pigment epithelial cells. Zeaxanthin at 30 M significantly
(P < 0.05) decreased the cell viability in 2 (SP6.5 and C918) of 3 uveal melanoma
cell lines tested and very significantly decreased the cell viability in all uveal
melanoma cell lines to 12-44% of the controls (melanoma cells not treated with
zeaxanthin) at concentrations of 100 M (P < 0.01). Annexin V-FITC staining
showed that numerous apopototic cells were present in melanoma cells treated with
100 M zeaxanthin.
Conclusions: Zeaxanthin inhibited proliferation of melanoma cells at 30 M and
caused apoptosis at 100 M, but did not influence the cell viability of normal uveal
melanocytes and retinal pigment epithelial cells at concentrations of 30-100 M.
These findings suggest that zeaxanthin has a specific cytotoxic effect on uveal
melanoma cells.
Commercial Relationships: Dan-Ning Hu, None; Richard B. Rosen,
OPKO/OTI/Optos C, Clarity-C, OD-OS-C (F); Min Chen, None; Steven A.
McCormick, None
Support: : Supported by the New York Eye and Ear Infirmary Pathology Research
Fund , the Ophthalmology Chairmans Research Fund, the Bendheim-Lowenstein
Family Retina Center research Fund and Zeavision LLC.
Intraocular Treatments of a New Orthotopic Primary Human Retinoblastoma
Xenograft
Nathalie Cassoux1A, franck Assayag1B, Olfa Chouchane-Mlik1C, fariba Nemati1D,
Aurelie Thuleau1D, Jean-Jacques Fontaine2, Isabelle Aets1E, Laurence
Desjardins1A, francois Doz1F, Didier Decaudin1G. AOphthalmology, BLaboratory of
preclinical investigation, Cdpt of pathology, DLab of preclinical investigation, EDpt
oncopediatry, FDpt of oncopediatry, Glab of preclinical investigation, 1Institut
Curie, Paris, France; 2Dpt pathology, Veterinary school, Maison Alfort, France.
Purpose: The treatment retinoblastoma could require enucleation in unfavorable
prognostic disease. In order to allow pharmacological assessment of innovative
intraocular treatments in more relevant and predictive models, we have developed
preclinical orthotopic primary retinoblastoma (Rb) models xenografted into SCID
immunodeficient mice
Methods: Orthotopic models of human Rb have been developed from two panels
of 3 subcutaneous xenografts model (RB102, RB111, and RB200) previously
established and characterized in the laboratory (Aerts et al 2010). One of them,
RB200, was used for intraocular therapeutic experiment. Mice bearing xenografts
were sacrificed and tumors were dissected to obtain a suspension of fresh tumor
cells at a concentration of 8000 cells/l in DMEM serum-free medium. Under
intraperitoneal anesthesia, 2l of cell suspension was injected into the subretinal
space of the right eye for 4 groups of mice using a 32G needle via a Hamilton
syringe. Each group was constituted by 6 SCID mice. After sub-retinal injection,
ophthalmic examination of the mice was performed weekly. When tumor cells
started to invade vitreal cavity, the mice were treated by intravitreal injection (one
injection/week for 4 weeks) of BSS (balanced salt solution) (control), bevacizumab
(25mg/kg), melphalan (500 g/kg), and carboplatine (100g/kg). Fifteen days after
end of treatment, mice were sacrificed for pathological analyses.
Results: All transplanted mice have developed a retinal tumor 6 weeks after
subretinal tumor cell injection. In the control group, pathological analyses showed
enormous tumors of undifferentiated Rb cells with massive infiltration of vitreous
cavity and anterior chamber, and extrascleral extension in 4/6 mice. In the
bevacizumab group, pathological analyses showed massive infiltration of the eye
by Rb cells in 6/6 mice with extrascleral extension in 2/6 mice. In contrast, in the
melphalan group, we have observed complete regression of the tumor in 2/6 mice.
For the 4 remaining mice, pathological analyses showed the absence of vitreal cells
and a reduction of the tumor mass of approximately 50% compared to the control
group. Finally, in the carboplatine group, we have shown a complete regression of
the tumour in 5/6 mice with a reduction of the tumour volume in the 6 th mouse
compared to the control group.
Conclusions: Using intraocular administration in an orthotopic xenografted Rb
model, we have observed a high efficacy of carboplatin (83% of responses),
intermediate efficacy of melphalan (33%), and no efficacy of bevacizumab (0%).
Such experiment suggests that new therapies could therefore be tested in an

intraocular setting alone or in combination with standard chemotherapies.
Commercial Relationships: Nathalie Cassoux, None; franck Assayag,
None; Olfa Chouchane-Mlik, None; fariba Nemati, None; Aurelie Thuleau,
None; Jean-Jacques Fontaine, None; Isabelle Aets, None; Laurence Desjardins,
None; francois Doz, None; Didier Decaudin, None
Support: None
RXRG Agonist Bexarotene Suppresses Retinoblastoma Growth by Enhancing
TRB1 and p53 Tumor Suppressor Activity
Xiaoliang L. Xu1A, Renbing Jia1A,2, Hongyan Huang1A, Walter Joseph1A, Nengyi
Zhou1A, David H. Abramson1B, Xianqun Fan2, Suresh C. Jhanwar1A. ADepartment
of Pathology, BOphthalmic Oncology Service, 1Memorial Sloan Kettering Cancer
Center, New York, NY; 2Department of Ophthalmology, Shanghai Jiaotong
University, Shanghai, China.
Purpose: RB1 is often mutated in retinoblastoma, but is hyperphosphorylated in
KRAS mutant cancers. We previously reported that thyroid hormone receptor beta
2 (TRB2) and RXRG were important in retinoblastoma tumorigenesis, and more
recently found they may regulate Phospho-Rb, TRB2, and Emi1 based S phaseassociated complex (SPC), which was essential for S-phase progression, but it
suppressed G2-M transition. In this study, we try to clarify the RXRG role in the
cell cycle control and targeted therapy of these cancers.
Methods: RXRG was knocked down in retinoblastoma to check cell signaling
changes. Retinoblastoma and KRAS mutated tumor cells were also treated with
RXRG agonist Bexarotene and antagonist HX531 to check cell growth curve and
cell cycle changes. Western blot, real time PCR, and immunofluorescence were
utilized to test specific signaling response related to cell cycle and apoptosis. We
further tested the therapeutic effect of bexarotene on animal xenograft model of
retinoblastoma.
Results: RXRG KD caused SKP2 and PTTG1 activation in retinoblastoma. RXRG
agonist bexarotene promoted SPC dissociation by enhancement of TRB1 activity,
causing growth suppression in retinoblastoma; whereas RXRG antagonist HX531
prevented TRB2-SPC dissociation by enhancement of TRB2 activity, resulting in
growth promotion in retinoblastoma, but not in KRAS mutated cancers. Bexarotene
treatment in retinoblastoma caused Emi1 inactivation and SKP2 degradation
resulting in S phase arrest. RXRG could also bind to p53, thus Bexarotene
treatment could promote p53-targeted gene expression including MDM2, p21, 143-3, and GADD45, and p53-mediated apoptosis in retinoblastoma, but not in
KRAS-mutated tumors. We found that subconjunctival injection or oral
administration of bexarotene could significantly suppress tumor growth in
retinoblastoma xenograft model.
Conclusions: RXRG agonist Bexarotene can promote TRB1 and p53 tumor
suppressor activity and cause cell cycle arrest and apoptosis in retinoblastoma, but
not in KRAS mutated cancers. Retinoblastoma and KRAS mutated cancers have
distinct cell cycle signaling and require different treatment
strategies.
Commercial Relationships: Xiaoliang L. Xu, None; Renbing Jia,

None; Hongyan Huang, None; Walter Joseph, None; Nengyi Zhou, None; David
H. Abramson, None; Xianqun Fan, None; Suresh C. Jhanwar, None
Support: Gerber Foundation; Development Funds of Pathology in MSKCC
The Protein Kinase C (PKC)/Protein Kinase D (PKD)/Steroid Receptor
Coactivator (SRC)-3 pathway is an important therapeutic target in G-mutant
Uveal Melanomas

Vassiliki Poulaki1, Sue Anne Chew2A, Bin He2A, Vijay Kumar Eedunuri3, Diego
Jacinto Bedoya2A, Martine J. Jager4, Bert W. O'Malley2B, Nicholas Mitsiades2A.
1
Ophthalmology, VA Boston Healthcare System, Boston University, Boston, MA;
A
Medicine/Molecular and Cellular Biology, BMolecular and Cellular Biology,
2
Baylor College of Medicine, Houston, TX; 3Adrienne Helis Malvin Medical
Research Foundation, New Orleans, LA; 4Ophthalmology, Leiden University Med
Center, Leiden, The Netherlands.
Purpose: Activating somatic mutations in the G protein subunits Gq and G11
(encoded by GNAQ and GNA11, respectively), occur, in a mutually exclusive
pattern, in ~80% of uveal melanomas (UMs). As G-mutant UMs are resistant to
all currently available therapies, including B-Raf inhibitors, novel agents are
urgently needed to inhibit Gq/11 and their downstream signaling pathway in UM.
We explored this pathway in UM cell lines in order to identify novel druggable
therapeutic targets.
Methods: Using RNA interference (RNAi), small molecule inhibitors (SMIs) and
adenoviral-mediated gene transfer in UM cell lines, we studied the effect of
different modulators of the Gq/11 signaling cascade on cell survival (MTT),
oxygen consumption rate (Seahorse XF24 analyzer), mitochondrial membrane
potential (JC1), autophagy (detection of LC3BII accumulation by immunoblotting;
intracellular localization of LC3B by deconvolution fluorescence microscopy),
MEK/ERK signaling and caspase cleavage (immunoblotting).
Results: GNAQ-mutant (but not BRAF-mutant) UM cell lines were very sensitive
to Gq depletion by RNAi, undergoing early mitochondrial dysfunction and
inhibition of MEK/ERK signaling, followed by caspase-independent death
exhibiting hallmarks of autophagy (LC3BII accumulation and association with
perinuclear autophagosomes). RNAi-based screening for kinase mediators
identified PKC and PKD1 as necessary for MEK/ERK activation and cell viability
downstream of Gq. GNAQ-mutant and GNA11-mutant UM cell lines were also
more sensitive than BRAF-mutant cell lines to SMIs of PKC (Bisindolylmaleimide
I, G6983, rottlerin, midostaurin/PKC412) and PKD (kbNB142-70), again
undergoing caspase-independent death (exhibiting hallmarks of autophagy) that
was enhanced by concurrent treatment with the MEK inhibitor AZD6244 or the
Akt inhibitor MK2206. Depletion of Gq or PKC or PKD1 (by RNAi), or SMIs
of PKC or PKD, promoted the proteasomal degradation of SRC-3, a potent
oncogenic transcription coactivator that is frequently overexpressed in cancer and
confers poor prognosis. Depletion of SRC-3 via RNAi induced cell death in Gmutant, but not in G-wild type UM cell lines. Exogenous overexpression of SRC3 protected GNAQ-mutant UM cells from cell death induced by GNAQ RNAi or
PKC inhibition.
Conclusions: The PKC/PKD/SRC-3 pathway is a downstream effector of mutant
G proteins in UM, driving MEK/ERK activation, cell growth and survival. Our
results highlight important novel druggable therapeutic targets for UM. In
particular, PKC/PKD SMIs and their combinations with MEK and Akt SMIs
appear very promising approaches for treatment of G-mutant UM.
Commercial Relationships: Vassiliki Poulaki, None; Sue Anne Chew,
None; Bin He, None; Vijay Kumar Eedunuri, None; Diego Jacinto Bedoya,
None; Martine J. Jager, None; Bert W. O'Malley, None; Nicholas Mitsiades,
None
Support: Adrienne Helis Malvin Medical Research Foundation through its direct
engagement in the continuous active conduct of medical research in conjunction
with Baylor College of Medicine.
Periocular Tissue Concentration of Propranolol after Delivery with a Gelforming Solution
Michael B. Yang1A, Jinsong Hao1B, Hongzhou Liu1B, S Kevin Li1B. AAbrahmson
Pediatric Eye Institute/Ophthalmology, Cincinnati Children's Hospital, College of
Medicine, BDivision of Pharmaceutical Sciences/Winkle College of Pharmacy,
1
University of Cincinnati, Cincinnati, OH.
Purpose: Oral propranolol is an effective treatment for periocular infantile
capillary hemangiomas (ICH). We previously reported higher concentrations of
propranolol in periocular tissues and lower peripheral blood levels of drug after
topical, ocular instillation of propranolol as compared with oral delivery (Hao et al
2011). The delivery of propranolol in a gel-forming solution (GFS) may further
increase the local tissue concentration of propranolol while decreasing systemic
exposure to the drug as compared with a non-GFS. We compared the periocular
tissue concentration and plasma concentration of propranolol after ocular
instillation of propranolol 0.5% in GFS and in non-GFS
Methods: Propranolol was delivered by topical, ocular instillation of 0.15 ml (50 l
x 3 doses) of propranolol (0.5%) in phosphate buffered saline solution or
propranolol (0.5%) in 1% sodium alginate gel-forming solution (GFS) for total of
0.75 mg in the treated eye of New Zealand male white rabbits (2 to 2.5 kg body
weight). Rabbits were sacrificed at 1, 4, 8 and 24 hours after drug instillation.
HPLC was used to analyze peripheral blood and periocular tissues, which were
dissected and separately analyzed as upper lid inner, upper lid outer, lower lid
inner, and lower lid outer layers; bulbar conjunctiva; superior rectus and oblique;
inferior rectus and oblique; medial and lateral rectus; and periocular fat. Peripheral
blood samples were also analyzed by HPLC.

Results: At 1 h post dosing, propranolol in GFS resulted in similar or higher
concentrations of propranolol in most periocular tissues (except inferior rectus and
oblique) as compared with propranolol in non-GFS. By 8 h, most tissue types had
higher concentrations of drug in the GFS group. The plasma concentration of drug
was lower in the GFS group at 1h, and both GFS and non-GFS groups had
undetectable drug levels in plasma by 4 to 8 h.
Conclusions: Ocular delivery of propranolol in a GFS resulted in similar or higher
concentration of propranolol in most periocular tissues as compared to non-GFS.
The finding of higher tissue concentrations with GFS may be due to prolonged
retention of drug on, and decreased clearance of the drug from, the ocular surface.
Lower systemic exposure to propranolol when using GFS may decrease the risk of
systemic side effects from the drug.
Commercial Relationships: Michael B. Yang, None; Jinsong Hao,
None; Hongzhou Liu, None; S Kevin Li, Aciont (C)
Support: Unrestricted Grant from Research to Prevent Blindness (to James J.
Augsburger, chairman), Faculty Research Grant to J. Hao (University of Cincinnati
College of Pharmacy)
Outcomes Of Rho-kinase Inhibition On The Metastatic Profile Of A Uveal
Melanoma Cell Line
Aizhan Tapenbayeva, Julia Lke, Matthias Lke, Salvatore Grisanti, Aysegul Tura.
Ophthalmology, University of Lbeck, Lbeck, Germany.
Purpose: Uveal melanoma is the most common primary intraocular tumor in adults
with a high rate of mortality due to metastasis. Rho-kinase is an intracellular
signalling cascade effector, which is activated in a wide variety of tumor types. In
this study, we evaluated the outcomes of Rho-kinase inhibition on the viability,
proliferation and migration of the uveal melanoma cell line 92.1 as well as the
expression of several metastatic proteins.
Methods: The 92.1 cells were cultured in DMEM/F-12 supplemented with 10%
serum and treated with the specific pharmacological Rho-kinase inhibitor H1152 at
a concentration range of 0.1-10 M. Viability and proliferation were assessed by
Live-Dead staining, MTT assay, and Ki-67 immunostaining. The organization of
the actin cytoskeleton was evaluated by phalloidin staining. The extent of Rhokinase activity was determined by an in vitro kinase assay. Migration was analyzed
by the scratch assay on the confluent monolayers of cells grown in 6-well plates.
The levels of beta-catenin, N-cadherin, and vimentin were determined by
immunostainings on cells grown on fibronectin coated 8-well slides and by
Western blot.
Results: H1152 resulted in a dose-dependent reduction in the Rho-kinase activity
at the concentrations of 0.1 and 1 M, whereas the administration of the inhibitor at
10 M weakened this effect possibly due to the interference with other signalling
pathways and resulted in a more aberrant, oncotic morphology together with a
slight decrease in viability. H1152 at 1 M significantly suppressed the
proliferation of the 92.1 cells by 50% (p<0.05) without exerting any toxicity and
interfered with the migration of the cells into the wound area after 2 days. The
levels of vimentin and beta-catenin underwent a significant reduction of 20-30%,
respectively, in response to 1 M H1152 already after 1 day of incubation.
Moreover, the weak immunoreactivity to N-cadherin was further reduced by 20%
in the cells treated with 1 M H1152 for 1 day.
Conclusions: These findings provide evidence to the involvement of Rho-kinase in
the proliferation and migration of the 92.1 cells and demonstrate the efficacy of
Rho-kinase inhibition in suppressing the metastatic potential of these cells as a
novel alternative for the treatment of uveal melanoma.
Commercial Relationships: Aizhan Tapenbayeva, None; Julia Lke,
None; Matthias Lke, None; Salvatore Grisanti, None; Aysegul Tura, None
Support: None
Nicotinamide Treatment Decreases Secretion of Angiogenic and Inflammatory
Cytokines in Uveal Melanoma Cell Lines
Shawn C. Maloney, Shriya Hari, Tamara Granner, Emilia Antecka, Cristina
Miyamoto, Miguel N. Burnier, Jr.. Ophthalmology, McGill University, Montreal,
QC, Canada.
Purpose: Attenuation of angiogenic and inflammatory signalling in tumor cells is
an important objective in many cancers, including uveal melanoma (UM).
Nicotinamide (NIC) is a compound that has been shown to have anti-angiogenic
properties in some cancer cell types. This study aimed to investigate the effects of
NIC on the secretion of pro-angiogenic and pro-inflammatory cytokines in UM cell
lines.
Methods: Two human UM cell lines (92.1 and OCM-1) were treated with NIC
(10mM) or control media for 48 hours in culture. The supernatant from control and
NIC conditions was used in sandwich ELISA-based angiogenesis and inflammation
arrays to evaluate the effects of NIC on secretion of 20 pro-angiogenic and pro-

inflammatory proteins. Samples were assayed in quadruplicate and a Students ttest with a significance cutoff of p<0.05 was used to determine statistical
significance.
Results: Seven pro-angiogenic cytokines were detected in control conditions for
both UM cell lines while three were undetectable. Treatment with NIC caused a
significant decrease in secretion of the following cytokines: Angiogenin, ANG2,
EGF and VEGF-A in 92.1 cells; bFGF and HB-EGF in OCM-1 cells; PIGF in both
cell lines. On inflammation arrays, MCP-1 and IL-8 had the highest levels of
expression in both untreated cell lines and were significantly decreased in both cell
lines following NIC treatment. No significant change was seen for the remaining
eight inflammatory cytokines on the arrays.
Conclusions: Treatment of UM cell lines with NIC caused a reduction in secretion
of several pro-angiogenic cytokines and differences were observed between cell
lines. Additionally, NIC treatment significantly reduced expression of MCP-1 and
IL-8 without up-regulating the other inflammatory cytokines assayed. These
findings suggest that NIC has anti-angiogenic and anti-inflammatory effects on UM
cells. This data provides the basis for further studies of NIC as a possible
therapeutic agent in UM.
Commercial Relationships: Shawn C. Maloney, None; Shriya Hari,
None; Tamara Granner, None; Emilia Antecka, None; Cristina Miyamoto,
None; Miguel N. Burnier, Jr., None
Support: None
Two Years Report: New Experience With Ranibizumab Against Choroidal
Melanoma
Peter E. Liggett1A, Veronica A. Kon Jara1B, Gregory Haffner1A. AOphthalmology,
B
Retina, 1New England Retina Associates, Hamden, CT.
Purpose: To evaluate the safety and efficacy of SHIP + High dose Ranibizumab
versus SHIP + standard Ranibizumab for the treatment of Choroidal Melanoma.
Methods: The study was done in a clinical practice setting. Prospective,
randomized, off label study.
Ten eyes of 10 patientes with small or medium size choroidal melanomas were
selected. All patients were treated with Simultaneous hyperthermia and ICGenhanced Photodynamic Therapy (SHIP). Patients were subdivided to receive 0.5
mg or 2 mg of intravitreal Ranibizumab. SHIP was performed using an infrared
light delivered from a diode laser. Ranibizumab was injected in a standard sterile
fashion. All patients were evaluated to rule out metastasis and underwent a
complete ophthalmological evaluation including: Dilated fundus examination,
fluorescein angiography (FA), SD-Optical coherence tomography (OCT) and A/Bscan ultrasonography (US). Tumor activity was evaluated with FA and US. SDOCT was performed to rule out macular complications. Main outcome measures
were visual acuity, local control of the tumor and rate of complications.
Results: Ten patients were studied with a mean age of 68.5 years (range from 5578). All patients received SHIP (transpupillary thermotherapy + ICG enhanced
photodynamic therapy). Six patients were treatment nave, and 4 patients had a
recurrent tumor, which was initially treated with SHIP (series of 3). The mean time
for recurrence in these 4 patients was 5.3 years. In the group receiving 0.5 mg of
Ranibizumab, the mean number of SHIP was 3. In the group receiving 2 mg of
Ranibizumab, the mean number of SHIP was 6.7. All the patients received a
cumulative dose of 6 mg of Ranibizumab as adjuvant treatment.
Local control of the tumor was achieved in all cases. />Complications included
subconjunctival hemorrhage, cystoid macular edema, peripheral branch retinal vein
occlusion, epiretinal membrane and rhegmatogenous retinal detachment. All the
complications were related to the laser treatment and not to the intravitreal
injection. Their incidence did not affect the control of the disease.
Visual acuity was variable with no statistically difference in both groups. In the
high dose group the baseline VA was 20/50 and 20/25 in the standard dose group.
Conclusions: Ranibizumab did not appear to decrease the number of SHIP
treatments required for local erradication of the tumor. However, patients treated
with adjunctive intravitreal ranibizumab had a significant better visual acuity
results than those treated with SHIP alone, regardless of the dose used.
Commercial Relationships: Peter E. Liggett, None; Veronica A. Kon Jara,
None; Gregory Haffner, None
Support: None
Therapeutic Efficacy By Targeting Correction Of Notch1-induced Aberrants
In Uveal Tumors
Xiaolin Huang1, Li Wang2, He Zhang2, Renbin Jia1, Haibo Wang2, Xiaoping
Zhao1,2, Guanxiang Qian2, Arun D. Singh3, Shengfang Ge1, Xianqun Fan1.
1
Department of Ophthalmology, Ninth Peoples Hospital, Shanghai Jiaotong
University School of Medicine, Shanghai, P.R., China; 2Department of
Biochemistry and Molecular Biology, Shanghai Jiaotong University School of

Medicine, Shanghai, P.R., China; 3Cole Eye Institute, Cleveland, OH.
Purpose: Uveal melanoma (UM) is the most common primary intraocular
malignant tumor in adults. Deregulation of the Notch pathway is implicated in
carcinogenesis. We extended this potential role to UM, observing high expression
of Notch1 in the UM cell lines OCM1 and VUP. The recombinant oncolytic
adenovirus H101 has been approved by the Chinese State Food and Drug
Administration. We seek to explore a potential synergy of inhibiting Notch
signaling combined with H101 oncolytic adenovirus therapy on UM.
Methods: The chemosynthetic Notch1-siRNA (siNotch1) and control siRNA
(siNC) oligonucleotide were synthesized and purified by Shanghai Genepharma
(Genepharma, Shanghai, China). Recombinant oncolytic adenovirus H101 was
kindly provided by Shanghai Sunway Biotech (Sunwaybio, Shanghai, China). The
expression of Notch1 after transfection with siNotch1 and/or H101 was determined
by RT-PCR and Western blot. Appropriate multiplicity of H101 infection and cell
proliferation were measured by MTT assay. Cell cycle and apoptotic activity of
UM cells were analyzed by flow cytometry.
Results: Semiquantitative RT-PCR and Western blot for Notch1 confirmed
suppression by siNotch1 but not siNC or by H101 alone. Notch1 expression was
markedly inhibited by siNotch1 or siNotch1 plus H101. Combined treatment with
siNotch1 and H101 (H101-Notch1-siRNA) produced substantial growth inhibition
on UM cells. The combined treatment H101-Notch1-siRNA greatly enhanced
apoptosis and cell cycle arrest as compared to treatment with H101 or siNotch1
alone.
Conclusions: Notch pathway deregulation could be a feature of UM, and could
also be a promising therapeutic target. Current observations are consistent with the
previous analyses we demonstrated that blocking Notch1 signaling via RNA
interference inhibited HeLa cell growth (2007 Int J Gynecol 17: 511-516). We
previously used a double target approach to antitumor therapy by combining
H101 with siRNA that targeted Bcl2 (2009 Mol Ther 17: 57-64). In this study, we
explored the potential synergy of inhibiting Notch signaling combined with H101
oncolytic adenovirus therapy on UM cell lines OCM1 and VUP. This is the first
report of this combination treatment of UM cell lines.
Commercial Relationships: Xiaolin Huang, None; Li Wang, None; He Zhang,
None; Renbin Jia, None; Haibo Wang, None; Xiaoping Zhao, None; Guanxiang
Qian, None; Arun D. Singh, None; Shengfang Ge, None; Xianqun Fan, None
Support: CR: N. Shanghai Leading Academic Discipline Project S30205, National
Key Program for Basic Research of China 2010CB529902, National Natural
Science Foundation of China 0979034 and 81001008
Towards a Novel Therapy for Uveal Melanoma: Targeting Oncogenic Gq
Timothy W. Corson, Kamakshi Sishtla. Glick Eye Institute, Department of
Ophthalmology, Indiana University School of Medicine, Indianapolis, IN.
Purpose: To develop a peptide ligand for an oncoprotein found in uveal melanoma.
Recently, oncogenic mutations were identified in more than 80% of uveal
melanoma in the genes GNAQ or GNA11, encoding heterotrimeric G-protein
subunits Gq and G11, respectively. These mutations lock the proteins in the
active, GTP-bound conformation, causing constitutive mitogenic signaling.
Selective blockade of
the function of these oncoproteins offers an appealing route to inhibition of
proliferation in uveal melanoma, a chemoresistant cancer currently lacking targeted
therapies.
Methods: Phage display was used to find peptide ligands selective for the common
Q209L mutant of human Gq. Recombinant wild type and Q209L Gq-glutathione
S-transferase fusions were expressed in E. coli, and folding of the wild type protein
was confirmed by binding of BODIPY-GTPS. Recombinant proteins were used
for phage display panning using a 12-mer M13 phage library, and binding
confirmed by anti-M13
enzyme-linked immunosorbent assays.
Results: Multiple phage clones were isolated that showed preferential binding to
Gq Q209L over wild-type protein, while several clones bound preferentially to
wild-type Gq. Sequencing and consensus peptide identification is underway, to be
followed by validation with synthetic peptides.
Conclusions: We have identified peptide-expressing phage that bind Gq Q209L.
Validated, selective peptide ligands for Gq Q209L can be used in fluorescence
polarization displacement assays to find small-molecule ligands with similar
binding modes, tested for cytotoxicity in GNAQ mutant cells, and/or linked to
signals for targeted protein degradation. Selective targeting of oncogenic Gq
Q209L will open an important
new avenue for development of novel therapies for uveal melanoma.
Commercial Relationships: Timothy W. Corson, None; Kamakshi Sishtla,
None
Support: IUPUI Research Support Funds Grant

Topical Timolol for the Treatment of Benign Vascular Periocular Lesions
Raymond G. Areaux, Jr., David Yoo. Ophthalmology, Loyola University Chicago,
Maywood, IL.
Purpose: Benign vascular tumors of the eyelid are common causes of ocular
morbidity. Capillary hemangiomas are the most common orbital tumors of
childhood occurring in 1-3% of term newborns. Corticosteroid therapy of benign
vascular lesions risks sight-threatening complications including central retinal
artery occlusion and significant systemic morbidity. Alternatively, oral and
intravenous beta-blockers have been reported to induce regression of these lesions.
One recent report documented the efficacy of topical timolol in treating a large
capillary hemangioma of the eyelid in a child. Topical application reduces the
potential for systemic side effects of beta-blockers including bradycardia,
hypotension, heart block, and bronchospasm. However, there are no reports on the
effects of beta-blockers for similar lesions in adults. This study investigates
whether topical Timolol 0.5% solution applied twice daily causes significant
regression of benign vascular periocular lesions in adults.
Methods: Prospective case series of patients with benign vascular periocular
lesions from Loyola University Health System. Inclusion criteria: 1) Presence of a
benign vascular periocular lesion. Exclusion criteria: 1) Allergy or contraindication
to timolol or beta-blocker. 2) Intraocular pressure less than 10 mmHg. 3) Lesion
characteristics concerning for atypia or malignancy. 4) Patient less than 18-yearsold. Patients were enrolled, and photo documentation of lesion morphology was
obtained. Patients were given a 15 mL bottle of timolol 0.5% ophthalmic solution
to apply to the lesion(s) twice daily via topical massage. Patients were followed at
monthly intervals for 3 months or until lesion resolution. Photo documentation of
final lesion morphology was obtained. Pre-treatment and post-treatment lesion
sizes were compared.
Results: 14 lesions met criteria, were treated with topical timolol, and were
followed for 1 to 3 months. None of the lesions regressed with therapy. One patient
reported mild sensitivity with treatment. No other side effects were reported.
Conclusions: Our study refutes the hypothesis that Topical timolol 0.5% solution
applied twice daily facilitates regression of benign vascular periocular lesions in
adults. The lack of response to therapy of adult lesions in our trial differs markedly
from the responses reported in children with capillary hemangiomas and lends
further credibility to the hypothesis that lesion susceptibility to beta-blockers is due
to differing endothelial receptor expression in congenital and adult hemangiomas.
To our knowledge, this is the first report on the effect of timolol on adult vascular
periocular lesions.
Commercial Relationships: Raymond G. Areaux, Jr., None; David Yoo, None
Support: Illinois Society for the Prevention of Blindness
Association Of Ocular Findings And Preventive Therapy With Onset Of
Cerebral Involvement In Patients With Primary Intraocular Lymphoma
Noriyasu Hashida1, Kei Nakai1, Nobuyuki Ohguro2, Kohji Nishida2. 1Dept of
Ophthalmology, Osaka University, Suita, Japan; 2Department of Ophthalmology,
Osaka Koseinennkinn Hospital, Osaka, Japan.
Purpose: To investigate whether the site of the ocular lesions and prophylactic
treatment in patients with primary intraocular lymphoma (PIOL) are associated
with the time to onset of cerebral involvement.
Methods: We retrospectively studied 22 patients (5 men, 17 women; mean age, 66
12 years) with a diagnosis of PIOL at our hospital between January 2001 and
October 2011 and with at least 1-year follow-up after treatment. The Osaka
University Medical School Ethics Committee approved the study protocol, and all
patients provided informed consent. We analyzed the relation between the site of
the ocular lesions and the onset of cerebral involvement by classifying the PIOL
lesions into three types: vitreous opacity (VO) type, vitreous opacity of 2+ or
higher without retinal lesions; retina type, vitreous opacity of 1+ or less with retinal
lesions only; and combination type, with both. We also evaluated whether
prophylactic treatment inhibited the onset of cerebral involvement by administering
systemic chemotherapy or topical ocular treatments, such as intravitreal injections
of methotrexate and rituximab, in patients with PIOL without cerebral involvement.
Results: During a mean follow-up period of 38 21 months, 14 (64%) patients had
onset of cerebral involvement, including 3 (60%) of 5 patients with retina-type
lesions, 5 (50%) of 10 patients with VO-type lesions, and 6 (86%) of 7 patients
with combination-type lesions. There are no significant differences between the site
of the ocular lesions and the onset of cerebral lesions (P=0.285). In addition,
cerebral involvement occurred in 6 of 8 patients receiving prophylactic systemic
chemotherapy and 8 of 14 patients receiving topical ocular treatments, with no
significant difference between the two groups (P=0.649). The time to onset of
cerebral involvement in the systemic chemotherapy group (50.2 23.2 months)
was significantly longer than that in the topical ocular treatment group (15.6 13.6
months) (p = 0.004).
Conclusions: While prophylactic systemic chemotherapy did not inhibit the onset
of cerebral involvement in patients with PIOL, it significantly prolonged the time to

cerebral onset of disease.
Commercial Relationships: Noriyasu Hashida, None; Kei Nakai,
None; Nobuyuki Ohguro, None; Kohji Nishida, None
Support: Ministry of Education, Culture, Sports, Science and Technology Grant.
C23592604
Syringes Design Used for Intravitreal Injection Influences Dosing Accuracy
and Drug Wastage
David M. Krumholz. Clinical Sciences, SUNY College of Optometry, New York,
NY.
Purpose: A 1cc tuberculin syringe with a half-inch long 30 gauge needle is
typically used for intravitreal injection of a 0.05cc dose of Lucentis for wet AMD
management. A previous study showed that this delivered a greater than intended
dose. The tuberculin syringe and needle assembly also contain considerable "dead
space", wasting medication. One-cc syringes are also manufactured with a half-inch
long 30 gauge needle integrated into the syringe body, leaving very little dead
space. This study compared dosing accuracy and drug wastage between the two
different designs of 1cc syringes that may be used for intravitreal injections.
Methods: Fifty samples each of two different 1cc syringe designs were tested. The
"integrated" design had the needle integrated into the body of the syringe. The
"separate" design was a 1cc tuberculin syringe with a separate 30g inch long
needle attached. Syringes were weighed empty, with needle attached but covers
removed, on an analytical balance with 0.1mg resolution. Each syringe was filled
as accurately as possible with 0.05cc of sterile water, and weighed again after
blotting the needle tip. The contents of the syringe was ejected, weighed, and
converted to ccs. The ejected volume was compared with that from an Eppendorf
pipette, which served as a control. Each syringe was weighed again to determine
the volume retained. Data from the two syringe designs were compared using a
Students T test.
Results: The Eppendorf pipette control delivered a mean volume of 0.0500cc (+/0.0007cc). The integrated syringe design delivered a mean volume of 0.0516cc (+/0.0030cc), significantly different from the control (t=5.56, p=0.000). The separate
syringe design delivered a mean volume of 0.0548cc (+/- 0.0027cc), also
significantly different from the control (t=12.36, p=0.000). The integrated and
separate designs delivered mean volumes that differed significantly from each other
(t=5.60, p=0.000). The integrated design retained a mean residual volume of
0.0007cc (+/- 0.0050cc) while the separate design retained an average of 0.0790cc
(+/- 0.0013cc). This difference was significant (t=30.49, p=0.000).
Conclusions: The two syringe designs are not interchangeable, and both delivered
a volume in excess of that intended, although the integrated design was closer to
the intended dose. The integrated design also retained less volume after injection.
With Lucentis costing approximately $2000 per 0.2cc vial, $500 worth of
medication is actually injected into the eye while almost $800 worth remains in the
tuberculin syringe and is wasted. The integrated design retains only about $7 worth
of medication. Significant cost savings could be realized by re-packaging Lucentis
in pre-filled integrated design syringes.
Commercial Relationships: David M. Krumholz, None
Support: None
Precise Modeling of the Eye for Proton Beam Radiotherapy of Intraocular
Tumors
Michael B. Rueegsegger1A,1B, Jens H. Kowal1A,1B, Sebastian Wolf1B. AARTORG
Center Ophthalmic Technologies, BDepartment of Ophthalmology, 1University of
Bern, Bern, Switzerland.
Purpose: A challenging topic in ophthalmology is the treatment of intraocular
tumors by irradiation. Choroidal melanoma and other tumors are commonly treated
by charged particle beam irradiation or brachytherapy. For charged particle beams,
it is possible to shape the irradiation volume in a very precise and sharply
delineated way. This allows for an optimized tumor control rate, at the same time
sparing as much as possible eye structures at risk. In order to take advantage of
charged particle radiotherapy a precise treatment planning is crucial. State of the art
treatment planning is based on a parametric eye model, which is adapted to the
patients anatomy based on Euclidean distances, extracted from different image
modalities e.g. CT, MRT, US, etc. This approach is highly limited in accuracy and
strongly depends on the surgeon. In this study we evaluate the feasibility of a
Statistical Shape Model as a method for modeling the human eye to improve
treatment planning accuracy. An increase in planning accuracy will lead to higher
treatment efficacy and safety for the patient. At a later stage we plan to fuse the
different image modalities into a single patient specific eye model to enable multimodal treatment planning.
Methods: Statistical Shape Models (SSM) have been shown to be a powerful tool
to capture the large, but limited variability of organ shape in a compact manner. In

this study a 3D SSM of the human eye including Cornea, Lens, Sclera as well as
the position of the Optic Nerve Head was built. 18 manual segmented head CT
scans with a resolution of 0.39 x 0.39 x 0.6 mm has been used as training set for the
SSM construction as well as for validation purposes. In a first step an atlas is
constructed representing the mean eye shape. From this atlas, landmarks are
extracted in a second step and propagated to each shape in the training set via a
volumetric non-rigid registration technique. This approach enables the construction
of a SSM in presence of large shape variability and multiple objects. In order to
automatically fit the SSM into patient specific CT slice stacks an Active Shape
Model (ASM) has been built.
Results: To measure the performance of the SSM and ASM a Leave-One-Out
cross validation on the training set was carried out and similarity with the manual
segmentation done by an expert was measured by the Dice Similarity Measure
(DSM). The cross validation revealed a Dice coefficient of 95.22.2% for the
Sclera and Cornea and 91.42.1% for the Lens. An average model fit took 30s on a
regular desktop PC.
Conclusions: The presented method to model a patients specific eye has proven to
be highly accurate. Furthermore it has been shown that this model-based method
can be used to segment eye structures on CT scans with a high amount of
robustness and with a short execution time.
Commercial Relationships: Michael B. Rueegsegger, None; Jens H. Kowal,
None; Sebastian Wolf, None
Support: None
In Vivo Confocal Microscopy Study Of Conjunctival Intraepithelial Neoplasia
Treated With Interferon-alpha2b
Hyunjoo J. Lee1,2A, Robert Dunphy2B, Mary Daly2A,1, Donna Siracuse-Lee2A,1.
1
Ophthalmology, Boston Medical Center / Boston University School of Medicine,
Boston, MA; AOphthalmology, BOptometry, 2Veterans Affairs Boston Healthcare
System, Boston, MA.
Purpose: The diagnosis of conjunctival intraepithelial neoplasia (CIN) is usually
based on clinical appearance. The gold standard of treatment for CIN is excisional
biopsy with adjunctive cryotherapy. Studies have shown topical agents such as
interferon-alpha2b (IFN2b) to be efficacious for treating CIN, but it is not clear
exactly how long a patient should be treated with these agents in order to achieve
adequate treatment and prevent tumor recurrence. The purpose of this study is to
assess whether in vivo confocal microscopy (IVCM) can help distinguish CIN from
benign lesions, and whether serial IVCM in patients treated with IFN2b can
confirm treatment efficacy and help monitor for recurrence.
Methods: This is a retrospective chart review of consecutive cases of suspected
CIN treated with IFN2b and imaged using the Heidelberg Retina Tomograph III Rostock Cornea Module (Heidelberg, Germany) in vivo confocal microscope at the
Veterans Affairs Boston Healthcare System. A healthy normal volunteer was
included for comparison.
Results: Between January 2010 and July 2011, there were 9 cases in 9 patients of
suspected CIN treated with topical IFN2b. Two of the eyes were initially treated
with topical IFN2b, and subsequently underwent excisional biopsy due to
unsatisfactory clinical response. One of these lesions proved to be squamous cell
carcinoma, and the other lesion had histologic findings consistent with a severely
inflamed pterygium. The remaining 7 cases resolved clinically after IFN2b
treatment. With IVCM, 7 of 9 presumed CIN lesions were initially seen to have
cells with bright and prominent nuclei. In 7 of 9 cases, the cell borders were
indistinct, in contrast to the usually distinct cell borders in normal conjunctival
epithelium. After the CIN lesions were no longer apparent clinically, bright
prominent nuclei could occasionally be seen by IVCM. In 5 cases followed with
serial IVCM during IFN2b treatment, the cell nuclei generally became less
prominent and the cell borders more distinct and regular. In one case, there was
complete resolution clinically and by IVCM by 4 months. The pterygium also had
areas of bright nuclei and indistinct cell borders.
Conclusions: IVCM cannot replace the pathologic and clinical diagnosis of CIN,
but can aid in monitoring patients for disease regression or recurrence. Also,
treatment of CIN with IFN2b alone can lead to complete clinical and microscopic
resolution of disease.
Commercial Relationships: Hyunjoo J. Lee, None; Robert Dunphy,
None; Mary Daly, None; Donna Siracuse-Lee, None
Support: None
Expression Of N-glycolyl Gm3 In Retinoblastoma, A Promising Candidate For
Targeted Therapies
Ana Vanesa Torbidoni, Alejandra Scursoni, Sandra Camarero, Claudia Sampor,
Guillermo Chantada, Maria T. de Davila. Hospital de Pediatria Prof. Dr. Juan P.
Garrahan, Capital Federal, Argentina.
Purpose: The identification of molecules expressed selectively on the surface of

retinoblastoma cells, would allow applying targeted therapies to reach almost
exclusively tumor tissue. Gangliosides are potential antigens for cancer targeted
therapy, but they are expressed in normal cells. Humans lack the enzyme to
synthesize N-Glycolyl GM3 ganglioside (NGcGM3) which is not expressed in
human normal tissues. NGcGM3 has been detected in other pediatric
neuroectodermic tumors. We sought to characterize the expression of the NGcGM3
in retinoblastoma cell lines and in retinoblastoma tumor samples.
Methods: We studied WERI-Rb1 and Y79 cell lines and paraffin embedded
tumors from 20 children with retinoblastoma. Fourteen had unilateral disease, 5 of
them with extraocular dissemination. The others six had bilateral retinoblastoma. A
bone marrow from a metastatic retinoblastoma was also studied. A retinoblastoma
free eye was used as control. The 14F7 antibody (provided by Center of Molecular
Immunology, La Habana, Cuba) recognizes NGcGM3. Cell lines were fixed and
incubated with 14F7, washed and incubated with secondary antibody with FITC.
Sections of tumors were incubated with 14F7 and then with peroxidase conjugated
secondary antibodies and developed with 3,3-diaminobenzidine. NGcGM3
expression was evaluated analyzing the percentage of tumour positive cells and the
staining intensity.
Results: Both retinoblastoma cell lines showed immunoreactivity to NGcGM3 but
WERI-Rb1 presented higher levels of expression than Y79. All the tumors studied
showed strong immunoreactivity to NGcGM3, in either intraocular or extraocular
sites including those cases with optic nerve or extrascleral invasion. In children that
underwent bilateral enucleation we could evaluate NGcGM3 before and after
chemotherapy. The expression of NGcGM3 persisted unaltered after chemotherapy.
Normal eye and normal retina of affected eyes were completely negative,
confirming the absence of this molecule in normal human tissues. In metastatic
sites high levels of NGcGM3 were observed in retinoblastoma cells.
Conclusions: Our results indicate that NGc-GM3 is highly expressed in
retinoblastoma tumor and it is absent from control eye. Its expression did not
decrease after chemotherapy and is present in retinoblastoma cells in metastatic
sites. Our results highlight the possibility of using antibodies against this molecule
as targeted therapy.
Commercial Relationships: Ana Vanesa Torbidoni, None; Alejandra
Scursoni, None; Sandra Camarero, None; Claudia Sampor, None; Guillermo
Chantada, None; Maria T. de Davila, None
Support: None
Sulindac Protects RPE Cells Against Oxidative Damage but Enhances the
Killing of Retinoblastoma Cells Exposed to Oxidative Stress
Arunodoy Sur1A, Howard M. Prentice1B, Herbert Weissbach2, Janet C. Blanks1C.
A
Integrative Biology Phd Program, Dept of Biology, BCharles E Schimdt College
of Medicine, CCenter for Complex Systems & Brain Sci, 1Florida Atlantic
University, Boca Raton, FL; 2Center For Cellular and Molecualr Biology, Florida
Atlantic University, Jupiter, FL.
Purpose: The ability of sulindac to enhance killing of the retinoblastoma (RB) cell
line, Y79, was tested in combination with reagents that affect mitochondrial
function. Recent studies have shown that sulindac can enhance the killing of
multiple cancer cell lines, when used in combination with an oxidizing agent. This
effect of sulindac was unrelated to the non-steroidal anti-inflammatory activity.
Methods: The ability of sulindac to kill retinoblastoma cells was determined by
treating Y79 cells with sulindac for a period of 48 hours in combination with 3
different chemicals. The three reagents used with sulindac in this study were
hydrogen peroxide (H2O2), doxorubicin (DOX) and dichloroacetic acid (DCA).
The H2O2 was added for 2 hours after the cells were exposed to sulindac, whereas
DOX and DCA were added to the cells along with sulindac for 48 hr. DCA was
used at a range of 4uM to 24uM. The DOX concentration ranged from 50nM to
300nM while H2O2 concentration ranged from 250M to 1.25mM. After treatment
with the drugs cellular viability was determined using an assay that utilizes
tetrazolium compound (MTS). Conversion of tetrazolium to formazan by cellular
dehydrogenase in metabolically active cells was detected by colorimetric analysis
Results: The results show that exposing cultured Y79 cells to sulindac, combined
with anti-cancer agents that affect mitochondrial function, such as DCA or DOX,
causes increased death of cancer cells. The combination of the two drugs resulted in
death of about 30% of Y79 cells under conditions where sulindac or anti-cancer
agents that affect mitochondrial function alone caused no loss of cell viability.
Conclusions: We reported previously that sulindac protected RPE cells from
increased oxidative stress induced by oxidizing agents. In contrast, the killing of
cancer cells by sulindac and oxidizing agents involve mitochondrial dysfunction,
reactive oxygen species production and apoptosis. Evidence is presented that
sulindac enhances the killing of RB cells exposed to agents that affect
mitochondrial function. Future experiments will study the role of mitochondria and
ROS and elucidate the signaling mechanisms involved in this process. This
combination therapy using sulindac represents a novel therapeutic approach to treat
RB.

Commercial Relationships: Arunodoy Sur, None; Howard M. Prentice,
None; Herbert Weissbach, None; Janet C. Blanks, None
Support: Florida Atlantic University Priority Research Grant (JB)

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ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

114 AMD: New Drugs, Delivery Systems and Mechanisms

Conclusions: Olmesartan reduced the MCP-1 expression and the resultant

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Purpose: To evaluate three year follow up treatment outcomes with ranibizumab

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drug loading, controlled release of therapeutic over extended times, materials

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VEGF antibody intravitreal injections were decreased by 11% (p=0.1).

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

Commercial Relationships: Jennifer S. Wade, Genentech Inc (F); Tejal A.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

Conclusions: ECT intraocular implant is capable of sustained delivery of a VEGF

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

Program Number: 459 Poster Board Number: D1136

Commercial Relationships: li xingyi, None; chen hao, None

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

Commercial Relationships: Ankita Desai, Ocular Therapeutix, Inc. (E);

differences in PD in eyes treated with tropicamide by eye drops or by the

Commercial Relationships: Maria J. Del Sole, None; Paula Schaiquevich,

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

appropriate calibration solutions of melphalan and internal standard. A

Commercial Relationships: Julie E. Whitcomb, Allergan (E); Susan S. Lee,

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

SMVT Targeted Lipid Prodrug Of Cidofovir: Novel Treatment Strategy For

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ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

Commercial Relationships: Kongnara Papangkorn, Aciont Inc (E); Donald

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

Jorge A. Fortun1, Alex Gonzalez2, Cornelis Rowaan2, Mariela Aguilar2, William

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

Jay M. Stewart1A, Chris J. Diederich1B, Elliot Chan1A, Vasant Salgaonkar1B,

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

Commercial Relationships: Lingyun Cheng, None; Elizabeth Wu,

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

Vida Rahmani1A, Frances Lasowski1B, Heather Sheardown1A. AChemical

Commercial Relationships: Vida Rahmani, None; Frances Lasowski,

Commercial Relationships: Houman D. Hemmati, In-situ crosslinked CMC

Program Number: 501 Poster Board Number: D1178

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

Commercial Relationships: Qiongyu Guo, None; Ahmed Aly, None; Oliver D.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

Commercial Relationships: Charles D. Blizzard, Ocular Therapeutix, Inc. (E);

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

Efficient Transduction of Tyrosine-to-Phenylalanine Mutated AAV2 vectors

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

Purpose: Usher syndrome type 1B (USH1B) is a common and severe form of

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

Commercial Relationships: Anna Salas Torras, None; Andrea R. Carvalho,

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

transpositional insertion, leading to long term reduction of neovascularization in

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

Commercial Relationships: Scott Ellis, Oxford BioMedica Ltd (E); James

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

Methods: A human RDH12 cDNA construct previously shown to exhibit RDH