Original Investigation
Electron Microscopic Features of Nasal Mucosa Treated
with Topical and Circumostial Injection of Mitomycin C:
Implications in Dacryocystorhinostomy
Mohammad Javed Ali, F.R.C.S.*, Farhana Baig, M.D., D.N.B.†, Mekala Lakshman, Ph.D.‡,
and Milind N. Naik, M.D.*
*Dacryology Service, L.V. Prasad Eye Institute; †Department of Pathology, Global Hospital;
and ‡Ruska Laboratories, College of Veterinary Sciences, Hyderabad, India
Purpose: To evaluate the ultrastructural effects of topical and
circumostial injection of mitomycin C (COS-MMC) on nasal
mucosa and compare them with the controls. The study also
aimed at classifying the subcellular effects in detail.
Methods: The nasal mucosa of 6 patients were subjected to
0.02% of mitomycin C for 3 minutes (3 patients) and 0.02%
COS-MMC (3 patients) as per standard protocol, during
endoscopic dacryocystorhinostomy. Normal nasal mucosa from
untreated areas (2 each from topical and COS-MMC groups)
were taken as controls after harvesting the treated areas. Full
thickness tissues (5 mm × 5 mm) were collected for transmission
electron microscopy, and ultrastructural effects were evaluated.
Results: Both topical and COS-MMC showed significant
and distinct ultrastructural changes involving the epithelial,
glandular, vascular, and fibrocollagenous tissues compared
with the controls. There were profound changes within
fibroblasts with intracellular edema, pleomorphic and
vesicular mitochondria, dilated smooth and rough endoplasmic
reticulum, and chromatin condensation. In addition, COS-
MMC samples showed subepithelial hypocellularity with
limited disorganization of structure. The changes in both the
MMC groups were restricted to treated areas only.
Conclusions: Both topical and COS-MMC show profound
changes in nasal mucosa with more marked changes in COS-
MMC group. These changes being limited in nature may
help in enhancing the success of dacryocystorhinostomy
by preventing cicatricial changes of the ostium, especially
in high-risk cases such as revision and post-traumatic
dacryocystorhinostomy.
(Ophthal Plast Reconstr Surg 2015;31:103–107)
D acryocystorhinostomy (DCR) is a common surgery per-
formed for the management of nasolacrimal duct obstruc-
tion with a good success rate. However, the failure rates can
Accepted for publication April 12, 2014.
The authors have no financial or conflicts of interest to disclose.
This study has been reviewed by the ethics committee and has been
performed in accordance with the ethical standards laid down in the 1964
Declaration of Helsinki. Informed consent was obtained from the patients.
M.J.A. helped in concepts and manuscript writing; F.B. in concepts and
electron microscopic interpretation; M.L. in electron microscopy; and
M.N.N. in critical review.
Address correspondence and reprint requests to Mohammad Javed Ali,
F.R.C.S., L.V. Prasad Eye Institute, Road No 2, Banjara Hills, Hyderabad 34,
India. E-mail: drjaved007@gmail.com
DOI: 10.1097/IOP.0000000000000205
Ophthal Plast Reconstr Surg, Vol. 31, No. 2, 2015 103
range from 4% to 13%.1–3 The most common cause of fail-
ure is scarring and cicatricial closure of the osteotomy site.3,4
Therefore, increased success can be achieved using antifibrotic
agents, such as mitomycin C (MMC) that can inhibit scarring
and cicatrix formation through its wound modulatory effects.5–12
Ali et al.13 studied the effects of MMC on nasal mucosa
fibroblasts and found that the minimum effective concentration
of MMC was 0.2 mg/ml for a duration of 3 minutes. They found
that at this concentration, there is significant inhibition of cel-
lular proliferation among fibroblasts without causing significant
apoptosis and cellular death. Kamal et al.14 described a new
technique of injecting MMC called circumostial mitomycin C
(COS-MMC) to potentiate the wound modulatory effects of the
drug. They showed it to be safe and effective in high-risk cases
such as revision DCRs and surgeries for post-traumatic second-
ary acquired lacrimal duct obstruction (SALDO).
Ultrastructural effects of MMC in DCR have been stud-
ied once earlier by Ugurbas et al.,15 who demonstrated mito-
chondrial and nuclear changes following topical application of
0.5 mg/ml for 2.5 minutes. The current lacunae in ultrastructural
knowledge are lack of detailed drug effects, no data with the
current evidence of 0.02% MMC for 3 minutes, and no data on
the subcellular effects of COS-MMC. This article attempts to
fill this void and report detailed transmission electron micro-
scopic (TEM) effects of standardized MMC on nasal mucosa
using various approaches. These changes were also documented
by classifying them under 4 subheadings, namely epithelial,
glandular, vascular, and fibrocollagenous.
METHODS
The nasal mucosa of 6 patients were subjected to 0.02% of
MMC for 3 minutes (3 patients) and 0.02% COS-MMC (3 patients) as
per standard protocol, during endoscopic DCR.13,14 Normal nasal mu-
cosa from untreated areas (2 each from topical and COS-MMC groups)
were taken as controls after harvesting the treated areas. Full thickness
tissues (5 mm × 5 mm) were collected for TEM, and ultrastructural ef-
fects were evaluated. All patients were operated by a single surgeon
(M.J.A.). Institutional review board and ethics committee approval was
obtained. All patients provided an informed written consent.
TEM Protocol. The samples were fixed in 2.5% glutaraldehyde in 0.1-
M phosphate buffer (pH 7.2) for 24 hours at 4°C and postfixed in 2%
aqueous osmium tetroxide in 0.1-M phosphate buffer for 2 hours. The
tissues were dehydrated in a series of graded alcohols, infiltrated, and
embedded in Araldite 6005 resin or Spur resin. Ultrathin sections of
50 nm were made with a glass knife using the Ultra-microtome (Lieca
Ultracut UCT-GA-D/E-1/00, Wetzlar, Germany). The ultrathin sections
were then mounted on copper grids and stained with saturated aqueous
M. J. Ali et al. Ophthal Plast Reconstr Surg, Vol. 31, No. 2, 2015

uranyl acetate and counter stained with Reynold’s lead citrate. The sec-
tions were viewed under TEM (Hitachi H-7500, Tokyo, Japan) at ap-
propriate magnifications as per standard protocols.16
RESULTS
Both topical and COS-MMC showed significant ultrastructural
changes involving epithelial, glandular, and vascular changes compared
with the controls. Features of each category are described separately.
Controls or Normal Nasal Mucosa. The normal nasal mucosa
showed healthy epithelial cells with well-defined cellular boundaries
and microvilli toward the luminal side (Fig. 1A). The cytoplasm and
nucleus were normal, with numerous intracytoplasmic normal mito-
chondria reflecting an increased metabolism. Within the epithelium,
there were distinct goblet cells with a large lumen and nucleus to one
side (Fig. 1A,B). The glandular tissues showed cells with tight intra-
cellular junctions with numerous intracytoplasmic golgi bodies and
endoplasmic reticulum (Fig. 1C). There were numerous secretory ves-
icles with secretory granules within them reflecting secretory activity
(Fig. 1C). Subepithelial tissues had numerous collagen fibers, some
longitudinally cut (fiber like) and some cross-sectional (end-on dark
tips) with intervening normal fibroblasts with scanty cytoplasm, large
nucleus, and numerous normal mitochondria without gross chromatin
condensation (Fig. 1D).
Topical MMC (0.02%, 3 Minutes). The epithelium was attenuated
with changes up to the basal cells with gross inter- and intracellular ede-
ma (Fig. 2A). Intercellular junctions were either loose or indistinct. The
cytoplasmic organelles were indistinct with patchy chromatin condensa-
tion (Fig. 2A). The nuclei were pleomorphic with indistinct nucleoli and
gross marginal chromatin condensation. Few cells showed gross peri-
nuclear dilated areas (Fig. 2A).
The glandular cells showed distinct intracellular junctions but
were not tight. Cytoplasm showed permeable vesicles with scattered and
disorganized secretory granules (Fig. 2B). There were areas of intracy-
toplasmic edema with dilated cisternae of rough endoplasmic reticulum
(Fig. 2B). Nuclear changes were more marked with perinuclear dilated
areas, disrupted outer nuclear membranes, and peripheral chromatin
condensation abutting the inner nuclear membrane (Fig. 2B).
Vascular changes noted were dilated microcapillaries with lu-
men homogenously filled with blood cells (Fig. 2C). The endothelial
cells were dilated with irregular or indistinct nucleus with plenty of
chromatin condensation. There were deep indentations in smooth
muscle fibers with intervening edema, adjacent to the capillaries
(Fig. 2C).
Fibroblasts and collagen fibers showed the most profound ef-
fects. The collagen fibers were edematous with intervening fluid be-
tween fibers in some places (Fig. 2D). The fibroblasts showed gross
intracellular edema and indistinct cytoplasmic organelles but main-
tained its shape and cellular outline (Fig. 2D). There was scanty but
widespread patchy electron dense granular condensation (Fig. 3A).
The mitochondria were pleomorphic with altered size and shape.
Some mitochondria were predominantly vesicular without any mito-
chondrial matrix, reflecting loss of function(Fig. 3B). The nuclei were
indistinct. Endoplasmic reticulum was discontinuous with intervening
edema (Fig. 3A,B).

FIG. 1.  Ultrastructural features of normal nasal mucosa. Transmission electron micrograph (TEMG) showing the normal nasal mucosa
epithelium. Normal epithelial cells (E) are seen with tight junctions and normal nucleus (N) and nucleolus (NL). Intervening goblet cells
(G) and microvilli (M) can be noted (×2,895) (A). Large goblet cell with nucleus to one side (×3,860) (B). TEMG showing glandular
cells with tight intercellular junctions (T), plenty of golgi bodies (G), and numerous secretory granules (S) (×3,474) (C). TEMG showing
fibroblast (F) with surrounding collagen fibers (C) (×5,790) (D).

104 © 2014 The American Society of Ophthalmic Plastic and Reconstructive Surgery, Inc.
Ophthal Plast Reconstr Surg, Vol. 31, No. 2, 2015 Ultrastructural Effects of Mitomycin C on Nasal Mucosa

FIG. 2.  Ultrastructural features following topical mitomycin C treatment. Transmission electron micrograph (TEMG) showing epithelial
changes up to the basal cells (B) with inter- and intracellular edema (E), degenerating nuclei (N), peripheral nuclear chromatin condensa-
tion (C), and perinuclear dilatation (PND) (×6,755) (A). TEMG of glandular cells showing vesicular cytoplasm (V), dilated endoplasmic
reticulum (ER), nuclei (N) with widespread chromatin condensation (C), disruption of outer nuclear membrane (ONM), and perinuclear
dilatations (PND) (×7,720) (B). TEMG showing a dilated microcapillary (DC) with a lumen (L) filled with erythrocytes (B). The endothelial
cell (EC) is edematous with disorganized nucleus (N). Smooth muscle (SM) fibers are seen in the vicinity (×2,316) (C). Edematous colla-
gen fibers (C) with a swollen fibroblast (F) with scanty electron dense granules (ED) and vesicular mitochondria (VM) (×7,720) (D).

Circumostial Injection of MMC 0.02% (COS-MMC). The epithe- DISCUSSION

lium was grossly attenuated with very sparse microvilli (Fig. 3C). This
epithelium can be compared with the normal one (Fig. 1A). The nuclei
were vesicular with indistinct nucleoli and irregular chromatin con-
densation (Fig. 3C,D). The mitochondria were devoid of matrix, and
numerous such vesicular changes were noted (Fig. 3D). The basement
membrane of the epithelium was found to be discontinuous with ir-
regular collagen bundles and gross gaps within the bundles (Fig. 4A).
The highlight of COS-MMC was focal disorganization in the subepi-
thelial tissues (Fig. 4A).
Glandular tissues were disorganized with thickened septae and
diffuse inter- and intracellular edema (Fig. 4B). The secretory compo-
nents were hypoplastic with numerous empty secretory vesicles and
scanty secretory granules. Grossly disturbed but plenty of rough endo-
plasmic reticulum were seen (Fig. 4B).
The vascular changes were similar to that seen with topical
MMC (Fig. 2C), with no perceptible differences; however, the fibro-
collagenous tissues showed marked changes. The collagen fibers were
appearing scant because of severe edema of the fibers and intervening
spaces between collagen bundles (Fig. 4C). Few fibroblasts showed
gross degenerative changes with partly missing cellular outlines
(Fig. 4C,D). However, all the fibroblasts showed indistinct cytoplasm,
indistinct cytoplasmic organelles, loss of mitochondria, peripheral nu-
clear chromatin condensation, and patchy electron dense granular depo-
sition all over (Fig. 4C,D).
MMC is an antineoplastic antibiotic obtained from strep-
tomyces caespitosus and is commonly used in lacrimal surgeries.
It is a potent inhibitor of DNA and protein synthesis and acts by
covalently reacting with DNA forming cross-links between the
complementary strands.17 It is well-known that for any wound
healing, the proliferation and extracellular matrix production by
the fibroblasts play a central role and that low dose of MMC
induces cell cycle arrest at S phase and also at G2-M phase,
while increased MMC concentrations results in apoptosis.17
Desmouliere et al.18 conducted electron microscopic studies
of the granulation tissues and scar in female Wistar rats. They
found that fibroblast and myofibroblasts play an essential role in
wound healing and also in the scar formation.
Ugurbas et al.15 studied the histopathological effects of
MMC on nasal mucosa by both light microscopy and ultrastruc-
turally. They used 0.5 mg/ml for 2.5 minutes followed by a wash.
They harvested the nasal mucosa not only intraoperatively but
also many times postoperatively on day 15, 1, 3, and 6 months.
They concluded that effects of MMC were confined to treated
areas, and effects of MMC continued up to 6 months after sur-
gery. They observed attenuated epithelium, intracellular edema,
and abnormal mitochondria as the most prominent findings and
concluded that the topical use of MMC may enhance the success

© 2014 The American Society of Ophthalmic Plastic and Reconstructive Surgery, Inc. 105
M. J. Ali et al. Ophthal Plast Reconstr Surg, Vol. 31, No. 2, 2015

FIG. 3.  Ultrastructural features following mitomycin C application. Transmission electron micrograph (TEMG) high magnification of
fibroblast (F) shows retained cellular outline on 1 side with dilated endoplasmic reticulum (ER) and vesicular mitochondria (VM) with
peripheral chromatin condensation (C) (×13,510) (A). TEMG high magnification showing subcellular features of fibroblast, including
dilated endoplasmic reticulum (ER), pleomorphic mitochondria (M), VM with peripheral chromatin condensation (C), and scattered
electron dense (ED) granular material (×28,950) (B). TEMG of circumostial injection of mitomycin C (COS-MMC)–treated mucosa
showing attenuated epithelium (E) with vesicular nuclei (VN) and VM and sparse microvilli (M) (×3,860) (C). TEMG of COS-MMC–
treated epithelium in a higher magnification showing vesicular nuclei (VN) and VM (×4,825) (D).

of a DCR. The current study used standardized dose with differ-
ent approaches, but the authors did not believe the need for post-
operative collection of samples because of the current evidence
on duration,13 increased patient morbidity, increased trauma in
the crucial ostium areas, and undesirable stimulation of a heal-
ing wound. This study for the first time documented detailed
subcellular effects and classified the TEM findings as those of
epithelial, glandular, vascular, and fibrocollagenous. The cur-
rent study also looked at the effects of a new modality called
COS-MMC.
In conclusion, the authors found that the ultrastructural
effects of MMC are many but are restricted to the treated areas
only. Changes affected epithelial, glandular, and vascular tissues;
however, the effects on fibroblasts were marked in both the groups
with slightly more profound effects in the COS-MMC samples.
These changes may help in enhancing the success of DCR by pre-
venting cicatricial changes of the ostium, especially in high-risk
cases such as revision cases and post-traumatic DCR.
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106 © 2014 The American Society of Ophthalmic Plastic and Reconstructive Surgery, Inc.
Ophthal Plast Reconstr Surg, Vol. 31, No. 2, 2015 Ultrastructural Effects of Mitomycin C on Nasal Mucosa

FIG. 4.  Ultrastructural features following circumostial injection of mitomycin C (COS-MMC) treatment. Transmission electron micro-
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