Académique Documents
Professionnel Documents
Culture Documents
A thesis Submitted to
College of Medicine - Al-Nahrain University
By
Assistant Professor
Dr. Muthanna Abdul-Ameer Al-Kaabi*
M.B.CH.B., M.Sc., Ph.D.,
Dr. Fadhil Husam Ahmed
B.Sc.V.M.S, M.Sc.
December 2016
Ten adult male rats (Rattus Norvegicus Albinus) aged 3-6 months
were used to study the general histological morphology of ependymal cells
at the sulcus medianus organum and median eminence regions by
hematoxylin and eosin stain, and to explore the presence of tight junctions
and adhesion junctions with anti-ZO1 antibodies directly conjugated with
Fluorescein isothiocyanate (FITC), and unconjugated anti-pan-cadherin
primary antibodies with secondary antibodies labeled with tetra-methyl-
rhodamine isothiocyanate (TRITC), respectively, at these specified regions.
I
of the sulcus medianus organum in a way similar to what is found between
tanycytes of the median eminence, suggesting that tanycytes or tanycyte-
like cells were present in the ependymal lining of the sulcus medianus
organum. On the other hand, anti-pan-cadherin antibodies immunolabeling
showed that adhesion junctions between ependymal cells of the sulcus
medianus organum were present analogous to that between ependymal cells
lining the brain ventricles elsewhere.
II
Contents
Abstract I
List of Tables VI
Abbreviations X
III
1.6. Cell Junctional Complexes 23
1.6.1. Tight Junctions 24
1.6.2. Adhesion Junctions 26
1.7. Immunohistochemistry and Immunofluorescence 27
1.8. Aim of the Study 29
IV
3.2.1.1. Adhesion Junctions 46
3.2.1.2. Tight Junctions 49
3.2.2. Sulcus Medianus Organum 51
3.2.2.1. Adhesion Junctions 51
3.2.2.2. Tight Junctions 54
3.2.3. Controls 57
3.2.3.1. Pan-cadherin TR Antibodies 58
3.2.3.2. ZO1-FITC Antibodies 59
V
List of Tables
Table Page
VI
List of Figures
Figure Page
VII
Figure 3.5 Coronal section of the floor of the 4th
ventricle at the region of SMO. H. & E. stain.
44
Ependymal cells line the region of SMO as a
double layer.
VIII
Figure 3.14 Coronal section at the region of the SMO
showing pan-cadherin TR internal negative 53
control.
IX
Abbreviations
Abbreviate Meaning
3V 3rd ventricle
4V 4th ventricle
Ab Antibody
AP Area postrema
C Degree centigrade
EC Ependymal cells
IR Infundibular recess
ME Median eminence
NH Neurohypophysis
Pi Pineal gland
SM Sulcus medianus
ZO Zonula occludens
XI
Chapter 1
Introduction &
literature review
Chapter 1 Introduction & literature review
1.1. Embryology of Ependymal Cells
In the rat brain, ependymal cells (ECs) derived from redial glial
neuroepithelial cells before day 10 of embryonic development and start to
proliferate after the neural plate formation (Korr, 1980, Ohata and Alvarez-
Buylla, 2016). The generation of ependyma proceeded along a caudal to rostra1
gradient beginning in the fourth ventricle and cerebral aqueduct on embryonic
day 14, the third ventricle on day 15 and the lateral ventricles on day 17. The
proliferation of ependyma in the rat brain continued until the end of gestation
reaching a peak on different days in different sites and then declining
(Mirzadeh et al., 2017).
In overall study of the origin and development of the lining of the rat
brain ventricles, Altman and Bayer (1978b) observed that ependyma was first
formed on day 16 of embryonic development but reported that it was not
completed in some regions until the second postnatal week. Whereas the bulk
of common ECs within the ventricle form on embryonic days 16-18, the
specialized tanycytic epithelium is generated mainly postnatal and succeeds the
already late development of neighboring nuclei of the hypophysiotropic zone.
Ventrally placed tanycytic epithelium of the hypophysiotropic area begins to
arise on embryonic day 19 and is produced mostly during the first postnatal
week (Altman and Bayer, 1978a).
2
Chapter 1 Introduction & literature review
epithelial cells (figure 1.1). However, there is no basal lamina. Instead, the
basal ends of EC are elongated and extend branching processes into the
adjacent neuropil area (Mescher, 2013, Del Bigio, 2010).
Figure 1.1 4th ventricles floor, ependymal cells are columnar or irregular
in shape with extend cilia and microvilli from the apical
surfaces into the ventricle while basal ends have extending
processes extend into the adjacent neuropil area. 100X. Stain:
H&E. E: ependymal cell, N: nueropil area, V: ventricle.
(Mescher, 2013)
ECs lie at the interface between the ventricular cavities and the brain
parenchyma. They are highly active metabolically and are involved in the
propulsion of the cerebrospinal fluid (CSF) through ventricular cavities.
Ependymal cells also take an active role in insulating the brain from potentially
harmful substances present in the CSF (Kuchler et al., 1994). It was proposed
that such ependymal cells may be involved in absorption, hormonal secretion
and transport between hypothalamic neurons and CSF of the third ventricle
(Bleier, 1971). Tanycytes, cuboidal and radial ependymal cells considered
three different subtypes of ECs have been report based on morphology
(Gurout et al., 2014).
3
Chapter 1 Introduction & literature review
1.2. Tanycytes
It has long been recognized that the ependymal lining of the ventricles of the
brain consist of morphologically different cell types (Gurout et al., 2014). In
spite of the fact that these investigators recognized that some ependymal cells
have long basal processes that prolonged into the substance of the brain, the
term tanycyte was not applied to these cells until the description of EC in
Selacians by Horstmann (1954), which described that the elongated bipolar
ependymal cells lining the infundibular recess of the third ventricle, which the
proximal pole of these cells in the ventricular wall and a distal pole contacting
the pituitary portal vessels. Because of their shape, Horstmann called these
cells tanycytes (from the Greek word tanus, elongated) (Mirzadeh et al.,
2017)
A special structural feature of tanycytes is that they have a single, long
basal process that project to discrete regions of the hypothalamic area. This
lead Lofgren (1959) to suggest, for the first time, that tanycytes may link CSF
to neuroendocrine events. Thus, it is possible that the ependyma in various
parts of the ventricular wall, especially the tanycyte ependyma, allows
communication between the CSF and subependymal neuropil through its
processes. Morphological studies of tanycytes have revealed close
relationships of the basal processes (shafts) to neural and vascular elements in
the adjacent nueropil (Rizzoti and Lovell-Badge, 2016, Rodrguez et al., 2005).
line the floor of third ventricle and their basal processes end on the ME portal
capillaries. According to ultrastructure; tanycytes has cylindrical cell
body, ovoid nucleus, with abundant euchromatin. The cytoplasmic region
contain the Golgi complex, less developed rough and smooth endoplasmic
reticulum, many mitochondria, polyribosomes, and all the endocytic
components; 1 tanycytes are cylindrical cells with elongated nuclei lying at
various levels, giving the region a stratified appearance. The cytoplasmic area
contain a welldeveloped Golgi apparatus, a few cisternae of the rough
endoplasmic reticulum, many polyribosomes, mitochondria, and endocytic
components. Apically, 1 tanycytes are joined by zonula adherens adhesion
5
Chapter 1 Introduction & literature review
junction and there are no zonula occludens tight junction; 2 tanycytes are
elongated cell body line the floor of third ventricle, microvilli projects from
cytoplasmic surface, specific ultrastructure features, they are apically joined
together by adhesion junction and tight junctions. According to functions thus,
(1, 2) tanycytes do not have barrier properties, whereas 1 tanycytes
establish a barrier between the ARH and the median eminence, and 2
tanycytes form a barrier between the CSF and the median eminence neuropil;
There are also important differences in the neurontanycyte relationships, (1,
2) tanycytes are innervated by peptidergic and aminergic neurons; at contrast,
(1, 2) tanycytes appear to lack innervation (Rodrguez et al., 2005, Rizzoti
and Lovell-Badge, 2016).
6
Chapter 1 Introduction & literature review
capillaries are lined by fenestrated endothelial cells indicative of a defective
bloodbrain barrier (BBB) to macromolecules (Paxinos, 2014).
In CVOs where exchange between the specialized neurons and the blood
needs to be maintained (neuropeptide secretion to blood; chemosensitivity to
monitor blood composition), the capillary endothelium is permeable, but
these regions do not form a leak across the BBB, by virtue of tight junctions
between specialized ECs in CVOs, and between the processes of tanycytes
and astrocytes glia that isolate the CVOs from brain parenchyma (Abbott,
2005).
The ECs framing the ventricular surfaces of the CVOs are distinctive
in appearance from the usual cuboidal ependyma covering whatever remains
of the ventricular surface. ECs of the CVOs can be columnar, elongated or
irregular in shape. They are either nonciliated, or have few cilia on their
luminal surface (McKinley et al., 2003).
8
Chapter 1 Introduction & literature review
The specialized ECs (tanycytes) of these brain areas, are connected by
tight junctions (TJs) between their apices, near their ventricular surface. The
hemal milieu of the perivascular spaces and the CSF-milieu of the ventricle
are here distinctly separated by the apical TJ of the tanycytes (Nakai et al.,
1977).
The TJ protein zonula occludens-1 (ZO-1) and epithelial cadherin (E-
cadherin) is expressed by ependymal as opposed to fenestrated endothelial
cells, suggesting that the barrier has been shifted from the vascular to the
ventricular side. Bloodcerebrospinal fluid barrier (BCSFB), composed of the
choroid plexus epithelial cells (ependymal cell) and the endothelial BBB,
composed of the highly specialized central nervous system (CNS)
microvascular endothelial cells. These barriers prevent paracellular diffusion
of destructive elements into the CNS by presence of highly complex TJ and
adherence junctions (AJs) of the BCSFB and BBB which are maintain brain
hemostasis (Wolburg and Paulus, 2010, Tietz and Engelhardt, 2015).
The ECs lining the OVLT have TJs close to their apical surfaces which
make them distinct from the cuboidal EC lining the rest of the ventricular
system, which lacks TJs and subsequently offers no barrier to the flow of
substances between the CSF and the brain parenchyma (McKinley et al.,
1987). The most noticeable morphological feature of the OVLT is that it has
a rich specialized vascular plexus which displays fenestrated endothelium
(Duvernoy and Risold, 2007). The activity of TJs between the specialized ECs
(tanycytes) at the ventricular surface, and between basal processes reaching
out to the boundaries of the OVLT is shown by the spread of blood-borne
substances, for example, horseradish peroxidase, through specific parts of the
OVLT but not into the encompassing neuropil or ventricular CSF (Krisch et
al., 1987).
The arterial blood to the fenestrated plexus of capillaries in the OVLT
most commonly arises from one or two horizontally directed arterioles, which
branch off the anterior communicating artery, and occasionally via an
12
Chapter 1 Introduction & literature review
arteriole that branches from an anterior cerebral artery, at the rostral margin
of the organ (Ambach et al., 1977).
There are previous data, showed that neurons and glial cells within the
OVLT respond with intracellular calcium signals to lipopolysaccharide,
tumor necrosis factor-, or interleukin-1 or interleukin 6 (Ott et al., 2010).
16
Chapter 1 Introduction & literature review
processes to the vascular walls, where they make contact through end feet
specializations (Paxinos, 2014).
Magnocellular neurons which give origin to the unmyelinated fibers of
this tract are from the supraoptic and paraventricular nuclei of the
hypothalamus; they contain vasopressin and oxytocin. In addition to that,
nerve terminals are available in the inner zone. Catecholaminergic and
peptidergic neurons in the brain stem or arcuate nucleus give rise to these
terminals (Paxinos, 2014).
Glial cells, ECs, and nerve terminals like that portrayed for the inner zone
are present in the outer zone, despite the fact that terminals are particularly
neurohemal. Essentially, there is a fenestrated vascular system called the
primary portal plexus that is in close relationship with a plenitude of terminals
containing neurohormones and releasing factors (Page, 1986). There are no
TJs in this vascular endothelium and consolidated with the fenestrations, the
hypophysiotropic neurosecretory products of the nerve terminals in the ME
quickly diffuse into the portal vessels supplying the anterior pituitary. These
vessels are found in the pars tuberalis that frames the ventral surface of the
outer zone (Norsted et al., 2008).
The arterial vessels that supply the posterior pituitary emerge completely
from the internal carotid artery. To the contrary, the anterior pituitary, does
not get a direct arterial blood supply but rather is perfused solely by long
portal vessels exuding from the primary portal plexus, and to a lesser degree
from short vessels arising from the posterior pituitary (Paxinos, 2014).
This is a basic feature of the structure and function of the
adenohypophysis, since it is by means of these portal vessels that
hypophysiotrophic hormones reach the adenohypophysis. This anatomical
structure, together with the absence of nerve terminals, drove early scientists
to recommend that the glandular pituitary is controlled by humoral factors
17
Chapter 1 Introduction & literature review
discharged into the region of the portal vasculature (Paxinos, 2014). Since
these early findings, significant accentuation has been set on characterizing
the nature and origins of the different hypophysiotrophic elements released
into the portal vessels, and at last in charge of the release of adenohypophysis
hormones, including thyroid stimulating hormone, follicular stimulating
hormone, prolactin, luteinizing hormone, adrenocorticotropic hormone and
growth hormone. Supraoptico-neurohypophysial tract fibers in inner zone are
derived from the supraoptic nucleus, and to a lesser degree from the
hypothalamic paraventricular nucleus and accessory neurons in the
hypothalamus. Other afferent sources of input which may contain
hypophysiotrophic factors, emerge from the parvocellular hypothalamic
paraventricular nucleus, arcuate nucleus, periventricular hypothalamus,
preoptic region, OVLT, diagonal band of Broca, brain stem, and medial
septum (Wiegand and Price, 1980). Immunohistochemical and tracing
studies, for instance, have located gonadotropin releasing hormone neurons
that project to the ME in the medial septum, preoptic area around the OVLT,
diagonal band of Broca, and lateral hypothalamus (Silverman et al., 1987).
This anatomical overlapping of areas supplying afferents to the ME with the
hypophysiotrophic factors, proposes the possible origins of such factors. For
instance, the diagonal band of Broca gives origin of thyrotropin releasing
factor, arcuate nucleus, medial septum, and hypothalamic paraventricular
nucleus (Paxinos, 2014). The arcuate nucleus produces dopamine, which has
an inhibitory impact on prolactin release, just like the cells of origin of
filaments in the outer lamina containing growth hormone releasing factor,
galanin, neurotensin, neurokinin B, dynorphin and kisspepetin (Krajewski et
al., 2010).
18
Chapter 1 Introduction & literature review
located in more lateral parts of the outer zone (Meister et al., 1989).
Somatostatin-containing cells distribution is widespread, however, those cells
that project specifically to the ME are present in the periventricular
hypothalamus (Paxinos, 2014).
19
Chapter 1 Introduction & literature review
parts: a superficial Pi at the brain surface, and a deep Pi near the 3rd ventricle.
The Pi does not contain neurons, yet has numerous secretory cells known as
pinealocytes in addition to other cell types such as interstitial cells that look
like glia, phagocytic cells situated in the perivascular spaces and possibly
peptidergic neuron-like cells (Moller and Baeres, 2002).
20
Chapter 1 Introduction & literature review
choroid plexus epithelium which secretes the specialized CSF into the
cerebral ventricles, and (3) the arachnoid epithelium separating the blood
from the subarachnoid CSF (Abbott, 2005, Serlin et al., 2015).
The BBB components include the endothelial cells layer and its
basement membrane, adjoined by tight cell-to-cell junction proteins with
specific transport mechanisms and pinocytic vesicles. The endothelium is
surrounded by cellular elements including pericytes and astroglial foot
processes, forming an additional continuous stratum that separates blood
vessels from brain tissue. Around penetrating vessels and venules there is
some distance between endothelial cells and brain tissue forming the
Virchow-Robin space in which perivascular macrophages, executing some of
the immune functions of CNS. The intimate contact between neurons,
astrocytes, microglia, pericytes and blood vessels, and the functional
interactions and signaling between them form a dynamic functional unit,
known as the neurovascular unit (Serlin et al., 2015).
The CNS is tightly sealed from the changeable milieu of blood by the
BBB and the BCSFB. While the BBB is considered to be localized at the level
of the endothelial cells within CNS microvessels, the BCSFB is established
by choroid plexus epithelial cells. The BBB inhibits the free paracellular
diffusion of water-soluble molecules by an elaborate network of complex TJs
that interconnects the endothelial cells (Engelhardt and Sorokin, 2009).
22
Chapter 1 Introduction & literature review
of the cilia and the microvilli over the surface of the ependyma. they also
reported the presence of intraventricular elements near this surface, on which
they are seen to lie as polymorphous protrusions, beaded supraependymal
fibers resembling axons and different types of supraependymal cells
(Alvarez-Morujo et al., 1992)
23
Chapter 1 Introduction & literature review
membranes and establishing an effective paracellular barrier to diffusion of
fluid and solutes. The cytosolic plaque proteins are responsible for organizing
and positioning of the AJC (Tsukita et al., 2001, Matter and Balda, 2003).
The ZO proteins ZO-1, ZO-2, and ZO-3A are a vital group of TJs
molecules. These proteins belongs to the membrane-associated guanylate
24
Chapter 1 Introduction & literature review
kinase-like homologs (MAGUK) family and are portrayed by three N-
terminal PDZ domains, a SH3 and a GUK domain. These proteins can interact
specifically with occludin and claudins by means of their PDZ domains,
though their C-end can relate to actin, in this way giving an immediate
connection the cytoskeleton (Schneeberger and Lynch, 2004).
26
Chapter 1 Introduction & literature review
2003). Inactivation of different cadherins in a variety of organisms have
shown their importance in tissue morphogenesis (Halbleib and Nelson, 2006).
27
Chapter 1 Introduction & literature review
The main application of fluorescence histochemistry are auto-
fluorescence (a substance that shows fluorescence without any chemical
treatment or staining such as lipofuscin, vitamin A and porphyrin), induced-
fluorescence (tissue substances are converted into fluorophore by special
chemical treatment), fluorochromy (use of fluorescent dyes, such as acridine
orange, eosin, fluorscene isothiocynate (FITC), thioflavin and tetra-methyl-
rhodamin (TRITC), immuno-fluorescence and marker labeling with
fluorescent material (FITC and TRITC) and enzyme-produced fluorescence.
Fluorescence histochemistry can detect fine amounts of substances.
Fluorescence can also be used in quantitative histochemistry (fluorometry).
The main disadvantage are fading of fluorescence (Al-Salihi, 2011, Ferenk,
2012).
28
Chapter 1 Introduction & literature review
1.8. Aims of the Study
This study aims at investigating for the presence of tanycytes or tanycyte-like
cells in the region of the SMO by using general histological staining and
immunofluorescence labeling were target to achieve the following aims:
29
Chapter 2
Materials & Methods
Chapter 2 Materials & Methods
2.1. Animals and Housing
A sample of 10 adult male rats (Rattus Norvegicus Albinus) aged 3-6 months
were taken from the animal house of the High Institute for Infertility
Diagnosis and Assisted Reproductive Technologies Al-Nahrain University
during the years 2015-2016 on basis of being apparently active and healthy,
with 300 50 g body weight. Animals were kept on fresh trefoil diet for 2
weeks prior to dissection.
Table 2.1: Equipment used in research
Equipment Company
Hot oven Heraeus
An electric wax dispenser Lipshaw, Model No. 222
Rotary electric microtome Richert-Jung, 2030 MOT Biocut
Light microscope Polivar
Fluorescent microscope AXIO-ZEISS
2 Micropipette (10l + 100l )+tips (100l) GILSON
Materials Company
Formaldehyde 40% USA Fisher scientific
Distilled water Our department
Ethyl alcohol Solvochem Netherlands
Xylene Solvochem Netherlands
Paraffin wax Germany MEDITE PURE Paraffin
Harris modified hematoxylin Riedel-de Haen
Eosin-Y Fisher scientific
Mounding Medium (DPX) SYRBIO
UltraCruz mounting medium (DAPI) Santa Cruz (catalog # SC-24941)
Positive charge slides BIOCARE MEDICAL
Disposable microtome blades ERMA INC.
31
Chapter 2 Materials & Methods
2.2. Collection of Samples
Animal euthanized with chloroform impregnated cotton wool in an air tight
chamber for 5 minutes. Then, animals were pinned on a dissection board in
the prone position and the skull was opened dorsally by a strong pair of
scissors starting from the foramen magnum to the nasal bones. The parietal
and temporal bones had to be cut in order to expose the entire contents of the
skull. The cranial nerves were severed and the brain was delivered in one
piece. Next, the brain was fixed in 10% neutral buffer formalin (NBF) for 36
hours (according to trials study) preparation for dissection into two part under
dissecting lens, one part contained the ME and the other part included the
cerebellum, 4th ventricle and SMO.
ME SMO
specimen specimen
Figure 2.1 Sagittal scheme represent the brain, ME specimen extend from
optic chiasm anteriorly to rostral part of brain stem posteriorly.
SMO specimen extend anteriorly from anterior surface of
cerebellum and pons to rostral part of spinal cord posteriorly.
ME: median eminence, SMO: sulcus medianus organum, AP,
area postrema, NL: neurohypophysis, OVLT: organum
vasculosum lamina terminals, SFO: subfornical organ, PI: pineal
gland, SCO: subcommissural organ
32
Chapter 2 Materials & Methods
Table 2.3: Chemicals used in research with its formula.
Chemicals Formula
Sodium phosphate monobasic monohydrate
(BDH AnalaR) NaH2PO4.H2O
2.3.1. Fixation
Tissue samples were fixed for 36 hour at room temperature with 10% NBF
(trials optimization) which was prepared according to (Bancroft and Gamble,
2008) by mixing the following:
2.3.2. Dehydration
Tissue was transferred into increasing concentrations of ethyl alcohol
(Solvochem) as follows (according to trials optimization):
33
Chapter 2 Materials & Methods
70 % ethanol for 24 hours
90 % ethanol for 6 hours
100 % ethanol for 2 hours
2.3.3. Clearing
Specimen were cleared with two exchanges of xylene (Solvochem) each for
20 minutes to ensure good tissue transparency (according to trials
optimization).
2.3.5. Sectioning
Tissue blocks were mounted on the holder of an electric microtome (Richert-
Jung, 2030 MOT Biocut). Sections of 5 m thickness from the ME and SMO
were cut and floated on 45 C water bath for 20 seconds before attaching the
sections to slides (1-2 sections per -slide). Two sets of slides were made: one
for staining with hematoxylin & eosin by using normal glass slides and the
other for immunohistochemistry by using positive charge slides. These slides
are incubate for 24 hours in 40-42 C.
2.3.6. Dewaxing
Slides were transferred into 60 C hot air oven for about 15 minutes, left to
cool down for 15 minutes (to liquefy the paraffin in tissue and increase the
34
Chapter 2 Materials & Methods
tissue section attachment) then dewaxed in two exchanges of xylene, each for
5 minutes (according to trials optimization).
2.3.7. Hydration
This was done with decreasing concentrations of ethyl alcohol (100%,
90%, 70%, and distal water) 3 minutes for each (according to trials
optimization).
2.4. Staining:
2.4.1. Hematoxylin and Eosin (H&E)
Harris alum hematoxylin (Riedel-de Haen) and Eosin-Y (Fisher
scientific) were prepared according to (Bancroft and Gamble, 2008) with
trials optimization modification: staining and preparation procedure was as
follows:
2- Slides were stained with eosin for 1 minutes. (Dye solution prepared by
0.5 g of eosin-Y dissolved in 20% ethyl alcohols with 0.5 ml acetic acid).
Slide were put in a coplin jar containing sodium citrate buffer (antigen
retrieval solution) which was prepared according to (Taylor et al., 2006, and
Renshaw, 2007): and as follows:
Solution A: Citric acid monohydrate (C6H8O7.H2O or C6H10O8) 10.505 g in
500 ml of distilled water (0.1 M)
9 mL of solution A
Mixed in 500 mL of distilled water and the pH was
adjusted to 6
41 mL of solution B
Then the coplin jar transferred to an autoclave (120 C, 1.2 Bar) for 30
minutes and left to cool down to room temperature for 20 minutes (according
to trials optimization). Slides were washed in running tap water for 5 minutes,
distilled water for 5 minutes, Tris buffer-Tween 20 (pH 8) for 5 minutes and
finally rinsed 3 time with Tris buffer (pH 8).
Antibody Company
2.4.3.3. Controls
Negative internal control slides were prepared from the regions of the SMO
and ME, while negative external controls were obtained from kidney sections.
The same staining procedure was performed except for the primary antibody.
38
Chapter 3
Results
39
Chapter 3 Results
3.1. General Morphology with H. & E. Staining
3.1.1. Median Eminence
The ME, located at the ventral side of the 3 rd ventricle, was lined with a
single layer of ependymal cells similar to those lining the cerebral
ventricles elsewhere. The nuclei of these cells stained blue-violate in color
while their cytoplasm stained pink. No specific features could be identified
with H. & E. staining to mark these EC as tanycytes (figures 3.1-2).
Figure 3.1 Ependymal cells lining wall and floor of 3rd ventricle in rat.
Coronal sections. H. & E. stain. (A) 40X. (B) 100X. 3V: 3rd
ventricle. LV: lateral ventricle. ME: median eminence. ARH:
arcuate hypothalamic nucleus.
40
Chapter 3 Results
Figure 3.2 Ependymal lining of the 3rd ventricular walls and floor
(arrows). Ependymal cells appear to be squamous to irregular
shape, lack to cilia. H. & E. stain. 400X. 3V: 3rd ventricle. ME:
median eminence. ARH: arcuate hypothalamic nucleus.
41
Chapter 3 Results
3.1.2. Sulcus Medianus Organum
The SMO was seen as a median longitudinal cleft in rostral part of
the floor of 4th ventricle. This furrow was lined with a single layer of EC
except at the deepest part of the furrow where a double layer of these cells
were observed. Their nuclei stained blue violate and the cytoplasm stained
pink with H. & E. stain (figures 3.3-6).
Figure 3.3 Sagittal section through the 4th ventricle (4V) and SMO
(arrows). H. & E. stain. 40X.
42
Chapter 3 Results
B
Cerebellum
Medulla Oblongata
Figure 3.4 Coronal section of the floor of the 4th ventricle (4V) at the
region of SMO (arrow). H. & E. stain. (A) 40X. (B) 100X.
43
Chapter 3 Results
AA
Figure 3.5 Coronal section of the floor of the 4th ventricle (4V) at the
region of SMO. H. & E. stain. (A) 400X. (B) 1250X. (A)
Ependymal cells (arrows) line the region of SMO with a single
layer which appear cuboidal in shape and have cilia in
ventricular surface (head arrows). (B)Which are double layer
features could be identified to mark these ependymal cells as
tanycytes.
44
Chapter 3 Results
Figure 3.6 Coronal section at the region of SMO. The deepest part of the
sulcus medianus shows 2-3 layers of cells which appear as
ciliated pseudostratified cuboidal epithelium (arrows). Cilia
present in the ventricular surface (head arrows). H. & E.
stain. 1250X. SMO: sulcus medianus organum.
45
Chapter 3 Results
3.2 Immunofluorescence Labeling
3.2.1 Median Eminence
3.2.1.1 Adhesion Junctions
Pan-cadherin immunolabeling marked the 3rd ventricle walls and
floor where ME located as a continuous red fluorescent line (figure 3.7).
Higher magnifications showed the AJs to surround the EC between their
borders and on their apical surfaces (figure 3.8). An internal negative
control is seen in (figure 3.9).
Figure 3.7 Coronal section at the 3rd ventricle. Pan-cadherin labeling for
AJs is seen as a continuous red line along the ependymal lining
of the 3rd ventricle (arrows). The section was also labeled with
DAPI nuclear counterstaining (blue). 3V: 3rd ventricle. 200X.
TRITC filter (619 nm) and DAPI filter (435 nm). The TRITC
and DAPI images were superimposed by Image-J program
46
Chapter 3 Results
Figure 3.8 Higher magnification of the inset shown in figure 3.7. AJs
between ependymal cells of the 3rd ventricular wall are seen as red
fluorescence between the cells and on their surfaces (arrows).
DAPI nuclear counterstaining shows cells nuclei as bright blue
fluorescence. 3V: 3rd ventricle. 400X. TRITC filter (619 nm) and
DAPI filter (435 nm). The TRITC and DAPI images were
superimposed by Image-J program
47
Chapter 3 Results
Figure 3.9 Coronal section at the region of the 3rd ventricle showing pan-
cadherin TR internal negative control. No labeling is seen along
the ependymal lining of the 3rd ventricle. DAPI nuclear
counterstaining is seen as bright blue fluorescence along the
nuclei of 3rd ventricle ependyma (arrows). 3V: 3rd ventricle.
400X. TRITC filter (619 nm) and DAPI filter (435 nm). The
TRITC and DAPI images were superimposed by Image-J
program.
48
Chapter 3 Results
3.2.1.2. Tight Junctions
ZO1 immunolabeling was seen as green fluorescence surrounding
tanycytes of 3rd ventricular floor where the ME located. This labeling of
TJs was clearly seen in a diffuse rather than a honeycomb pattern on the
apical surface and lateral borders of these tanycytes (figures 3.10). An
internal negative control is seen in (figure 3.11).
ME
Figure 3.10 Coronal section at the wall (ARH) and floor (ME) of 3rd
ventricle showing TJs as green fluorescence around tanycytes
(arrows). DAPI nuclear counterstaining appears as blue
fluorescence.3V: 3rd ventricle. ARH: arcuate hypothalamic
nucleus, ME: median eminence. 400X. FITC filter (519 nm)
and DAPI filter (435 nm). The FITC and DAPI images were
superimposed by Image-J program
49
Chapter 3 Results
ME
Figure 3.11 Coronal section at the region of the wall (ARH) and floor
(ME) of 3rd ventricle showing ZO1 FITC internal negative
control. No labeling is seen along tanycytes, ependymal lining
of the 3rd ventricle. DAPI nuclear counterstaining is seen as
bright blue fluorescence along the nuclei of 3rd ventricle
ependyma (arrows). 3V: 3rd ventricle, ARH: arcuate
hypothalamic nucleus, ME: median eminence. 400X. FITC
filter (519 nm) and DAPI filter (435 nm). The FITC and DAPI
images were superimposed by Image-J program.
50
Chapter 3 Results
3.2.2. Sulcus Medianus Organum
3.2.2.1. Adhesion Junctions
Pan-cadherin immunolabeling marked the floor of the 4 th ventricle
at the region of the SMO as a continuous red fluorescent line (figure 3.12).
Higher magnifications showed the AJs surround the ECs between their
borders and on their apical surfaces (figure 3.13). An internal negative
control is seen in figure (3.14). Pan-cadherin of AJs present along all of the
ECs lining the floor and roof of the 4th ventricle (figure 3.15).
Medulla Oblongata
Figure 3.12 Coronal section at the SMO. Pan-cadherin labeling for AJs is
seen as a continuous red line along the ependymal lining of the
4th ventricle (arrows). The section was also labeled with DAPI
nuclear counterstaining (blue). 4V: 4th ventricle. 200X. TRITC
filter (619 nm) and DAPI filter (435 nm). The TRITC and DAPI
images were superimposed by Image-J program
51
Chapter 3 Results
Figure 3.13 Higher magnification of the inset shown in figure 3.12. AJs
between ependymal cells of the SMO are seen as red
fluorescence between the cells and on their surfaces (arrows).
DAPI nuclear counterstaining shows cells nuclei as bright blue
fluorescence. 4V: 4th ventricle. 400X. TRITC filter (619 nm)
and DAPI filter (435 nm). The TRITC and DAPI images were
superimposed by Image-J program
52
Chapter 3 Results
Figure 3.14 Coronal section at the region of the SMO showing pan-cadherin
TR internal negative control. No labeling is seen along the
ependymal lining of the 4th ventricle. DAPI nuclear
counterstaining is seen as bright blue fluorescence along the
nuclei of 3rd ventricle ependyma (arrows). 4V: 4th ventricle.
200X. TRITC filter (619 nm) and DAPI filter (435 nm).
4V
Figure 3.15 Coronal section at the region of the SMO showing pan-cadherin
TR along the ependymal lining the floor and roof of 4th ventricle
(arrows). DAPI nuclear counterstaining shows cells nuclei as
bright blue fluorescence. 4V: 4th ventricle. 200X. TRITC filter
(619 nm) and DAPI filter (435 nm). The TRITC and DAPI
images were superimposed by Image-J program.
53
Chapter 3 Results
3.2.2.2. Tight Junctions
Findings of TJs labeling in ependyma of the SMO were similar to
those seen in tanycytes of the ME. This labeling of TJs was clearly seen in
a diffuse rather than a honeycomb pattern on the apical surface and lateral
borders of these tanycytes (figures 3.16-17). An internal negative control
is seen in (figure 3.18).
4V
54
Chapter 3 Results
AA
B B
Figure 3.17 Higher magnifications of the inset shown in figure 3.16. TJs
between tanycytes of the SMO are seen as green fluorescence
between the cells and on their apical surfaces (arrows). DAPI
nuclear counterstaining shows cells nuclei as bright blue
fluorescence. 4V: 4th ventricle. (A) 400X (B) 630X. FITC filter
(519 nm) and DAPI filter (435 nm). The FITC and DAPI images
were superimposed by Image-J program.
55
Chapter 3 Results
Figure 3.18 Coronal section at the region of the SMO showing ZO1 FITC
internal negative control. No labeling is seen along tanycytes
of the 4th ventricle ependymal lining. DAPI nuclear
counterstaining is seen as bright blue fluorescence along the
nuclei of 4th ventricle ependyma (arrows). 4V: 4th ventricle.
200X. FITC filter (519 nm) and DAPI filter (435 nm). The
FITC and DAPI images were superimposed by Image-J
program.
56
Chapter 3 Results
3.2.3. Controls
3.2.3.1. Pan-cadherin TR Antibodies
Positive controls of AJs between cells of renal tubules were labeled
with pan-cadherin antibodies (figure 3.19). Negative controls were
prepared by following the same immunohistochemical labeling procedure
except for the omission of the primary antibodies (figure 3.20).
57
Chapter 3 Results
58
Chapter 3 Results
3.2.3.2. ZO1-FITC Antibodies
Positive controls of TJs between cells of renal tubules were labeled
with ZO1 antibodies (figure 3.21). Negative controls were prepared by
following the same immunohistochemical labeling procedure except for
the omission of the primary antibodies (figure 3.22).
Figure 3.21 ZO1 positive control in kidney section. Renal tubules show
intense green immunofluorescence at TJs between their cells.
DAPI nuclear counterstaining is seen as bright blue
fluorescence at cell nuclei. 200X. FITC filter (519 nm) and
DAPI filter (435 nm). The FITC and DAPI images were
emerged by superimposed program.
.
59
Chapter 3 Results
Figure 3.22 ZO1 negative control in kidney section. Renal tubules do not
show TJs immunofluorescent labeling between their cells.
DAPI nuclear counterstaining is seen as bright blue
fluorescence at cell nuclei. 200X. FITC filter (519 nm) and
DAPI filter (435 nm). The FITC and DAPI images were
superimposed by Image-J program.
60
Chapter 4
Discussion
Chapter 4 Discussion
4.1. Morphological Considerations with H. & E. Stain
4.1.1. Median Eminence
The neurohypophysis is organized into three parts, the ME (infundibulum),
the infundibular stem, and the neural lobe (infundibular process). The ME
shows a characteristic organization of glial and nervous elements arranged in
three layers: ependymal cells lining the infundibular recess, an internal layer,
and an external layer. Nerve terminals of the neurohypophysis, ME and neural
lobe, release hypothalamic hormones that are carried via blood vessels
(hypothalamic portal system) to adenohypophysial cells or to peripheral target
organs. In addition, the neurohypophysis contains tanycytes and pituicytes
which are specialized glial cells besides nerve terminals and vessels
(Wittkowski, 1998, Paxinos, 2014).
The inner layer (or zone) of the ME is just adjacent to the ependyma
and comprises the subependymal glial cells, pituicytes, the radially arranged
tanycytic processes, and most significantly, filaments of the supraoptico-
neurohypophysial tract that are on the way to the neurohypophysis (Daniel
and Pritchard, 1975, Page, 1986).
62
Chapter 4 Discussion
ventricular wall and a distal pole contacting the portal vessels. Because of
their shape, Horstmann called these cells tanycytes.
63
Chapter 4 Discussion
4.1.2. Sulcus Medianus Organum
The sulcus medianus (SM), passes from the caudal end of the midbrain
(mesencephalic) cerebral aqueduct to the inferior part of rhomboid fossa of
4th ventricle (calamus scriptorius). The sulcus medianus of the 4 th ventricle
was found to contain an overlooked CVO (Collins, 1989).
In the most coudal part of the SM lies the AP which is one of the well
known CVOs. The AP is bilateral eminences on either side of the 4th ventricle.
It is located at the caudal end of the calamus scriptorius in the dorso-medial
medulla oblongata (figure 1.3). In many species it lies at the beginning of the
central canal. In rat, these eminences have blended so that in coronal sections
the AP shows up as an area of tissue dorsal to the nucleus of the tractus
solitarius (Wilson, 1906). In the work of Langlet et al. (2013) on AP
64
Chapter 4 Discussion
parenchyma, vimentin immunolabeling uncovered long and slim strands
stretching out from the ECs bodies towards MECA 32-positive fenestrated
vessels, where they shaped a thick network encompassing these vessels.
These outcomes propose that the ventricular lining of the AP are framed by
process-bearing ECs that connect the ventricular and fenestrated vessels
compartments, as portrayed in the ME. Researchers, however, did not
elaborate on the rostral part of the SM where little attention is paid for the
presence of the SMO as one of the CVOs.
Collins (1989) noticed in her study that cells in SMO when examined
under electron microscope were ciliated with predominant microvilli. The
apical surface of each cell, instead, had characteristic microvillous projections
with pinocytotic vesicles. In addition, cell bodies were found to be elongated,
with basal process. In such context, the general morphology of the cells in the
SMO was similar to that of tanycytes found in other CVOs. In our work,
however, we could not decide whether such observations were true for the
cells under study since they were examined with light microscope.
65
Chapter 4 Discussion
4.3. Immunohistochemical Characterization of Junctional
Complexes at Tanycytes
Previous works studied the presence of tanycyte-like cells in the CVOs by
analyzing the morphology of vimentin-positive ependymal cells covering
their ventricular walls, and the relationship of these cells with fenestrated
vessels. Strong vimentin immunoreactivity was seen all through the coating
of the 3rd and 4th ventricles, including the ependymal cells flanking the CVOs.
The portrayal of these tanycyte-like cells was reached out by an investigation
of tight junctions along the ventricular walls of the CVOs (Langlet et al.,
2013a&b).
Mullier et al., (2010) and Abbott et al. (2010) studied the expression
and distribution of important three tight junctional proteins which have
essential functional roles in CNS diffusion barriers. These proteins are the
ZO-1, occludin, and claudin1. ZO-1 and occludin immunolabeling was
detected at the cells that line the 3rd ventricle and the AP of the 4th ventricle,
in other words, those specialized ECs forming the ventricular walls of the
CVOs (Langlet et al., 2013b).
66
Chapter 4 Discussion
rich capillary plexus with a fenestrated endothelium (Schulz and Engelhardt,
2005).
ECs have well-developed AJs at the apicallateral interface. Cadherins
are transmembrane cellcell adhesion molecules conserved among all
organisms, which play essential roles in tissue morphogenesis and
homeostasis (Del Bigio, 2010). In this study, AJs were seen along all
ependymal cells lining the 3rd and 4th ventricles (figures 3.7 and 3.15).
The work of Collins (1989), and before that Cummings & Felten (1979)
revealed the present of tanycyte-like cells in the floor of 4th ventricle,
suggesting that the SMO is another CVO. However, immunohistochemical
methods were not used to characterize cell population in that region. In this
study we aimed to label ECs in the MS with anti-ZO1 and anti-pan-cadherin
antibodies to explore the junctional complexes between these cells.
68
Chapter 4 Discussion
to the examining eye. This might reflect our observation with H. & E. staining
that ependymal cells in the depth of the furrow were arranged in more than
one layer, leading to emission of more fluorescence than the single-layered
ependyma of the 4th ventricle.
69
Chapter 4 Discussion
Raphe nuclei are important areas for the tonic and reflex control of
arterial blood pressure (Dampney et al., 1982) as arterial blood pressure
depends upon the activity of medullary and pontine vasomotor centers
(Uvnas, 1975). In these regions, corresponding to areas that contain
serotonergic and noradrenergic cell bodies and axon terminals, alteration of
monoamines can produce modifications in blood pressure (Fuxe, 1965).
71
Chapter 5
Conclusions &
Recommendations
Chapter 5 Conclusions & Recommendations
5.1. Conclusions
2. These tanycytes are arrange in more than one layer in the depth of the
MS at the region of the SMO.
5. The SMO is another CVO in the 4th ventricle at a strategic location near
the brainstem nuclei, to link CVOs on both sides of the cerebral
aqueduct.
73
Chapter 5 Conclusions & Recommendations
5.2. Recommendations:
74
Chapter 6
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78
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80
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84
.
.
.
) (ZO1
) (Pan-Cadherin
( ) .
.
( )
.
.
) (ZO1-FITC
( )
.
) (Pan-Cadherin-TRITC
.
.
.
/