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Republic of Iraq

Ministry of Higher Education and


Scientific Research
Al-Nahrain University
College of Medicine

Expression of ZO-1 and Pan-Cadherin at


Tanycytes of Sulcus Medianus Organum in Rats

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

* Al-Nahrain University College of Medicine Department of Human


Anatomy Iraq
Abstract
Tanycytes are considered as highly specialized ependymal cells that
line the circumventricular organs. And are connected by zonula-occludens
tight junctions between their apices, near the ventricular surface, to form a
bloodcerebrospinal fluid barrier at the regions of these organs, thus
connecting the cerebrospinal fluid to neuroendocrine event.

This study investigated the presence of zonula occludens 1 (ZO1)


tight junctions proteins and pan-cadherin adhesion junctions proteins
between ependymal cells of the sulcus medianus organum in the floor of the
4th ventricle and the median eminence of the hypothalamus, which is a one
well known circumventricular organs in the brain.

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.

General histological examination of cells in the sulcus medianus


organum and median eminence showed linings of ependymal cells similar
to those seen elsewhere in the brain ventricles. These ependymal cells,
however, were arranged in 2-3 layers in the depth of the median sulcus at
the sulcus medianus organum region. These results were compared with
positive internal and external controls: median eminence and kidney tissue,
respectively.

Immunofluorescent labelling of ependymal cells with anti-ZO1


antibodies revealed the presence of tight junctions between ependymal cells

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.

We concluded that the sulcus medianus organum contained tanycytes


or tanycyte-like cells that exhibited tight junctional complexes. This unique
cell population added a strong evidence for the presence of a
circumventricular organ in the rostral part of the median sulcus, networking
the circumventricular organs on both sides of the cerebral aqueduct.

II
Contents

Abstract I

List of Tables VI

List of Figures VII

Abbreviations X

Chapter one: Introduction


1.1. Embryology of Ependymal Cells 2
1.2. Histology of Ependymal Cells 2
1.2.1. Tanycytes 4
1.3. Circumventricular Organs 6
1.3.1. Subfornical Organ (SFO) 9
1.3.1.1. Afferent Neural Connections of SFO 10
1.3.1.2. Efferent Neural Connections of SFO 11
1.3.1.3. Neural Functions of SFO 11
1.3.2. Vascular Organ of Lamina Terminals (OVLT) 12
1.3.2.1. Afferent Neural Connections of OVLT 13
1.3.2.2. Efferent Neural Connections of OVLT 13
1.3.2.3. Neural Functions of OVLT 13
1.3.3. Area Postrema 14

1.3.3.1. Afferent Neural Connections of AP 15


1.3.3.2. Efferent Neural Connections of AP 15
1.3.3.3. Neural Function of AP 15
1.3.4. Median Eminence and Neurohypophysis 16
1.3.5. Pineal Gland 19
1.4. Blood Brain Barrier and Blood Cerebrospinal Fluid Barrier 20
1.5. Sulcus Medianus of the 4th Ventricle 22

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

Chapter Two: Materials & Methods


2.1. Animals and Housing 31
2.2. Collection of Sample 32
2.3. Preparation of Paraffin Sections 33
2.3.1. Fixation 33
2.3.2. Dehydration 33
2.3.3. Clearing 34
2.3.4. Paraffin Infiltration and Embedding 34
2.3.5. Sectioning 34
2.3.6. Dewaxing 34
2.3.7. Hydration 35
2.4. Staining 35
2.4.1. Hematoxylin and Eosin (H&E) 35
2.4.2. Immunohistochemical Staining 36
2.4.2.1. Antigen Retrieval 36
2.4.2.2. Immunofluorescent staining 36
2.4.2.3. Controls 37
2.5. Examination of Slides 37

Chapter Three: Results


3.1. General Morphology with H. & E. Staining 40
3.1.1. Median Eminence 40
3.1.2. Sulcus Medianus Organum 42
3.2. Immunofluorescence Labeling 46
3.2.1. Median Eminence 46

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

Chapter Four: Discussion


4.1. Morphological Considerations with H. & E. Stain 62
4.1.1. Median Eminence (ME) 62
4.1.2. Sulcus Medianus Organum (SMO) 64
Immunohistochemical Characterization of Junctional
4.2. 66
Complexes at Tanycytes
Inspection of Immunofluorescence Reaction in the Median
4.2.1. 66
Eminence
Immunofluorescence Labeling of Junctional Complexes in
4.2.2. 67
the Region of the Sulcus Medianus Organum
4.3. Tanycytes of the Fourth Ventricle 69

Chapter Five: Conclusions & Recommendations


5.1. Conclusions 73
5.2. Recommendations 74

Chapter Six: References


6.1. Reference 76

V
List of Tables

Table Page

Table 2.1 Equipment used in research 31

Table 2.2 Materials used in research 31

Table 2.3 Chemicals used in research with its formula. 33

Table 2.4 Antibodies used in immunoflourscent staining 37

VI
List of Figures

Figure Page

Figure 1.1 floor of 4th ventricles showing shape of


ependymal cells and related structure 3

Figure 1.2 3rd ventricle area showing locations of tanycytes 5


types.

Figure 1.3 Location of the circumventricular organs


shown on a sagittal section through 3rd and 4th 8
ventricles of the rat brain.

Figure 1.4 Scheme of the types of tight junctions and 25


adhesion junctions proteins.

Figure 1.5 Scheme of the adhesion junctions, gap


26
junction and desmosome.

Figure 2.1 Sagittal scheme of brain, showing region of


ME specimen and region of SMO specimen 32

Figure 3.1 EC lining ME in rat. Coronal sections. H. & 40


E. stain.

Figure 3.2 Ependymal lining of the 3rd ventricle walls 41


(arrows). H. & E. stain.

Figure 3.3 Sagittal section through the 4th ventricle and


42
SMO. H. & E. stain.

Figure 3.4 Coronal section of the floor of the 4th


ventricle at the region of SMO. H. & E. stain. 43

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.

Figure 3.6 Coronal section at the region of SMO. The


median sulcus is lined with a single layer of
ependymal cells. The deepest part of the 45
sulcus shows 2-3 layers of ependymal cells.
H. & E stain.

Figure 3.7 Coronal section at the ME showing pan-


46
cadherin labeling for AJs.

Figure 3.8 Higher magnification of the inset shown in


figure 3.7 showing AJs between ependymal 47
cells of the ME.

Figure 3.9 Coronal section at the region of the ME


showing pan-cadherin TR internal negative 48
control.

Figure 3.10 Coronal section at the ME showing ZO1


49
labeling for TJs between tanycytes.

Figure 3.11 Coronal section at the region of the ME


50
showing ZO1 FITC internal negative control.

Figure 3.12 Coronal section at the SMO showing pan-


51
cadherin labeling for AJs.

Figure 3.13 Higher magnification of the inset shown in


figure 3.14 showing AJs between ependymal 52
cells of the SMO.

VIII
Figure 3.14 Coronal section at the region of the SMO
showing pan-cadherin TR internal negative 53
control.

Figure 3.15 Coronal section at the region of the SMO


showing pan-cadherin TR along the
53
ependyma, lining the floor and roof of 4th
ventricle.

Figure 3.16 Coronal section showing TJs as green


fluorescence around tanycytes of the SMO.
54
TJs are seen on the apical surface and borders
of these cells.

Figure 3.17 Higher magnifications of the inset seen in


figure 3.18 showing TJs between tanycytes 55
of the SMO.

Figure 3.18 Coronal section at the region of the SMO


showing ZO1 FITC internal negative 56
control.

Figure 3.19 Pan-cadherin positive control in kidney


57
section.

Figure 3.20 Pan-cadherin negative control in kidney


58
section.

Figure 3.21 ZO1 positive control in kidney section. 59

Figure 3.22 ZO1 negative control in kidney section. 60

IX
Abbreviations

Abbreviate Meaning

3V 3rd ventricle

4V 4th ventricle

Ab Antibody

Ad2 Adrenergic Neurons 2

AP Area postrema

AJs Adherence junctions

AJC Apical junctional complex

NA2 Noradrenergic Neurons 2

ARH Arcuate hypothalamic nucleus

BBB Bloodbrain barrier

BCSFB Bloodcerebrospinal fluid barrier

C Degree centigrade

CCK-1 Cholecystokinin receptor


CNS Central nervous system

CSF Cerebrospinal fluid

CVO Circumventricular organ

DAPI 4',6-Diamidino-2-Phenylindole, Dihydrochloride

DPX Dibutyl phthalate and xylene

EC Ependymal cells

E-cadherin Epithelial cadherin

FITC Fluorscene isothiocynate

GABA Gamma amino butyric acid

GLP-1 Glucagon-like peptide-1 neuropeptide


X
Ghrelin-GHSR Growth hormone secretagogue receptor

H&E Hematoxylin and eosin

IR Infundibular recess

JAM Junctional adhesion molecule

ME Median eminence

MECA 32 Mouse Endothelial Cells Antibody

MAGUK Membrane-associated guanylate kinase

NH Neurohypophysis

NBF Neutral buffer formalin

NK-1 neurokinin receptor


OVLT Organum Vasculosum Lamina Terminalis

Pi Pineal gland

SFO Subfornical organ

SM Sulcus medianus

SMO Sulcus medianus organum

SEM Scanning electron microscope

SCO Subcommissural organ

TRITC Tetra-methyl-rhodamin isothiocynate

TEM Transmission electron microscope

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).

In a more recent study also reported that genesis of tanycytic ependyma


in the rat ventricle began on the late gestation period of embryonic life and the
differentiation of these cells terminate postnatal (Rizzoti and Lovell-Badge,
2016).

1.2. Histology of Ependymal Cells


The common ECs are cuboidal or columnar in shape and have numerous cilia
in apical surface joined apically by junctional complexes similar to those of

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).

Tanycytes are originate in the infundibulum, from neuroepithelial glial


cells through the last 2 days of gestation and the first postnatal days, make their
full differentiation through the first month of life (Rizzoti and Lovell-Badge,
2016).
Tanycytes that line the third ventricle do not comprise a homogenous cell
population; different types of tanycytes have been described based on their
location, morphology, cytochemistry, ultrastructure, and function. So, there are
4
Chapter 1 Introduction & literature review
four types of tanycytes, (1, 2)
and (1, 2) can be noted. These
subtypes differentiate according
to Location; such as 1
tanycytes line the area of the
ventromedial and dorsomedial
hypothalamic nucleus, and
project their basal processes to
these nuclei; 2 tanycytes line
the arcuate hypothalamic
nucleus (ARH) area and project
their processes within this
nucleus; 1 tanycytes line the
lateral side of the third ventricle
Figure 1.2 Area of 3rd ventricle. Showing
and project their processes to the locations of (1, 2) and (1, 2) tanycytes.
perivascular space of the portal (1, 2) tanycytes are strongly reactive
throughout the cell processes. CE, ciliated
capillaries located in the lateral ependyma. -catenin Immunocytochemistry
region of the ME; 2 tanycytes X85 (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).

Tanycytes are considers specialized ependymal cells, which connected


by tight junctions, form a complex network that seal the CNS from the
circumventricular organs (CVOs), creating a distinct bloodCSF barrier which
have been reported on the floor of the fourth ventricle as well as on the walls
and floor of the third ventricle (figure 1.1-2). These cells are reported in the
CVOs by examining the morphology of vimentin positive ECs lining their walls
of ventricle, and the association of these cells with fenestrated capillaries.
Immunoreactivity of vimentin was seen throughout the lining of the third and
fourth ventricles, including the ECs bordering the CVOs. (Mullier et al., 2010,
Langlet et al., 2013b, Mirzadeh et al., 2017).

1.3. Circumventricular Organs


Adjacent to the median ventricular cavities (third ventricle, cerebral aqueduct,
and fourth ventricle) are several specialized regions of ependymal origin called
circumventricular organs. The common vascular, ependymal, and neural
organization of these structures differs from that found in typical brain tissue.
They are referred to as being in the brain, but not of it, in part because their

6
Chapter 1 Introduction & literature review
capillaries are lined by fenestrated endothelial cells indicative of a defective
bloodbrain barrier (BBB) to macromolecules (Paxinos, 2014).

Most of the brains circumventricular organs (median eminence (ME),


neurohypophysis (NH), vascular organ of the lamina terminalis (OVLT),
subfornical organ (SFO), pineal gland (Pi), subcommissural organ (SCO),
are located at various positions in the wall of the third ventricle, while the area
postrema (AP) is situated in the wall of the fourth ventricle (figure 1.3). They
are a group of specialized structures within the brain, so named because they
occupy strategic positions along the surface of the brain ventricles (Hofer,
1958, Noback et al., 2005, and Paxinos, 2014).

Figure 1.3 Location of the circumventricular organs shown on a sagittal


section through 3rd and 4th ventricles of the rat brain. Stain:
cresyl violet/luxol fast blue. (AP: area postrema, ChP: choroid
plexus, ME: median eminence, NH: neurohypophysis, OVLT:
organum vasculosum lamina terminals, SFO: subfornical
organ, SCO: subcommissural organ, Pi: pineal gland)
(Paxinos, 2014)
From a utilitarian perspective, presumably the most fascinating part of
the vascular morphology is the capillary vessels having fenestrated endothelial
7
Chapter 1 Introduction & literature review
cells in all CVOs aside from the SCO (Morita and Miyata, 2012). Thus, the
BBB is lacking in all except one: the SCO of the CVOs and, unlike many
regions of the brain, there can be bidirectional movement of polar particles
between the blood and the neural environments of the CVOs. Nevertheless,
there are variations in the barrier properties of each of the CVOs, with the ME
and AP expressing less proteins related to endothelial tight junctions than SFO,
OVLT and SCO (Willis et al., 2007, Maolood and Meister, 2009, Morita and
Miyata, 2012), thus allowing more access of circulating polar particles into
and out of the interstitium. Kuhlenbeck (1970) Subdivided the mammalian
CVOs into two groups. One group, comprised of the subcommissural organ
and choroid plexus, is characterized by modified or vascularized ECs. The
second group, comprised of the SFO, OVLT, NH, ME, Pi, and AP, are
characterized by subependymal elements which differ frequently from the EC.
This second group has been further categorized by Johnson and Gross (1993)
into sensory (SFO, OVLT, and AP) and secretory CVOs (NH, ME, and Pi).

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).

1.3.1. Subfornical Organ


It is an unmistakable component in Nissl-stained sections of rat
cerebrum. The subfornical organ (SFO) of rat, located in the anterior dorsal
part of the 3rd ventricle, at the confluence of the two interventricular foramina
and the 3rd ventricle, is ventral to the hippocampal commissure and caudo-
ventral to the intersection of the two fornical columns (Figure 1.3). Its dorsal
limit associates with the tela-choroidea, though the ventral stalk of the SFO
converges into the median preoptic nucleus. Rostrally, the septal triangularis
nucleus limits the SFO. Two important sub-regions can be characterized
taking into account their neural connectivity, density of receptors, calcium
binding proteins, vessels and function as appeared by c-fos expression. These
are an internal "ventromedial center" and a fringe "external shell" (McKinley
et al., 2003).
9
Chapter 1 Introduction & literature review
Likewise with different CVOs, the SFO is rich with vessels, having a
particular vascular arrangement. The arterial blood supply is from branches
of the subfornical artery which emerges from the anterior cerebral artery and
passes behind the splenium to the SFO. It contains an extensive system of
vessels appearing as sinusoids and capillary loops (Duvernoy and Risold,
2007). The perivascular space is broad and overly complex, in which both
fenestrated and non-fenestrated capillary endothelia are seen. Administration
of horseradish peroxidase, fluorescein or Evans blue into vessels causes them
to enter the intercellular space of the SFO, which demonstrates that the whole
organ is exposed to the hemal compartment, albeit large molecular weight
markers (e.g., Dextran 70,000) have generally less access from blood to
intercellular spaces of SFO than smaller particles (Morita and Miyata, 2012).
The ventromedial core of the SFO shows the highest density of vessels and
neurons (Sposito and Gross, 1987) which are distributed all through the SFO
and are arranged into two types in view of vacuoles (Dellmann, 1998).
Ependyma is flat and for the most part non-ciliated in the center of the SFO,
while isolated ependyma around the margin shows tufts of cilia (McKinley et
al., 2003)

1.3.1.1. Afferent Neural Connections of Subfornical Organ


The median preoptic nucleus provides the richest afferent connection to the
SFO (Saper and Levisohn, 1983). Approximately 20% of neurons in the
median preoptic nucleus with projections to the SFO exhibit collateral
branches projecting to the supraoptic nucleus (Oldfield et al., 1992). The
OVLT also has neurons projecting to the SFO (McKinley et al., 2003), with
up to 30% showing collaterals connecting to the supraoptic nucleus . Other
afferent inputs to the SFO come from the paraventricular and dorsomedial
hypothalamic nuclei (Honda et al., 2003). Thalamic neurons in the zona
incerta and reuniens nucleus likewise have projections to the SFO alongside
10
Chapter 1 Introduction & literature review
the perifornical region of the lateral hypothalamus. They seem to end in the
ventromedial center of the SFO, plus they contain angiotensin II (Lind et al.,
1984). The more caudal afferent connections originate from the locus
coeruleus (Kawano and Masuko, 2001), lateral parabrachial nucleus (Gu and
Ju, 1995), laterodorsal tegmental nucleus, dorsal and middle raphe nuclei
(Gross, 1987), caudal and rostral ventrolateral medulla and nucleus of tractus
solitarius (Kawano and Masuko, 2001).

1.3.1.2. Efferent Neural Connections of Subfornical Organ


Three major efferent projections from SFO neurons founds by anterograde
tracing systems; these were the supraoptic nucleus, the OVLT, and the median
preoptic core. These discoveries have been affirmed by retrograde transport
of neuroanatomical tracers, and associations with both parvocellular and
magnocellular parts of the hypothalamic paraventricular nucleus, the dorsal
perifornical area of the lateral hypothalamus, the anterior periventricular
preoptic and ventrolateral preoptic regions, the dorsomedial hypothalamic
nucleus, the periaqueductal grey and the midbrain dorsal raph were
additionally recognized (Uschakov et al., 2009, Kawano and Masuko, 2010).

1.3.1.3. Neural Functions of Subfornical Organ


Angiotensin II activates the SFO receptors, stimulating neural pathways
linked to water drinking, pressor reactions and salt voracity in rat (Fitts et al.,
2004). Autonomic, behavioral and neuroendocrine reactions to stress are
intervened by means of parvocellular parts of the hypothalamus, which has
neural contribution from angiotensin-sensitive neurons inside the SFO
(Kawano and Masuko, 2010, Krause et al., 2011). Relaxin hormone may
cause oxytocin emission in pregnant rats by means of receptors in the SFO
(Summerlee et al., 1987). In addition, receptors for various circulating
hormones that impact food intake and energy balance have been recognized
in the SFO which entails its connection to the AP (Pulman et al., 2006).
11
Chapter 1 Introduction & literature review
Physiological studies through electrical stimulation of the SFO can start food
intake, a reaction that prove the role of the SFO in feeding behavior (Smith et
al., 2010).

1.3.2. Vascular Organ of Lamina Terminals


The organum vasculosum lamina terminals (OVLT) forms the ventral part of
the midline anterior wall of the third cerebral ventricle (figure 1.3). First
described by Behnsen (1927), organum vasculosum laminae terminalis, from
the Latin and is also known as the supraoptic crest, medial prechiasmatic
gland, or optic recess organ in older literature. In the rat, it extends dorsally,
approximately 1 mm from the optic chiasm and roof of the optic recess, the
small anteroventral extension of the third ventricle. It is bounded both
rostrally and caudally by liquid spaces, namely, the prechiasmatic cistern and
the optic recess of the third ventricle, respectively (Behnsen, 1927).

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).

1.3.2.1. Afferent Neural Connections of Organum Vasculosum


Lamina Terminals
Gonadotropin releasing hormone terminals and dendritic endings arise from
cell bodies in the adjacent preoptic area (Herde et al., 2011). Similarly,
oxytocin fibers arise from cell bodies in the areas surrounding the OVLT
(Weindl and Sofroniew, 1985). Vasopressin fibers project to the OVLT from
parent cell bodies located in the suprachiasmatic nucleus (Buijs, 1978).

1.3.2.2. Efferent Neural Connections of Organum Vasculosum


Lamina Terminals
The major efferent pathway from the OVLT passes to the medial forebrain
bundle, in spite of the fact that a lesser number of fibers go along the
periventricular hypothalamic region. The OVLT has reciprocal connections
with the SFO, hypothalamic paraventricular nucleus and median preoptic
nucleus (Gu and Simerly, 1997). There is additionally direct neural
contributions to the supraoptic nucleus that emerge mostly from a compact
gathering of neurons in the dorsal part of the OVLT. These neurons are
stimulated by systemic hypertonicity or dehydration. Thus, they may form an
important group of physiological osmoreceptors controling vasopressin
discharge (McKinley et al., 2004). Efferent associations with parvocellular
regions of the hypothalamus are likewise present (Gu and Simerly, 1997), and
it is likely that the larger part of these fibers originates from the lateral zone
of the OVLT (McKinley et al., 2003).

1.3.2.3. Neural Functions of Organum Vasculosum Lamina Terminals


The OVLT is rich in fibers and terminals containing gonadotropin releasing
hormone. They originate from cell bodies situated in the rostral preoptic
territory close to the OVLT. They are enacted amid the gonadotropin
13
Chapter 1 Introduction & literature review
releasing hormone/luteinizing hormone surge, and their dendrites are exposed
and react to circulating humoral signals, for example, kisspeptin, proposing
that they may assume a part in the control of fertility (Herde et al., 2011).

The OVLT is rich in binding sites/receptors for some circulating factors


like amylin (Sexton et al., 1994), relaxin (Osheroff and Phillips, 1991),
angiotensin II (Allen et al., 2000), atrial natriuretic peptide (Zorad et al.,
1992), calcitonin gene related peptide (Sexton et al., 1986), and estrogen
(Somponpun et al., 2004). Neurons in the OVLT might be affected by these
hormones as neurons of the OVLT act as physiological osmoreceptors
controlling vasopressin secretion and thirst (McKinley et al., 2004).

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).

1.3.3. Area Postrema


The area postrema (AP), first described in detail by (Wilson, 1906), is
situated at the apex of the calamus scriptorius in the dorso-medial medulla
oblongata (figure 1.3). In many species it shows up as two-sided rounded
elevations on either side of the 4th ventricle at its passageway to the central
canal. In rat, these elevations have blended so that in coronal sections the AP
shows up as a bulge or quadrant of tissue dorsal to the nucleus of the tractus
solitarius.
In the AP there are small cells surrounded by astrocytes cell bodies and
their processes forming axodendritic and axosomatic synapses (Armstrong et
al., 1982). The AP is covered by flattened ECs while internally it appears
spongy because of its rich blood supply (substantial sinusoidal vessels and
smaller capillaries). Duvernoy and Risold (2007) reported gatherings of
vessels that are winding in structure with numerous subependymal loops.
Many of its capillaries are lined by fenestrated endothelium, implying that the
14
Chapter 1 Introduction & literature review
BBB is absent. It was found that capillaries lack TJs proteins, for example,
occludin 1 and claudin (Maolood and Meister, 2009). In addition, there were
perivascular spaces that contain large neurons and processes (Armstrong et
al., 1982).

1.3.3.1. Afferent Neural Connections of Area Postrema


Most afferent contribution to AP originates from the hypothalamic region
near the hypothalamic paraventricular and dorsomedial nuclei. This
connection seems, by all accounts, to be a continuous group of cells reaching
out from the perifornical region and dorsomedial nucleus of the hypothalamus
to the lateral part of the hypothalamic paraventricular nucleus. This grouping
of cells and dendrites shows up as oval rings reaching out from a
periventricular to a perifornical area at each extremity in horizontal sections
(Shapiro and Miselis, 1985). In rat, vagal and glossopharyngeal fibers were
seen to end in the AP (Kalia and Sullivan, 1982).

1.3.3.2. Efferent Neural Connections of Area Postrema


In the dorsal part of the medial subnucleus, neurons of AP connect to regions
which are mostly next to the nucleus of the tractus solitaries (van der Kooy
and Koda, 1983). A denser terminal field is found in the Ad2 (adrenergic
neurons) that are halfway between the AP and the nucleus of the tractus
solitarius. NA2 (noradrenergic neurons) are likewise surrounded by a thick
terminal field of fibers from the region of AP. Prominently, efferent
projection of the AP outside the medulla is to the dorsal pontine area, mainly
targeting the middle third of the lateral parabrachial nucleus (Stein and
Loewy, 2010).

1.3.3.3. Neural Function of Area Postrema


It was found that rat AP has noradrenaline-and serotonin-containing neurons
(Armstrong et al., 1982). Likewise, immunohistochemical techniques
15
Chapter 1 Introduction & literature review
identified neurons containing GABA, serotonin, substance P, enkephalin,
cholecystokinin, neurotensin, orexin and dynorphin (Guan et al., 2005,
Mangano et al., 2012). Autoradiography restricting studies,
immunohistochemistry and in situ hybridization strategies, give bits of
knowledge into conceivable hormonal impacts from the systemic flow that
might be included in the functions of the AP. Additionally, the AP contains
binding sites/receptors for angiotensin II, atrial natriuretic peptide, amylin,
GLP-1, GhrelinGHRS receptor, Cannabinoid receptor (CB1),
cholecystokinin receptor (CCK-1), purinergic receptors (P2X2 and P2X7),
neuropeptide Y, peptide YY and pancreatic polypeptide-Y1, Y2, Y4 and Y5
receptors, substance P neurokinin receptor (NK-1), somatostatin,
vasopressin, vasoactive intestinal polypeptide, and insulin (Paxinos, 2014).

Angiotensin II and vasopressin have impacts on AP (Hasser et al.,


2000) where neurons in may coordinate a baroreflex and influence
cardiovascular control (Ferguson, 1991).

1.3.4. Median eminence and Neurohypophysis


The median eminence (ME) locates in the midline, just caudal to the
optic chiasm and rostral to the mammillary bodies. The internal lamina of the
ME is continuous with the neurohypophysis (NH), sharing embryological and
functional features. The ECs of the ME frames the floor of the 3rd ventricle
caudal to the optic chiasm. They are irregular or flat non-ciliated cells, with
intercellular junctions that form a barrier for the entry of substances to and
from the CSF (Petrov et al., 1994).
The ME contains tanycytes that constitute the specialized
ependymoglial cellular sheet that lines the floor of the third ventricle. A
dominant feature of tanycytes is their marked polarization: tanycyte cell
bodies are located in the ventral border of the 3rd ventricle, but they also send

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).

Neurons of the arcuate nucleus which contain neuropeptide Y have


terminals in the inner zone of the ME, while enkephalinergic terminals are

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).

Hypothalamic paraventricular nucleus has parvocellular parts that


contain several neuronal groups which send fibers to the outer lamina of the
ME. They include neurons containing vasopressin and corticotrophin
releasing hormone that control the release of adrenocorticotropic hormone
and cholecystokinin (Paxinos, 2014). Hypothalamic paraventricular and
dorsomedial hypothalamic nuclei in a several animal species, including rat,
contain the gonadotropin-inhibitory hormone, GnIH. Such neuronal terminals
are not found in the outer lamina of the ME like several other mammals
(Rizwan et al., 2009, Smith et al., 2012).

It is worth noting regarding the peptidergic neurons and fibers


mentioned above that supply the ME, is that the same terminals often contain
several peptides or monoamines like dopamine, neurotensin, galanin,
vasopressin, corticotrophin releasing hormone, neurokinin B, dynorphin and
kisspeptin (Krajewski et al., 2010).

1.3.5. Pineal Gland


It is a melatonin-secreting endocrine gland that has been a subject of interest
for long time. It is a midline, cone-like structure situated in the dorso-caudal
roof of the 3rd ventricle in mammals. It is a thickening of the ependyma around
the 3rd ventricle Pi recess, connected by a peduncles to the habenular and
posterior commissures (Korf et al., 1998). In the rat, the Pi is mostly
superficial with a long stalk relatively, as most of its cells have migrated in a
dorso-caudal direction (figure 1.3). The stalk has parenchymal cells, nerve
fibers, vessels and connective tissue (Pvet, 1983). The rat Pi consists of two

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).

Pinealocytes stimulation of melatonin depends on an intact sympathetic


innervation (containing noradrenaline and NPY) from the superior cervical
ganglion. This simulation occurs in darkness while light inhibits melatonin
secretion (Korf et al., 1998). The superior salivatory nucleus and nucleus of
the tractus solitarius send signals to relay in the sphenopalatine and otic
ganglia. This parasympathetic stimulation flows to the Pi. In addition, there
are direct central innervations from the hypothalamic paraventricular nucleus,
perifornical region (containing orexin A), lateral hypothalamic area, lateral
geniculate nuclei and dorsal raph that enter the gland via the pineal stalk
(Moller and Baeres, 2003).

The suprachiasmatic nucleus is considered to be the master circadian


clock. Signals from this nucleus stimulate the Pi to release melatonin. Light
signals from the retina relay in the suprachiasmatic nucleus and cause inhibition
of melatonin release. While melatonin secretion into the circulation
synchronizes the many tissues activities, including the master clock itself.
Secretion from the adrenal cortex, reproduction and sleep are only three of
numerous functions impacted by melatonin (Reiter et al., 2009, Pevet and
Challet, 2011).

1.4. Blood Brain Barrier and Blood Cerebrospinal Fluid Barrier


Three barrier layers contribute to the separation of the blood and neural
tissues: (1) a highly specialized endothelial cells layer comprising the BBB
and partitioning the blood and brain interstitial fluid, (2) The BCSFB with the

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).

Tanycytes are a highly specialized population of ECs that forms a blood


CSF barrier at the CVOs. They have well-organized TJs, a marker of CNS
barriers that is absent in the fenestrated portal capillaries of the CVOs. This
shift of barrier properties from the vascular to the ventricular side permits the
dissemination of blood-borne particles into the parenchyma of the CVOs
while tanycyte TJs control their dispersion into the CSF, keeping up brain
homeostasis. The ECs that border the CVOs are different from the cuboidal
21
Chapter 1 Introduction & literature review
ependyma; they have long processes that pass to the parenchyma of the CVOs
to contact their fenestrated capillaries. These tanycyte-like cells show typical
TJs around their cell bodies. Permeability studies demonstrate that this
ependymal layer functions as a diffusion barrier. Tanycytes are a marker of
all CVOs and they are involved in controlling the exchange between the
blood, the brain, and the CSF throughout these "brain windows" (Langlet et
al., 2013b).

The ME is considered as one of the CVOs; it is a neurohemal organ


located in the tuberal region of the hypothalamus ventral to the 3rd ventricle
and adjacent to the arcuate nucleus. The immunohistochemical and
permeability studies performed on this organ revealed the barrier properties
of a class of highly specialized ECs, or tanycytes, which line the floor of the
3rd ventricle. These properties are opposite to those of the fenestrated portal
vessels of the ME. (Mullier et al., 2010, Langlet et al., 2013a, Langlet et al.,
2013b, Myers, 2013).

1.5. Sulcus Medianus of the 4th Ventricle


The sulcus medianus (SM), passes from the caudal end of the midbrain
(mesencephalic) cerebral aqueduct to the inferior part of rhomboid fossa of
fourth ventricle (calamus scriptorius). The SM of the fourth ventricle in the
rabbit contains a previously undescribed CVO. The cells in this region are
predominantly nonciliated. The apical surface of each cell has characteristic
microvillous projections with pinocytotic vesicles. The cell body is elongated,
with basal process. The general morphology of the cells is similar to tanycytes
found in other CVOs (Collins, 1989).

Studies on the ependyma of the floor of fourth ventricle in rat with


scanning electron microscope (SEM) and transmission electron microscope
(TEM) demonstrating the existence of important variations in the distribution

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)

Examination of the floor of fourth ventricle with SEM revealed


microvilli of different sizes and shapes could be seen covering the apical pole
of the EC; the density of the microvilli varied from one EC to another. The
fourth ventricle lacking in cilia could also exhibit small spherical or ellipsoid
formations lying freely on the ependymal surface; these structures comprising
a stalk emerging from the apical pole of the EC, which joined it to its dilated
end (Alvarez-Morujo et al., 1992).

1.6. Cell Junctional Complexes


Intercellular adhesion in mammalian epithelia is intervened by cellcell
junctions that include (TJ) (ZO), (AJ) (zonula or fascia adheren) and
desmosomes (macula adheren). In simple epithelia, TJ and AJ are nearly
situated in the apical part of the lateral plasma membrane and are referred to
as the apical junctional complex (AJC). It is generally believed that the AJC
plays a key role in formation and maintenance of epithelial barriers. (Tsukita
et al., 2001, Blaschuk and Rowlands, 2002).

The TJ appears as a continuous network of intramembranous structures


or fibrils obliterating the intermembranous space. The underlying AJ is
distinguished ultra-structurally as a prominent electron-dense plaque along
the plasma membrane (Miyaguchi, 2000).

Both the TJ and AJ consist of transmembrane and cytosolic proteins. The


transmembrane proteins physically interface with restricting accomplices on
membrane of adjacent cells providing a mechanical link between two plasma

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 transmembrane proteins of epithelial TJ include occludin, members


of the claudin family and junctional adhesion molecule (JAM)-A. E-cadherin
and members of the nectin family represent the major transmembrane
components of epithelial AJ (Matter and Balda, 2003, D'Atri and Citi, 2002).

1.6.1. Tight Junctions


The TJs within the CNS are primarily located in the capillary endothelium
and the epithelium of the choroid plexus. They are also expressed by
tanycytes and EC lining the ventricles of the brain (Tietz and Engelhardt,
2015).
Several proteins associated with TJs have been identified, including Zo-
1 (Stevenson et al., 1986), cingulin (Citi et al., 1988), Zo-2 (Gumbiner et al.,
1991) and 7H6 (Zhong et al., 1993). Zo-1 is a high molecular mass (~ 225kD)
phosphoprotein peripherally associated with the cytoplasmic surface of the TJ
in epithelial and endothelial cells both in vitro and in vivo (Stevenson et al.,
1989).
The TJs mark the border between the apical and basolateral membrane
area. They act as an intramembranous diffusion barrier and as a paracellular
seal (Tsukita et al., 2001).

Three types of structural components of cell membrane that are present


at TJs have the potential to mediate cellcell adhesion: the IgG like group of
JAMs, the claudin, and occludin (Schneeberger and Lynch, 2004, Furuse and
Tsukita, 2006).

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).

Figure 1.4 Scheme showing types of tight junctions and adhesion


junctions proteins. TJ: tight junction, AJ: adhesion junction,
JAMs: junctional adhesions molecules, AF: Afadin protein,
ZO: zonula occludens.

Confinement of ZO-1 to TJs requires its actin-binding domain. What's


more, ZO-1 proteins can likewise specifically interact with JAMs and form
homodimers or heterodimers with either ZO-2 or ZO-3. Just recently, it was
demonstrated that either ZO-1 or ZO-2, yet not ZO-3, are critical for
clustering of claudins, strand formation, and barrier function (Umeda et al.,
2006).
The ZO-1, ZO-2, and ZO-3 proteins are related to the signaling and
scaffolding proteins. The assembly and regulation of the TJs in epithelial cells
are believed to be regulated by cytosolic proteins such as ZO-1 (Niessen,
2007).
25
Chapter 1 Introduction & literature review
1.6.2. Adhesion Junctions
The AJ consists of two basic adhesive units: the nectin-afadin complex and
the classical cadherincatenin complex (Figures 1.4-5). Both nectins and
cadherins are multimember families and the cell-specific expression of
cadherins and nectins ultimately determine the strength and adhesive
specificity of the AJs. The nectin family of IgG-like adhesion receptors
consists of four members, Nectin-1 to- 4. Nectins forms a structural adhesive
unit with the actin-binding protein afadin, also known as AF-6, providing
these adhesion molecules with a direct link to the cytoskeleton. Cadherin are
transmembrane cellcell adhesion molecules conserved among all organisms,
which play essential roles in tissue morphogenesis and homeostasis (Niessen,
2007).

Figure 1.5 Scheme showing adhesion junctions, gap junction and


desmosome. Adhesion junctions composed of Cadherin-
Catenin and nectin-afadin complexes. Also see (Figure 1.4)

Cadherins are considered homophilic adhesion molecules although


recent data indicate that binding can be more promiscuous. (Duguay et al.,

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).

1.7. Immunohistochemistry and Immunofluorescence


Immunohistochemistry is an approach to the selective staining of tissue
components. The methods employ specific antibodies which bind selectively
to tissue antigens. Antigens-antibody complex formed is invisible. However
various labels applied directly or indirectly produce colored compounds at the
site of the initial antibody attachment, with proper control, this technique can
be considered specific for tissue component which are not readily visualized
by conventional histochemical approaches (Al-Salihi, 2011).

Antibodies can be used to distinguish between polypeptides that differ


only by single amino acid; monoclonal antibodies are used for this purpose.
Monoclonal antibodies refer to a preparation of antibody molecules produced
by single colony or (clone) of cells. They are a single species of antibody
molecules. Hybridoma, which is a combination of malignant melanoma cell
and normal antibody producing cell, can be cultured and grown rapidly,
producing large quantities of monoclonal antibodies. In order to make use of
antibody-antigen binding in histochemical way, this should be visible in
histological section. The immuno-fluorescence method is one way for
detecting this complex, in which an antibody is conjugated with a fluorescent
dye and visualized under ultraviolet light (Al-Salihi, 2011, Ferenk, 2012)

Fluorescent histochemistry is a technique where by the tissues shows


fluorescence by illuminating it with a light of short wave length (ultra-violate
light). The substance undergoes excitation by absorption of light and start
emission (fluorescence) usually at longer wave length than that of the
absorbed light (Al-Salihi, 2011, Ferenk, 2012).

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:

1. To demonstrate the general morphology of ependymal lining in the


SMO in comparison to that of the ME.
2. To detect adhesion junctions between ECs of the SMO and the ME
through immunofluorescent labelling with anti-pan-cadherin
antibodies.
3. To localize tanycytes or tanycyte-like cells in the region of the SMO
by recognition of tight junctions between ECs using anti-ZO1
immunofluorescent labelling in comparison to ependyma of the ME.

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

Table 2.2: Materials used in research

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

Sodium phosphate dibasic anhydrous (BDH


AnalaR) Na2HPO4

Sodium citrate Na3C6H5O72H2O


Citric acid monohydrate C6H8O7.H2O or C6H10O8
[2-amino-2-(hydroxymethyl)-

Tris buffer (BDH AnalaR ) propane-1,3-diol,(tris)]
Polyoxyethylene (20) sorbitan

Tween 20 (BDH AnalaR ) monolaurate

2.3. Preparation of paraffin Sections


Paraffin sections were prepared according to (Bancroft and Gamble, 2008)
with modifications as follows:

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:

Formaldehyde (Fisher scientific) 40% 100 ml

Distilled water 900 ml

Sodium phosphate monobasic monohydrate (NaH2PO4.H2O) 4g

Sodium phosphate dibasic anhydrous (Na2HPO4( 6.5 g

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).

2.3.4. Paraffin Infiltration and Embedding


The cleared specimens were infiltrated two times (2 hour for each one) with
melted paraffin wax (MEDITE PURE Paraffin) that was kept liquefied at
56-58 C in hot oven (Heraeus). Then, tissue blocks were made using
stainless steel mold (paraffin base mold). An electric wax dispenser
(Lipshaw, Model No. 222) was used for embedding the specimen. When the
blocks were completely solidified, they were separated from their molds and
kept in refrigerator 4 C until sectioning (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:

1- After dewaxing and hydration, slides were immersed in Harris alum


hematoxylin for 1 minutes and washed gently in running tap water for 3-5
minutes. (Dye solution prepared by 5 gm of hematoxylin dissolved in 50
ml ethyl alcohol in 56 C oven or water bath. 100 g of K+ or NH4+ alum
dissolved in 950 ml of distal water with gentle heating and stirring, after
complete dissolving add alcoholic hematoxylin to the hot alum solution
with stirring and boiling followed by addition of 2.5 g mercuric oxide
(carefully & slowly). Cool the solution quickly in a cold water container
and add 40 ml acetic acid and filter).

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).

3- Finally, the slides were dehydrated in increasing concentrations of ethyl


alcohol (70, 90 and 100%), 1 minutes for each, cleared in xylene (3-5
minutes) and mounted with DPX and cover slips.
35
Chapter 2 Materials & Methods
2.4.2. Immunohistochemical Staining:
2.4.2.1. Antigen Retrieval
Hydrated slides were processed for antigen retrieval as follows:

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)

Solution B: Sodium citrate (Na3C6H5O72H2O) 14.704 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).

2.4.2.2. Immunofluorescent staining


Tissue sections were then encircled using wax pen, rinsed 3 times in Tris
buffer (pH 8) and incubated in a humid chamber with the primary antibodies
for 2 hours at room temperature (25C). The primary antibodies were diluted
at (1:100) in Tris buffer (pH 8) (According to trials optimization).

Rabbit anti-Zo1 primary antibodies conjugated with FITC (Biorbyte


catalog # orb16515) where they needed no further labeling procedure.
Sections labeled with mouse anti-pan cadherin primary antibodies
36
Chapter 2 Materials & Methods
(USBiological catalog # C0107-03B) were rinsed by Tris buffer (pH 8) and
incubated in the dark humid chamber for 1 hour with goat anti-mouse TRITC
conjugated secondary antibodies (Santa Cruz catalog # SC-2781) diluted
(1:100) in Tris buffer (pH 8) (according to trials optimization).

Table 2.4: Antibodies used in immunoflourscent staining

Antibody Company

Rabbit anti-Zo1 primary antibody directly Biorbyte


conjugated with FITC catalog # orb16515

Mouse anti-pan cadherin primary antibodies USBiological


catalog # C0107-03B
Goat anti-mouse TRITC secondary Santa Cruz
antibodies catalog # SC-2781

Sections rinsed carefully by Tris buffer (pH 8) using micropipette for 3


times then add small drop of mounting medium (Santa Cruz catalog # SC-
24941) and cover slip (according to trials optimization).

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.

Positive external control slides were primed by using kidney sections


stained with anti-ZO1 and anti-pan cadherin antibodies in the same procedure.

2.5. Examination of Slides


Examination of slides stained with H&E was done under light microscope
(Polivar) with 4X, 10X and 40X objective lenses.

These slides labeled with (anti-ZO1-FITC) and (anti-Pan-cadherin-


TRITC) were examined after 2-3 days (store in 4C and dark chamber) with
37
Chapter 2 Materials & Methods
fluorescent microscope (AXIO-ZEISS) by (20X, 40X, and 63X) objective
lenses. Three UV filters were used: FITC filter (519 nm), TRITC filter (619
nm), and DAPI filter (435 nm) for bright green, red, and blue fluorescence,
respectively (according to AXIO-ZEISS and NIKON). Digital camera
(AXIO-ZEISS) mount to the fluorescent microscope was used to take
pictures. The scale bars added and superimpose made to these pictures by
using Image-J program (V.1.50i, java 1.8.0_77).

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

Figure 3.16 Coronal section showing TJs as green fluorescence around


tanycytes of the SMO (arrows). TJs are seen on the apical
surface and borders of these cells. DAPI nuclear
counterstaining is seen as blue fluorescence. The ECs of roof
are negative for ZO1 (head 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.

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).

Figure 3.19 Pan-cadherin positive control in kidney section. Renal


tubules show intense red immunofluorescence at AJs
between their cells. DAPI nuclear counterstaining is seen
as bright blue fluorescence at cell nuclei. 200X. TRITC
filter (619 nm) and DAPI filter (435 nm). The TRITC and
DAPI images were superimposed by Image-J program.

57
Chapter 3 Results

Figure 3.20 Pan-cadherin negative control in kidney section. Renal


tubules do not show AJs immunofluorescent labeling
between their cells. DAPI nuclear counterstaining is seen
as bright blue fluorescence at cell nuclei. 200X. TRITC
filter (619 nm) and DAPI filter (435 nm). The TRITC and
DAPI images were superimposed by Image-J program.

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).

The outer zone of the ME contains a combination of glial cells,


ependymal cells, and nerve terminals like that depicted for the inner zone,
despite the fact that terminals are particularly neurohemal. Critically, there is
a fenestrated vascular system called the primary portal plexus that is in close
relationship with excess of terminals containing neurohormones and releasing
factors (Page, 1986).

The ME is considered to be one of the CVOs in the brain that is located


in the wall of 3rd ventricle (Hofer, 1958, Noback et al., 2005, Paxinos, 2014).
In 1954, Horstmann described the elongated bipolar ependymal cells lining
the infundibular recess of the 3rd ventricle, with a proximal pole at the

62
Chapter 4 Discussion
ventricular wall and a distal pole contacting the portal vessels. Because of
their shape, Horstmann called these cells tanycytes.

The morphology of tanycytes has been thoroughly explored by means


of photonic observations as well as by TEM and SEM electron microscopy
(Flament-Durand and Brion, 1985)

The labeling by antibodies against glial fibrillary acidic protein (GFA)


revealed by the peroxidase-antiperoxidase (PAP) method clearly shows the
topography of tanycytes in rat and man brains (Guerra et al., 2010). At the
level of the ME, anti-GFA-PAP staining demonstrated the presence of
tanycytes linking the floor of the third ventricle to the portal vessels, and also
revealed the presence of fibrillary astrocytes whose stellate shape contrasts
with the elongated shape of the tanycytes. Langlet et al. (2013) investigated
the presence of tanycyte-like cells in the CVOs by examining the morphology
of vimentin-positive ependymal cells lining their ventricular walls, and the
association of these cells with fenestrated capillaries.

In our study, examination of sections stained with H. & E. under light


microscope showed squamous or irregular cells lining the walls and floor of
the 3rd ventricle (figure 3.2) where cilia were difficult to be identified.
Akmayev (1973) reported that the floor and ventrolateral walls of the 3rd
ventricle were characterized by tanycytic ependymal cells with intimate
topographical relationships to hypothalamic neurons and to blood vessels of
hypothalamus and pituitary. Also, Page (1986) and Petrov et al. (1994)
reported that ependyma at the ME that form the floor of the 3rd ventricle,
immediately caudal to the optic chiasm, were irregular in shape (generally
flattened), lack cilia, and had intercellular junctions that restrict the passage
of substances to and from the CSF.

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).

On light microscopic examination the epithelium of the SM is


pseudostratified; the cells have narrow necks and long basal processes, which
either course deep into the neuropil or end close to a blood vessel. The apical
surface of the cells is covered with microvilli; only a few cilia clumps are
present.

On TEM examination pinocytotic vesicles can be seen between the


microvilli on the cell surface. The lateral walls of the cells form complex TJs
interdigitations with those of their neighbors; this is most marked apically.
Characteristic features of this region are the electron-dense desmosomes
which appear at low magnification as a dark discontinuous line across the
ventricular surface just beneath the apices of the cells. Tight junctions are also
present in this region (Collins, 1989, Alvarez-Morujo et al., 1992).

On SEM studies of the ependymal surface of the floor of fourth


ventricle revealed zones very rich in cilia accompanied by territories with
fewer cilia with microvilli (Alvarez-Morujo et al., 1992).

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.

Examination with H. & E. stain showed aligned cuboidal or short


columnar cells each with a single round nucleus and identifiable cilia at their
surfaces that line the region of SMO (figures 3.4-6) while there are no cilia in
the other region of the floor of 4th ventricle. It is interesting to note that
ependymal cells at the depth of the SM in the region of the SMO were seen
to be pseudostratified in 2-3 layers compared to the single layer ependyma
on the MS banks and the floor of the 4th ventricle (figures 3.5 and 3.6). This
condensation of cells at the SMO may be due to the presence of supra-
ependymal cells on the ventricular aspect of ependyma CVOs (Collins, 1989),
or it can be a reflection of the presence of tanycytes or tanycyte-like cells in
the region of the SMO or may be this the area of mitosis and generation of
ECs because the nuclei appear to be daughter similar to each other.

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).

TJ protein ZO-1 and epithelial cadherin (E-cadherin) are expressed in


ependymal cells as opposed to fenestrated endothelial cells suggesting that
the barrier has been shifted from the vascular to the ventricular side. (Smith
and Shine, 1992, Vorbrodt and Dobrogowska, 2003). Also, AJs are involved
in the formation of TJs since cadherin system is established now to be critical
for well-organized TJ morphology and function (Engelhard and Valyi-Nagy,
2006).
4.3.1. Inspection of Immunofluorescence Reaction in the
Median Eminence
The ME part of the hypothalamus (physiologically), and considers one of
three parts of NH (anatomically); is one such CVO that typically contains a

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).

In tanycytes located at the ME, previous studies showed that ZO-1


immunoreactivity was organized around the cell bodies in a continuous belt,
giving rise to a honeycomb-like shape. In contrast, tanycytes lining the region
of the ventricle adjacent to the ARH exhibited a diffuse pattern of ZO-1 and
occludin immunoreactivity in the apical region of the cells. Similar to
tanycytes of the ME, an organized pattern of ZO-1 and occludin labeling was
observed in ECs dorsal to the ARH (Mullier et al., 2010).

In this study, we could demonstrate immunofluorescent labeling for


TJs and AJs between ependymal cells lining the 3 rd ventricle at the region of
the ME. This detection of TJs with rabbit anti-ZO1 primary antibodies
conjugated with FITC, and that of AJs with mouse anti-pan-cadherin primary
antibodies conjugated with TR was used as an internal control to validate the
results of immunofluorescent labeling of ependymal cells at the region of the
SMO. Our results, however, were more in a diffuse rather than a honeycomb
pattern, partly because of the fluorescence microscope resolution capacity,
and probably because, in case of ZO-1 immunolabeling, the type of directly
conjugated antibodies used in this study.

4.3.2. Immunofluorescence Labeling of Junctional Complexes


in the Region of the Sulcus Medianus Organum
In a previous study, Collins (1989) used light microscopy, scanning and
transmission electron microscopy to provide evidence for the concept of a
67
Chapter 4 Discussion
CVO in the rabbit 4th ventricle. On transmission electron microscope
examination, pinocytotic vesicles could be seen between the microvilli on the
cell surface. The lateral walls of the cells formed complex interdigitations
with those of their neighbors, most marked apically. Characteristic features
of the SMO region were the electron-dense desmosomes just beneath the
apices of the cells in addition to the TJs.

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.

Pan-cadherin immunolabeling of the ependyma at the SMO region


revealed the presence of AJs throughout the ependymal lining of the SM and,
as expected, elsewhere in the ependymal floor and roof of the 4 th ventricle
since AJs is part of the brain-CSF interface (figure 3.17) (Smith and Shine,
1992, Vorbrodt and Dobrogowska, 2003). In other words, pan-cadherin is not
a specific marker for tanycytes, rather it is still important for the formation of
TJs (Tietz and Engelhardt, 2015, Engelhard and Valyi-Nagy, 2006).

On the other hand, ZO1 immunolabeling in the SMO region was


confined to its cell lining, sparing other cells in the ependymal roof of the 4 th
ventricle (figure 3.18). This finding proved that the ependyma of the SMO
contain TJs which give them special features of tanycytes or tanycyte-like
cells and make them different from the remaining ependyma of the 4 th
ventricle.

In both pan-cadherin and ZO1 immunolabeling, cells in the depth of


the MS at the SMO region showed higher intensity of immunofluorescence

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.

4.4. Tanycytes of the Fourth Ventricle


Tanycytes were identified in the midline and paramedian regions of the 4 th
ventricles floor (Cummings and Felten, 1979). These tanycytes contributed
shafts into a large midline medullary dendrite bundle in the brain. The raphe
nuclei are collections of neurons, with poorly defined cyto-architecture. They
flank the midline, along the rostro-caudal extension of the brainstem, both in
animals (Taber et al., 1960) and human (Olszewski, 1954). Tanycytes shafts
abutted raphe perikarya and dendrites, and dendrites of reticular formation
neurons. These findings raised the possibility that 4th ventricular tanycytes
may constitute a communication channel between the CSF of the 4th ventricle
and neuronal elements of the medullary raphe dendrite bundle in a manner
similar to that reported for 3rd ventricular tanycytes (Knigge et al., 1976)

Raphe nuclei contain heterogeneous populations of neurons, with


unique morphologies, projections and neurochemical characteristics.
Serotonergic neurons constitute the major portion of the raphe nuclei, and
most of the literatures dealing with these nuclei refer to their serotonergic
neuronal components. The serotonergic neuronal groups may be allocated, on
the basis of their distribution and main projections, into two clusters: the
rostral group, present at the midbrain and rostral pons, with major projections
to the forebrain, and the caudal group, extending from the caudal pons to the
caudal part of the medulla oblongata, with major projections to the caudal
region of the brainstem and to the spinal cord (Hornung, 2003).

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).

The locus coeruleus can mediate both a pressor and a depressor


response over arterial blood pressure. Changes in the brainstem
monoaminergic nuclei can affect arterial blood pressure, and that
monoaminergic precursors injected into the 4th ventricle can influence this
response. Tanycytes may participate in this process through their anatomical
contact with monoaminergic neurons, through their role as the main
ependymal elements in contact with CSF adjacent to monoaminergic nuclei,
or through a theoretical transport role. These possibilities require careful
physiological studies to delineate the in vivo function of the 4th ventricular
tanycytes in relationship to monoaminergic nuclei (Ward and Gunn, 1976).

The apical surfaces and processes of the midline and paramedian


tanycytes are in contact with CSF. If these 4th ventricular tanycytes can
uptake substances from the 4th ventricle and transport them down the shafts
in a manner similar to the tanycytes on the floor of the 3rd ventricle, then a
transport conduit from the CSF to the receptor surfaces of the raphe neurons
would exist. This suggestion is based upon functional evidence from
tanycytes of the 3rd ventricle; at present, no functional studies have been done
to illustrate a transport mechanism from the 4th ventricle to any brainstem
nuclei through tanycytes shafts (Felten et al., 1981 with wabsite or pubmed
reveiws).

In context of the aforementioned facts, we may suggest that the


presence of tanycytes in the region of the SMO could explain the reason
70
Chapter 4 Discussion
beyond the existence of the hypothesized CVO at this location as it can bridge
the gap between the AP and the rostrally located CVOs, where non-neuronal
signal transmission may occur through CSF-contacting tanycytes that reach
the deeply seated nuclei.

71
Chapter 5
Conclusions &
Recommendations
Chapter 5 Conclusions & Recommendations
5.1. Conclusions

1. The SMO contains tanycytes or tanycyte-like cells at its ependymal


lining.

2. These tanycytes are arrange in more than one layer in the depth of the
MS at the region of the SMO.

3. Tight junctions are present between tanycytes of the SMO.

4. Whereas adhesion junctions are present along ependyma of the 4th


ventricle, tight junctions are present only in the SMO region.

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:

1. Morphological study of tanycytes at the SMO is needed to identify the


extension of cellular processes of this unique cell population.

2. Identification of the various types of tight junctions proteins between


tanycytes of the SMO.

3. Investigation of the BBB properties of the vessels in the SMO region


is important to correlate the existence of tanycytes with the presence of
fenestrated or capillary sinusoids vessels.

4. Exploring the features of astrocytes neuroglia cells, in the SMO region


to show the relationship between tanycytes and other cell populations
in the brainstem.

74
Chapter 6
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