Cellular correlates of compensatory axonal sprouting in the magnocellular neurosecretory system of the
rat
by John Andrew Watt
A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in
Biological Sciences
Montana State University
© Copyright by John Andrew Watt (1993)
Abstract:
Neuronal and non-neuronal correlates of compensatory sprouting in the neural lobe (NL) were
investigated using a unilateral hypothalamic knife-cut of the hypothalamoneurohypophysial tract to
partially denervate the rat NL. Metabolic, morphometric and immunocytochemical methods were
employed to investigate the cellular activity which accompanies the magnocellular neurosecretory
system's response to partial denervation. Analysis of the metabolic effects of partial denervation
indicate a transient urine hypoosmolality within the first 24 hours post-surgery (PS) followed by a
chronic urine hyperosmolality which persists throughout the 3 month investigative period. Daily water
intake (drinking) and urine excretion volumes demonstrate drop below control levels immediately
following surgery which persists throughout the investigative period. Morphometric measurement of
the magnocellular oxytocinergic and vasopressinergic neurons in the contralateral supraoptic nucleus
(SON) demonstrated an enlargement of both cell populations within 10 days PS which remained
significantly above control levels for at least 30 days PS. These data also suggest the oxytocinergic cell
population may enlarge more vigorously or at an earlier time than their vasopressinergic counterparts.
Cell counts of glial cell nuclei within 1 micron coronal sections of the NL revealed an increase in the
number of pituicyte nuclei by 2 days PS which remained above age-matched control values for at least
30 days PS. The number of microglial cell nuclei numbers increased by 2 days PS but returned to
normal by 10 days PS. Morphometric analysis of the NL vascular plexis revealed a strong temporal
correlation between the enlargement of the NL vascular plexis and the process of compensatory
collateral sprouting of neurosecretory axons in the NL. Immunocytochemical analysis of
bromodeoxyuridine (Brdu) substitution suggests that the microglial but not the pituicyte populations
within the NL are undergoing hyperplasia between 1 and 6 days following partial denervation. Analysis
of nerve growth factor receptor (NGFR) expression in the NL indicates a dramatic increase in the
proportional area immunoreactive for NGFR between 5 and 10 days PS, which then returns to normal
levels. Immunocytochemical comparison between NGFR, iC3b compliment receptor and leukocyte
common antigen (LCA) immunoreactivity suggest that a subpopulation of microglial cells may
up-regulate their expression and/or increase the numbers of cells expressing the NGF receptor during a
period when axonal degeneration and phagocytosis of neuronal debris is occurring. These data describe
the varieties of neuronal and non-neuronal activities which underlie the compensatory axonal sprouting
of intact magnocellular neurons within the partially denervated NL. CELLULAR CORRELATES OF COMPENSATORY AXONAL SPROUTING
IN THE MAGNOCELLULAR NEUROSECRETORY SYSTEM
OF THE RAT
by
John Andrew Watt
A thesis submitted in partial fulfillment
of the requirements for the degree
of
Doctor of Philosophy
in
Biological Sciences
MONTANA STATE UNIVERSITY
Bozeman, Montana
January 1993
APPROVAL
of a thesis submitted byJohn Andrew Watt
This thesis has been read by each member of the thesis committee and
has been found to be satisfactory regarding content, English usage,
format, citations, bibliographic style, and consistency, and is ready for
submission to the College of Graduate Studies.
(Z,/rn
Chairperson, Graduate Committee
D
Approved for the Major Department
14 Towogry
Date
Head, Biology Department
Approved for the College of Graduate Studies
Graduate Dean
ill
STATEMENT OF PERMISSION TO USE
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for
a doctoral
degree
at Montana State University,
I agree that
the
Library shall make it available to borrowers under rules of the Library.
I
further
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iv
TABLE OF CONTENTS
vo -J in u H
INTRODUCTION..............................................................I
Statement of Purpose........................... ..........
Plasticity in the Mammalian Central Nervous System......
The Magnocellular Neurosecretory System..................
The Fine Structure of the Neural Lobe....................
Development of the Vascular Plexus of the Neurohypophysis
Neuronal Reorganization and Recovery of Function.................... 12
Sprouting in the Magnocellular and Parvocellular
Neurosecretory Systems............................................ . •14
Nerve Growth Factor and its Receptors.............................. ..19
Justification, Rational a.nd Specific Objectives..................... 23
Role of Glia in Axonal Sprouting..................................... 23
Neuronal Activation in Axonal Sprouting.............................. 26
Specific Objectives............................................... 28
Non-neuronal Response to Injury................................... 28
Neuronal Activity................................................. 29
METHODS.................................................................. 30
Animals............................................................... 3^
Surgery............................................................... 30
Tissue Preparation................................................... 3I
Tissue Microtomy..................................................... 33
Immunohistochemistry.................................................. 33
Image Analysis and Morphometric Measurements.........................38
192-IgG Proportional Area Analysis................................ 38
SON Cellular Morphometry.......................................... 39
Glial Morphometry................................................. 40
Metabolic -Analysis...........
41
Statistical Analysis................................................. 41
RESULTS.................................................................. 42
Post-Mortem Observations Within the Hypothalamus.................... 42
Lesion-Induced Changes in Urine Osmolality,
Excretion and Daily Water Intake................................... 44
Hypertrophy of SON Somata and Nuclei following
Partial Denervation................................................ 46
Morphometric Assessment of the Neural Lobe
Vascular Plexus.................................................... 53
Low Affinity (p75) Nerve Growth Factor Receptor
Immunoreactivity in the N L ......................................... 59
Morphometric Assessment of Glial Cell Numbers........................66
DISCUSSION............................................................... 73
Summary..................................................
90
REFERENCES CITED........................................................ 92
V
LIST OF TABLES
Table
Page
1.
Size Distribution Bins of Vascular Morphometry...... ............. 55
2.
Results of Glial Cell Counts - Lesion Group Means........... ..... 67
3.
Results of Glial Population/Proliferation Estimates............... 71
-I
vi
LIST OF FIGURES
Figure
Page
1.
Oxytocinergic Perivascular Plexis
Within the Hypothalamus......................................... 43
2.
Effects of Unilateral Hypothalamic Lesion
on Urine Osmolality...........
45
3.
Effects of Unilateral Hypothalamic Lesion
on Daily Drinking Volume....................................... 46
4.
Effects of Unilateral Hypothalamic Lesion
on Daily Urine Excretion Volume............................... 47
5.
Photomicrograph Demonstrating Silver-Enhanced
Oxytocinergic SON Neurons....................................... 48
6. Hypertrophy of Vasopressinergic Neurons.........................
7.
.49
Hypertrophy of Nuclei in Vasopressinergic Neurons.................. 50
8. Hypertrophy of Oxytocinergic Neurons................................ 51
9.
10.
Hypertrophy of Nuclei in Oxytocinergic Neurons..................... 52
Effects of Lesion on Blood Vessel Density
in the N L ....................................................... 54
11. Photomicrograph of NL Vasculature.................................. 56
12.
Size Distribution of PS 2 Neural Lobe
Vasculature..................................................... 57
13.
Size Distribution of PS 90 Neural Lobe
Vasculature...................................................... 58
14.
Mean Proportional Area of NL Vasculature........................... 59
15.
Mean Proportional Area of NL Capillary Plexus..................... 60
16.
192-IgG Immunoreactivity in the N L ................................. 61
17.
Photomicrograph of NGFR immunoreactivity
patterns in the NL at 5 days P S .................................. 62
18.
Photomicrograph of NGFR Immunoreactivity
Patterns in the NL at 90 days P S .................... ............ 64
19.
Photomicrograph of Microglial Cells in the NL at 5 Days P S ........ 65
20.
Pituicyte Density in the N L ........................................ 68
21. Pituicyte Nuclei Cross-Sectional Area.............................. 69
22.
23.
Microglia Density in the NL Following Surgery.......
.70
Confocal Photomicrographs of GFAP/Brdu and
GSA Lectin/Brdu Labeling in the NL at 5 Days P S ................. 72
vii
ABSTRACT
Neuronal and non-neuronal correlates of compensatory sprouting in
the neural lobe (NL) were investigated using a unilateral hypothalamic
knife-cut of the hypothalamoneurohypophysial tract to partially denervate
the rat N L . Metabolic, morphometric and immunocytochemical methods were
employed to investigate the cellular activity which accompanies the
magnocellular neurosecretory system's response to partial denervation.
Analysis of the metabolic effects of partial denervation indicate a
transient urine hypoosmolality within the first 24 hours post-surgery (PS)
followed by a chronic urine hyperosmolality which persists throughout the
3 month investigative period.
Daily water intake (drinking) and urine
excretion volumes demonstrate drop below control levels immediately
following surgery which persists throughout the investigative period.
Morphometric
measurement
of
the
magnocellular
oxytocinergic
and
vasopressinergic neurons in the contralateral supraoptic nucleus (SON)
demonstrated an enlargement of both cell populations within 10 days PS
which remained significantly above control levels for at least 30 days PS.
These data also suggest the oxytocinergic cell population may enlarge more
vigorously or at an earlier time than their vasopressinergic counterparts.
Cell counts of glial cell nuclei within I micron coronal sections of the
NL revealed an increase in the number of pituicyte nuclei by 2 days PS
which remained above age-matched control values for at least 30 days PS.
The number of microglial cell nuclei numbers increased by 2 days PS but
returned to normal by 10 days PS.
Morphometric analysis of the NL
vascular plexis revealed a strong temporal correlation between the
enlargement of the NL vascular plexis and the process of compensatory
collateral
sprouting
of
neurosecretory
axons
in
the
NL.
Immunocytochemical analysis of bromodeoxyuridine (Brdu) substitution
suggests that the microglial but not the pituicyte populations within the
NL are undergoing hyperplasia between I and 6 days following partial
denervation. Analysis of nerve growth factor receptor (NGFR) expression
in the NL indicates a dramatic increase in the proportional area
immunoreactive for NGFR between 5 and 10 days PS, which then returns to
normal
levels.
Immunocytochemical comparison between NGFR, iC3b
compliment receptor and leukocyte common antigen (LCA) immunoreactivity
suggest that a subpopulation of microglial cells may up-regulate their
expression and/or increase the numbers of cells expressing the NGF
receptor during a period when axonal degeneration and phagocytosis of
neuronal debris is occurring.
These data describe the varieties of
neuronal and non-neuronal activities which underlie the compensatory
axonal sprouting of intact magnocellular neurons within the partially
denervated N L .
I
INTRODUCTION
Statement of Purpose
These studies were designed to further elucidate the cellular
correlates of the compensatory collateral sprouting which occurs within
the magnocellular neurosecretory system (MNS) of the rat (Watt and
Paden, 1991).
The objectives of this research were to investigate the
response of the MNS to partial denervation and to explore the non­
neuronal activity associated with the initiation and maintenance of
compensatory collateral sprouting of intact MNS axons following partial
denervation of the neural lobe (NL).
The goals were five fold.
I)
Determine if the observed increase in relative area of partially
denervated NL occupied by glia is due to cellular hypertrophy or
hyperplasia of resident pituicytes and/or perivascular cells.
2) Investigate the possibility of a differential response in cellular
activation/hypertrophy between oxytocin and vasopressin-immunoreactive
cells in the contralateral supraoptic nucleus (SON).
3) Investigate the
vascular response to partial denervation of the NL and its possible
correlations with degeneration and sprouting of neurosecretory axons.
4) Evaluate the neurosecretory activity of vasopressinergic (VP) neurons
by measuring alterations in drinking volume, urine excretion volume and
urine osmolality.
5) Investigate the expression and possible up-
regulation of nerve growth factor receptors (NGFR) on glial cells in the
NL.
The first objective was accomplished in part by investigating the
proliferative activity of pituicytes and/or perivascular cells.
Glial
involvement in central nervous system (CNS) disorders has been
repeatedly demonstrated to occur in Alzheimer's disease (Mandybur and
Chuirazzi, 1990), experimental allergic encephalomyelitis (Cammer et
al., 1990) and muscular sclerosis (Lee et al., 1990).
The data obtained
in the present investigation may provide clues regarding the mechanisms
2
regulating the glial response to denervation and ultimately to improve
our understanding of the glial role in CNS repair.
The second goal was
to investigate the role of neuronal activity in the sprouting response
by comparing the response of oxytocin vs vasopressin MNS neurons to
partial denervation of the N L .
This was accomplished by comparing the
extent and time course of cellular hypertrophy during the compensatory
sprouting of vasopressin magnocellular neurons.
The third goal was to
investigate the response of the NL vascular plexus following partial
denervation.
As will be described below, the growth of the NL capillary
plexus during axonal development and regenerative sprouting of severed
neurosecretory axons appears to be required for successful axonal and
terminal maturation.
Therefore, a morphometric analysis of NL
vasculature was undertaken to determine if growth of the NL capillary
plexus correlates with axonal sprouting.
The metabolic effects of
partial denervation of the antidiuretic system, the MNS, were explored
as a means of gaining an indirect estimate of circulating plasma VP
levels and the relationship of plasma VP levels to the induction of the
compensatory sprouting response of vasopressinergic axons.
Finally,
alterations in the expression of nerve growth factor receptors were
measured immunocytochemically as a means of determining the contribution
of perivascular microglial cells to the post-denervation response.
A brief historical review of the literature related to neural
regeneration within the mammalian central nervous system will be
presented next.
This section will be followed by a detailed description
of the structure and development of the neurosecretory system at both
the light and ultrastructural level.
This is intended to familiarize
the reader with the close association of the components of the MNS and
the interactions which underlie its development, function and inherent
plasticity.
This will be followed by a brief review of the different
forms of injury-induced reorganization known to occur in the
neurosecretory system and the contribution of nerve growth factor and
3
its receptors to CNS and neurosecretory function and response to injury.
Finally, justification, rationale and specific aims will be presented.
Plasticity in the Mammalian CNS
Plasticity is a neurobiological concept which generally refers to
the capacity of the central nervous system to modify existing synaptic
contacts in response to injury, hormonal perturbations or environmental
stimuli (Cotman et al., 1984).
The principal evidence for plasticity
comes from investigations of injury-induced responses (Cotman et al.,
1984).
Several forms of injury-induced axonal growth have been observed
within the brain; these differ in several ways.
First, regeneration
generally describes the regrowth of a single fiber from the proximal end
of the severed axon and is most frequently observed following injury to
peripheral nerves.
In the brain regenerative sprouting refers to the
outgrowth of multiple collateral fibers from the proximal end of the
severed axon.
Second, the distance over which regrowth may occur is
generally more extensive in regenerating axons than in sprouting axons
(Cotman et al., 1984).
Sprouts may arise from collaterals of intact
fibers sharing the partially denervated terminal field (compensatory
collateral sprouting), or from sustaining collaterals of injured axons
proximal to the site of injury (regenerative sprouting; Kiernan, 1971).
Injury-induced sprouting, often termed reactive synaptogenesis, may
occur in several different forms, e.g., sprouts arising from nodes of
Ranvier (nodal sprouting), from preexisting axon collaterals (collateral
sprouting), or from the terminal bouton of intact axons innervating a
partially denervated terminal field (terminal sprouting; Cotman et al.,
1984).
Although sprouting in any form can be extensive, it is generally
more limited in the distance over which it may occur (Cotman et al.,
1984), and tends to occur more rapidly (Murray, 1986) than regeneration.
Cellular events accompanying the sprouting process have also been
observed.
For example, cellular hypertrophy following injury has been
4
well documented.
Following hypophysectomy both somata and nuclei of SON
neurons hypertrophy paralleling the formation of a neural lobe-like
structure in the proximal infundibulum (Moll and DeWeid, 1962; Raisman,
1973).
Likewise, somata and cell nuclei of SON and paraventricular
(PVN) neurons hypertrophy during compensatory sprouting of intact
neurosecretory fibers in the NL (Watt and Paden, 1991).
Such a response
may be attributed to a heightened level of activity in the sprouting
neurons (Moll and De Weld, 1962).
Following unilateral ablation of the
left striatum and overlying cortex Pearson et al., (1987a) described, an
increase in cell size in the contralateral pars compacta and ipsilateral
pars reticulata of the substantia nigra, and the contralateral globus
pallidus.
These investigators also demonstrated hypertrophy of
cholinergic cells of the rat basal forebrain following damage to the
contralateral cortex.
This enlargement was apparent by 7 days post­
surgery and persisted for at least 300 days PS (Pearson et al., 1984).
The degree of hypertrophy observed was related to the age of the animal
(Pearson et al., 1985) and the degree of damage sustained (Pearson et
al., 1987b).
They went on to conclude that the hypertrophy represented
a cellular response accompanying distal axonal sprouting (Pearson et
al., 1987b).
Cotman et al. (1977) have reported that increase in cell
size is one of the earliest manifestations of the sprouting response.
In several cases a correlation has been demonstrated between cellular
hypertrophy and sprouting of intact efferents of the enlarged cells
contralateral to the site of injury (Goldschmidt and Steward, 1980;
Hendrickson, 1982; Headon, 1985; Pearson, 1987a), in a manner similar to
that previously reported by the author (Watt and Paden, 1991).
Thus,
cellular hypertrophy in response to direct axonal injury or in response
to partial denervation of a terminal field shared with other lesioned
neurons is a well described cellular event.
5
The Maanocellular Neurosecretory System
The magnocellular neurosecretory system (MNS) is composed of the
large oxytocin and vasopressin-producing neurons of the paraventricular,
supraoptic and accessory nuclei.
The axons which originate from these
neurons project through the ventromedial hypothalamus, median eminence
and infundibular stalk to the neural lobe where they form synaptoid
contacts with the outer basement membrane of the capillary plexus
(Silverman, 1983; Ju et al., 1986).
The supraoptic nucleus is comprised of a compact mass of
magnocellular neurons lying just anterolateral to the optic chiasm and
optic tract (termed the principle division)(Ju et al., 1986), and a
loose aggregation of magnocellular neurons positioned along the
dorsolateral aspect of the optic tract and ventral surface of the brain
at the level of the arcuate nucleus (termed the retrochiasmatic
division)(Rhodes et al., 1981).
There exists a partial segregation of
vasopressinergic and oxytocinergic (OT) neurons within the principle
division of the SON.
The VP neurons are located predominately in the
posteroventral region while the OT neurons are found predominately
within the anterodorsal region (Vandesande and Dierickx, 1977; Rhodes et
al., 1981; Swanson and Sawchenko, 1983).
The paraventricular nucleus, once thought to be a uniform
aggregation of magnocellular neurons (Christ, 1969) is now known to be
composed of 3 topographically distinct magnocellular and 5 parvocellular
subdivisions (Armstrong et al., 1980; Swanson and Sawchenko, 1983;
Silverman, 1983).
The three magnocellular subgroups are described as
the anterior, the medial and the posterior magnocellular subdivisions.
The five parvocellular subdivisions are comprised of the anterior,
medial, posterior, lateral and periventricular cell groups (Swanson and
Sawchenko, 1983).
A topographically distinct arrangement of VP and OT
neurons also exists in the PVN.
The anterior and medial magnocellular
subdivisions are comprised almost exclusively of OT neurons.
In the
6
posterior magnocellular subdivision the VP neurons are located
predominately within the posterodorsalateral region and the OT producing
neurons are found predominately in the anteroventromedial region
(Swanson and Sawchenko, 1983).
In addition to vasopressin and oxytocin, at least 30 other
neuropeptides have been immunocytochemically localized within the MNS
including somatostatin, dynorphin, enkephalin, gastrin, and luteinizing
hormone releasing hormone (Silverman, 1983; Swanson and Sawchenko, 1983;
Ju, et al., 1986).
Retrograde transport studies utilizing horseradish peroxidase
(HRP) injections into the neural lobe have further delineated the cell
groups contributing terminals to the NL (Armstrong et al., 1980;
Kelly and Swanson, 1980; Ju et al., 1986).
Almost all of the cells of
the SON are labeled following these injections.
SON axons were seen
rising in a fan-like array from the dorsal aspect of the principle
division of the SON before coursing ventromedialIy toward the internal
lamina of the median eminence (ME) and infundibular stalk.
Axons
arising from the more lateral regions of this division first course
anterolaterally while the medially placed cells send axons
anteromedially.
Both groups then streamed caudalIy over the optic tract
to join the fibers arising from the dorsal SON.
Those axons originating
from the retrochiasmatic division of the SON were seen to emerge from
the dorsal aspect and stream medially, dorsolaterally and
anterodorsally.
However, only the anterodorsal axons were traceable for
any distance and were seen to join their principle division counterparts
along the dorsal surface of the optic tract (Ju et al., 1986).
The cell groups most heavily labeled within the PVN following
retrograde transport of HRP from the NL are predominately concentrated
within the medial and posterior magnocellular subnuclei (Silverman,
1983), corresponding to the PVM and PVL of Armstrong et al. (1980).
However, scattered large and small neurons within several parvoeellular
7
subnuclei are also labeled, with the periventricular division being the
most substantial contributor (Swanson and Kuypers, 1980; Ju et al.,
1986).
The axons arising from the magnocellular divisions of the PVN
project ventroposterolaterally from the PVN to course around the dorsal
and ventral aspects of the fornix then sweep posteroventromedially to
occupy the lateral horns of the zona interna of the median eminence on
their way to the NL (Ju et al., 1986; Swanson and Sawchenko, 1983).
The magnocellular projections arising from the PVN and SON remain
topographically segregated throughout their course through the
hypothalamo-neurohypophysial tract.
The SON efferents occupy the
central two thirds of the zona interna, decussate within the median
eminence, then continue through the infundibular stalk to form a
widespread terminal distribution throughout the entire N L , with a higher
concentration in the central core.
The PVN fibers, on the other hand,,
maintain an ipsilateral distribution throughout their course through the
lateral zona interna and infundibular stalk, and terminate primarily in
the periphery of the NL (Silverman, 1983; Alonso and Assenmacher, 1981).
The functional correlates, if any, of this anatomical separation remain
unknown (Silverman, 1983).
Silverman et al. (1980) have also reported
reciprocal connections between oxytocinergic neurons of the PVN nuclei
and between the ipsilateral PVN and SON nuclei.
The regions receiving
afferents from the contralateral PVN appear to be the PVL and PVM
subnuclei (Silverman, 1983).
Anatomical and electrophysiological
evidence suggests the existence of bilateral polysynaptic (Takano et
al., 1990) neural connections between the SON nuclei which may
facilitate the sychronous firing of oxytocin and vasopressin neurons.
The Fine Structure of the Neural Lobe
The NL is traditionally described at the fine structural level as
comprised solely of pituicytes, perivascular microglial cells,
fenestrated vascular endothelia, the endothelial and parenchymal
8
basement membranes which delimit an extensive perivascular space, and
the nerve fibers and terminals of neurosecretory axons.
Within the
confines of the perivascular space occasional fibroblasts and collagen
fibrils may also be observed (unpublished observations).
Axons arising
from dopaminergic (Baumgarten et al., 1972; Saavedra et al., 1985)
epinephrinergic and norepinephrinergic (Saavedra et al., 1985) and
serotoninergic neurons (Saland et al., 1987) have also been reported in
the N L .
However, the oxytocin and vasopressin neurosecretory axons
comprise the vast majority of fibers pervading the N L .
In the adult NL
the neurosecretory axons are typically surrounded by the processes of
pituicytes or completely engulfed within the pituicyte cell body.
Rarely does one observe a "naked" axon within the N L .
Likewise, the
nerve terminal, when not in direct contact with the basement membrane,
is ensheathed by pituicyte or parenchymal microglial processes.
There is no agreement yet on whether pituicytes occur in different
forms or whether differing morphologies represent the same cell type in
different functional states (Fugita and Hartman, 1961; Dellman and
Owsely, 1969); Zambrano and De Robertis, 1968; Kruslovic and Brucknerr'
1969).
Dellman (1966) has described "dark" or fibrous pituicytes
surrounding thin caliber axon cylinders in the proximal NL and a second
"protoplasmic" pituicyte surounding axonal swellings and terminals
within the body of the N L .
The "dark" pituicyte is characterized
ultrastructurally by an electron dense cytoplasm, little perinuclear
cytoplasm, thin processes, and an electron dense nucleus with abundant
heterochromatin particularly clumped at the inner surface of the nuclear
envelope.
In contrast, the protoplasmic pituicyte has a distinctly
thicker perinuclear cytoplasmic region, an electron lucent cytoplasm,
thick processes, and a large round nucleus filled with euchromatin with
only small amounts of clumped heterocromatin attached to the nuclear
envelope.
It is possible that these dark pituicytes represent a
parenchymal microglial cell (see below) while the "protoplasmic
9
pituicytes" represent the only pituicyte population endogenous to the
NL.
This distinction may have important functional implications in the
partially denervated N L .
A third cellular element endogenous to the N L , located within the
perivascular space, is the perivascular microglial cell (OliviereSangiacomo, 1972).
These cells are characterized by an elongate or oval
nucleus displaying condensed heterochromatin clumped around the nuclear
envelope, an electron dense cytoplasm, extensive granular endoplasmic
reticulum, particularly in the perinuclear region but also extending
into the long thin fibrous processes, lysosomes, numerous Golgi
complexes and many free polyribosomes scattered throughout the cytoplasm
(Oliviere-Sangiacomo, 1972; Watt and Paden, 1991).
The long processes
extend throughout the perivascular space and are often observed to
surround or partially envelop neurosecretory axons which have penetrated
the outer basement membrane of the perivascular space (OliviereSangiacomo, 1972).
Perivascular cells often contain dense inclusions
and phagocytosed debris arising from the degeneration of neurosecretory
axons in the normal animal (Oliviere-Sangiacomo, 1972) and following
unilateral destruction of the PVN (Zambrano and De Robertis, 1968) or
SON (Watt and Paden, 1991).
Development of the Vascular Plexus of the Neurohvnonhvsis
As a neuroheamal organ, the neurohypophysis is characterized by an
extensive penetrating vascular plexus.
This plexus is composed in part
by the border vessels located between the dorsal surface of the NL and
the intermediate lobe (IL) and the superficial vessels which surround
the remaining lateral and ventral external surface of the gland (D'Amore
and Thompson, 1987; Eurenius, 1977).
The internal vascular plexus is thought to arise through an
extensive invasive sprouting from the external vascular network during
development of the N L .
This development occurs in three principle
10
stages.
The first stage lasts until fetal day 17, the second from fetal
day 18 until the IOth day following birth, and the third from day 10
through day 30 (Galabov and Scheibler, 1978; Galabov and Scheibler,
1983).
By 30 days of age the NL capillary plexus is thought to be fully
mature (Galabov and Scheibler, 1983; D 'amore and Thompson, 1987).
Stage
I is characterized by the development of the superficial and border
plexuses surrounding the NL anlage, followed closely by the ingrowth of
vascular sprouts at embryonic day 14 which arise from this peripheral
network (Galabov and Scheibler, 1983).
This vessel growth follows a
distinct periperal to central pattern of maturation.
This pattern is
opposite that of the maturation of neurosecretory axons which first
penetrate the internal core of the NL then sprout toward the periphery
(Galabov and Scheibler, 1978).
During the early stages of vessel
ingrowth, the perivascular space is typically very narrow and closely
applied to the vessel wall.
Ultrastructural analysis of the NL anlage
at the later stages of the first developmental period show the NL to be
packed with immature pituicytes.
No morphological indications of
functional activity, such as neurosecretory contacts or the presence of
granules within the axons or pituicytes, are apparent by fetal day 17.
Thus, in the first stage of NL development, the vascular development is
clearly more advanced than that of the neurosecretory axons or
pituicytes (Galabov and Scheibler, 1983).
The second stage of NL
vascular development is of particular interest as the first indications
of functional activity become apparent (Galabov and Scheibler, 1978;
Galabov and Scheibler, 1983).
The inward penetration of vascular
sprouts continues concurrent with the first appearance of endothelial
fenestrae.
Pituicytes show signs of continued maturation and functional
activity and the perivascular cells have increased in size and numbers.
The perivascular space, delimited by the parenchymal and endothelial
basement membranes, widens, branchs extensively, and extends deep into
the NL parenchyma.
The neurosecretory fibers in the central core of the
11
NL contain numerous granules and make contact with the outer basement
membrane of the perivascular space (Galabov and Scheibler, 1983).
During the ensuing days of fetal development, until the day of birth,
the density of the vascular plexus'increases considerably.
From the day of birth to the IOth day in the rat the vascular
network continues to develop.
Some immature capillaries are still
observed within the central region of the NL but are few in number.
There is an increased incidence of endothelial fenestrae and there are
more fibrillar structures apparent within the perivascular space.
At
the beginning of this stage the pituicytes cover a large portion of the
surface area of the perivascular basement membrane.
However, as the
second stage proceeds, these contact zones diminish as more
neurosecretory axons establish terminal contact with the perivascular
basement membrane (Galabov and Scheibler, 1983).
From 10-30 days of age the capillary network gradually attains the
appearance of the mature animal.
The perivascular cells appear to be
highly active, as indicated by increased Golgi apparati, and the general
appearance of the perivascular space corresponds to that seen in the
adult animal (Galabov and Scheibler, 1983).
The precise interrelationship between the development of the
capillaries, pituicytes and neurosecretory axons remains unclear;
however, it is of some importance to reiterate that the development of
the capillary plexus precedes the ingrowth of the neurosecretory axons.
Likewise, the appearance of endothelial fenestrae and widening of the
perivascular space does not occur until contact is made between the
neurosecretory axons and the basement membrane of the capillary network.
These observations may indicate an inductive influence of the axons upon
the final stage of maturation of the capillary plexus.
Whether the
initial growth of the capillary network has an inductive influence on
the subsequent axonal growth also remains unresolved.
12
Neuronal Reorganization and Recovery of Function
If regeneration and sprouting represent forms of anatomical
recovery from injury, the question arises whether or not such changes
coincide with some degree of functional recovery.
This is particularly
relevant to the MNS since the regeneration of the proximal infundibulum,
following hypophysectomy, leads to complete recovery of fluid balance.
Although numerous studies have described some form of axonal
reorganization following axotomy (eg., regeneration, sprouting of intact
collaterals or sprouting of injured axons), relatively few studies have
demonstrated a correlation between axonal growth and recovery of
function (Finger and Almli, 1985).
In fact, the associations made
between neural regrowth and functional recovery have been so sporadic
that several opposing hypotheses have arisen regarding the underlying
function of the reorganizational process.
Some investigators maintain
that the reorganizational process is strictly governed by the need of
the system to achieve a level of function close to that maintained prior
to the injury.
Others suggest that the neural changes associated with
injury are related to developmental processes and may result from an
alteration in the balance between growth stimulating and growth
suppressing factors (Finger and Almli, 1985).
Other investigators argue
that any recovery of function associated with the reorganizational
process is strictly coincidental.
Support for the developmental
hypothesis comes from studies of age-related factors and sprouting
(Kawamoto and Kawashima, 1985a, 1985b, 1987).
These studies have
demonstrated that the initiation of the sprouting response is most rapid
in the developing brain, decreases in the mature brain and is slower
still in the aged brain (Cotman et a l ., 1984; Finger and Almli, 1985).
If injury induced reorganization is related to a developmental
process, it follows that this reorganization would be most likely to
occur or to occur more rapidly during periods of rapid brain growth
(Gall et al., 1981; Tsukahara et al., 1981; Cotman and Lynch, 1984;
13
Tsukahara, 1985).
This does not, however, indicate that neural
reorganization cannot take place in adult tissue.
As mentioned above,
there is ample evidence suggesting that mature systems do retain this
ability but generally to a more limited extent (Steward et al., 1976;
Cotman et al., 1977, 1984; Kawamoto et al.,1985; Needles et al., 1986).
There are conditions under which sprouting in a previously denervated
zone may contribute to recovery of function.
Loecshe and Stewart (1975)
demonstrated that unilateral damage to the entorhinal cortex resulted in
initial impairment of performance of a trained task in rats;
subsequently, task performance improved.
Histologic examination
demonstrated that sprouting had occurred within the contralateral
entorhinal cortex and that the time course of sprouting was correlated
with that of behavioral recovery.
Wuttke et al. (1977) reported functional recovery paralleling
axonal regrowth in the anterior hypothalamus.
Serotonergic input to the
hypothalamus was interrupted using 5,7-dihydroxytryptamine (DHT)
chemical axotomy.
Initially there was a marked reduction in plasma
luteinizing hormone (LH) levels; however, two months following surgery
the LH levels returned to normal values.
Axonal regeneration was
evidenced by an increase in 3H-SHT uptake and release in the anterior
hypothalamus which paralleled the recovery of LH levels.
Functional recovery following unilateral chemical axotomy of the
descending serotonergic bulbospinal afferents was described by Nygen et
al. (1974).
Using the hindlimb extensor reflex as a measure of
recovery, these investigators demonstrated that the initial 5-HT
hypersensitivity observed following chemical axotomy disappeared within
three months following surgery.
This recovery was shown to closely
parallel the time course of sprouting.
The prolific regrowth observed following damage to the axons of
the magnocellular neurosecretory system has been repeatedly correlated
with functional recovery (Raisman, 1977; Kawamoto, 1985; Kawamoto and
14
Kawaahima, 1987).
Hypophysectomy, the removal of the pituitary gland
and distal infundibulum, is followed by the reestablishment of
neurosecretory contacts with the capillaries of the mantel plexus in the
zona externa of the median eminence (Raisman, 1977) and within the
pseudo-neural lobe formed proximal to the stalk section (Kawamoto and
Kawashima, 1987).
Coincident with the development of these
neurosecretory contacts, the polyuria and polydipsia associated with
hypophysectomy disappear.
The functional recovery achieved is such that
the animal not only maintains homeostatic hydration levels but is able
to resist moderate dehydration stress (Moll and DeWeid, 1962).
Polenov
(1974) found that salt loading hypophysectomized rats with a 1% NaCL
drinking solution results in a substantial decrease in the amount of
neurosecretory materials present within the proximal infundibulum.
These animals demonstrate a direct correlation between alleviation of
diabetes insipidus and release of secretory products from the
reorganized neurosecretory endings.
The functional competence achieved
by the reorganized stalk was such that animals were able to withstand a
twenty day salt loading regimen without deleterious effect.
Sprouting in the Maanocellular and Parvocellular Neurosecretory Systems
The magnocellular neurosecretory system has been the subject of
extensive investigations of axonal sprouting in a variety of
experimental preparations.
Interruption of the hypothalamo-
neurohypophysial tract either by hypophysectomy, neurolobectomy,
infundibular stalk transection or hypothalamic lesion will result in
morphological reorganization of the neurosecretory fibers proximal to
the site of lesion in a variety of species.
This effect was first
described by Stutinsky in the rat (1951, 1952) and eel (1957).
These
initial reports were followed by further studies in the rat (Billenstein
and Leveque, 1955; Moll, 1957; Moll and De Weid, 1962; Dellman and
Owsley, 1969; Raisman, 1973; Polenov et al., 1974; Polenov et al., 1981;
15
Yulis and Rodriguez, 1982; Kawamoto and Kawashima, 1986, 1987), mouse
(Kawamoto, 1985; Kawamoto and Kawashima, 1985), goat (Beck, et al.,
1969), ferret (Adams, Daniel and Prichard, 1969), rabbit (Gaupp and
Spatz, 1955), frog (Rodriguez and Dellman, 1970), Rhesus monkey (Antunes
et al., 1978), and human (Beck and Daniel, 1959).
Following hypophysectomy the magnocellular neurosecretory axons
which are normally confined to the internal zone of the median eminence
grow ventrally into the external zone.
Here they establish functionally
competent contacts with fenestrated capillaries.
These "synaptoid
contacts" differ morphologically from axoaxonic, axodendritic or
axosomatic synapses.
The contact occurs between the terminal ending of
the axon and the outer of two basement membranes enclosing the
perivascular space.
In an inactive state, these terminals are separated
from the basement membrane by the active inclusion of a pituicyte foot
process.
Upon metabolic demand, the pituicyte withdraws and contact is
reestablished.
Hence these synaptoid contacts are dynamic and display
an inherent functional plasticity in both the SON nuclei and
neurohypophysis (Theodosis and Poulain, 1987).
Ultrastructural
examination found these neurosecretory contacts to be identical to those
of the intact neural lobe (Raisman, 1977).
Various investigators found
that the neurosecretory axons reorganized into a miniature neural lobe­
like structure at the proximal end of the transected infundibulum
(Billenstein and Leveque, 1955; Moll and DeWeid, 1962; Kiernan, 1971;
Raisman, 1973; Polenov et al., 1974; Kawamoto and Kawashima, 1987).
Formation of new neuroheamal contacts in both the ME and proximal
infundibular stalk is accompanied by a general hypertrophy of the
capillary mantel plexus (Raisman, 1973).
A similar process has been
described in ferrets (Adams et al., 1969) and mice (Kawamoto and
Kawashima, 1985).
The axonal growth observed in ferrets represents the
most robust regenerative activity of neurosecretory axons yet observed.
Adams et al., (1969) severed the infundibular stalk in ferrets but
16
left the cut ends juxtaposedi
A Gomori stain for neurosecretory
material was then used to follow the Course of regeneration from two
weeks to twelve months.
By two weeks the proximal portion of the lower
infundibular stalk displayed clear indications of regenerating fibers
having penetrated the scar tissue barrier.
One to three months
following surgery, the regenerating axons had grown as far as the caudal
end of the infundibular stalk.
By five months the regenerating fibers
had invaded the neural lobe and in two animals sacrificed at one year
post-surgery the entire neural lobe had been reinnervated.
Several
animals also developed a neural lobe-like structure just distal to the
site of lesion.
This growth is similar to the pseudo-neural lobe-like
structure formed in the transected infundibular stalk following
hypophysectomy.
Regeneration of severed MNS axons was also reported in rats
following replacement of the neural lobe immediately after its removal,
by suction, from the sella turcica.
However, successful reinnervation
of the neural lobe was only observed in two cases where the proximal
infundibular stalk had not degenerated (Kiernan, 1971).
Silverman and Zimmerman (1982) have demonstrated a sprouting
response within the parvocellular neurosecretory system following
unilateral electrolytic lesion of the paraventricular nucleus in
adrenalectomized rats.
As described above, the parvocellular
neurosecretory axons projecting from each PVN normally remain
ipsilateral within their terminal field in the external zone of the
median eminence (Alonso and Assenmacher, 1981; Silverman and Zimmerman,
1982; Vandesande et al., 1977; Antunes et al., 1977).
Approximately 21
days following unilateral destruction of the PVN, however, both HRP
injections into the intact contralateral PVN and immunocytochemical
localization of vasopressin-neurophysin within the median eminence
revealed a bilateral innervation of the external zone.
The authors
concluded that these previously undetected fibers arose through a
17
compensatory
sprouting response of the undamaged terminals of the
contralateral PVN efferents (Silverman and Zimmerman, 1982).
Regenerative sprouting of neurosecretory axons has also been
demonstrated following interruption of the hypothalamo-neurohypophysial
(HNT) tract within the hypothalamus (Danilova and Polenov, 1977; Antunes
et al., 1979; Dellmann et al., 1985, 1986, 1987a, 1987b, 1988, 1989).
However, growth occurs to a more limited extent than observed following
interruption of the infundibular stalk (Danilova and Polenov, 1977;
Antunes et al., 1970; Dellman et al., 1987a, 1988).
In the intact animal the neurosecretory fibers comprising the HNT
tract normally do not form connections with the capillaries, venules or
arterioles that traverse the hypothalamus (Antunes et al., 1979; Dellman
et al., 1987a).
However, following transection of the HNT
neurosecretory axons, the proximal segments of these fibers will form
new neuroheamal contacts with newly formed vessels surrounding the
lesion tract (Danilova and Polenov, 1977; Antunes et al., 1979; Dellman
et al., 1987a) and with resident vessels of the hypothalamus riot
previously innervated by neurosecretory fibers (Antunes et al., 1979).
Antunes et al. (1979) have reported such regeneration arising from both
paraventricular and supraoptic neurosecretory axons, including oxytocinneurophysin and vasopressin-neurophysin positive fibers.
Exuberant
growth of neurosecretory axons has also been observed in the ventral
leptomeniges and surrounding pial vessels following disruption of the
ventral hypothalamic surface (Danilova and Polenov, 1977; Antunes et
al., 1979; Dellman et al., 1988).
However, Antunes et al. (1979)
reported observing hypothalamic sprouting no earlier than 8 weeks postsurgery (PS) while Dellman et al. (1987a) reported ultrastructural
indications (e.g. the presense of morphologically defined neurosecretory
granules with axon terminals) of perivascular sprouts surrounding
hypothalamic lesion tracts and within the ventral leptomeninges as early
as 5 days PS (Dellman et al., 1988).
These fiber plexes were observed
18
within connective tissue spaces seemingly devoid of vessels, within
arachnoid trabeculae and perivascular spaces, as well as surrounding the
leptomeningial (pial) blood vessels (Dellman et al., 1988).
Unfortunately, Dellman's group did not discriminate between the two
classes of neurophysins, precluding any direct comparison of sprouting
efficacy of fibers containing vasopressin vs those containing oxytocin
under these conditions.
Unsuccessful heterotypic tissue grafts placed within the
hypothalamus are characterized by the absence of capillaries (Dellman et
al., 1987b), underlying the essential nature of rapid revascularization
for graft survival (Weigand and Gash, 1988).
When NL allografts were
positioned within the HNT, neurosecretory axons were observed invading
the graft site within five days (Dellman et al., 1987b).
Variations in
the degree to which a graft was innervated were common and were
correlated with the vascular density of the graft.
In other words,
graft sites containing the highest densities of vessels contained the
highest numbers of neurosecretory fibers (Dellman et al., 1987b).
During the initial stages of axonal invasion of the graft sites both
terminal and preterminal neurosecretory fibers were observed.
Preterminal fibers were typically found deeply invaginated into the
perikaryon of pituicytes or completely enwrapped by pituicyte or
"microglial-Iike" cell processes (Dellman et al., 1987b).
Terminal
fibers were less intimately associated with the pituicytes and were
often observed abutting perivascular basement membrane (Wiegand and
Gash, 1988) much as would be seen in the normal N L .
However, as time
passed, the axon-pituicyte relationship became more and more extensive
(Dellman et al., 1987b).
Stereotaxic placement of intrahypothalamic allographs of sciatic
nerve, optic nerve and hypodermal connective tissue within the HNT
result in varying degrees of penetration by neurosecretory axons
(Dellman et al., 1985a, 1985b, 1986a, 1987).
The relative density of
19
invading neurosecretory axons appears to be dependent upon the degree of
vascularization.
Hence, NL allografts contain the highest number of
axonal profiles while optic nerve allografts contain the least (Dellman
et a l ., 1987b).
However, within optic nerve allografts the highest
density of neurosecretory fibers were always found in the immediate
vicinity of blood vessels (Dellman et al., 1989).
These observations
indicate that the vasculature must play a prominent role in the
attraction and maintenance of the neurosecretory axonal ingrowth.
Although these data do not exclude the possible, and likely,
contribution of pituicytes to the development and growth of
neurosecretory axons in the normal animal, they do indicate the
underlying importance of blood vessels and the various components of the
perivascular network to a regenerative and/or compensatory sprouting
response within the compromised neurosecretory system.
Nerve Growth Factor and its Receptors
Neuronal differentiation and survival in the developing and adult
CNS is mediated by a variety of neurotrophic factors most notable of
which is nerve growth factor (NGF).
NGF is known to promote the
survival and differentiation of neural crest-derived sensory neurons,
sympathetic neurons and magnocellular cholinergic neurons of the rat
basal forebrain (Williams et al., 1986; Taniuchi et al., 1986) and
striatum (Martinez et al., 1985; Mobely et al., 1985).
The highest
levels of NGF protein and its message are found in those brain regions
receiving the most dense cholinergic innervation: the cortex and
hippocampus (Springer, 1988).
Direct evidence indicating the
contribution of NGF to survival and function of basal forebrain
cholinergic neurons has emerged from studies of hippocampal plasticity.
Unilateral lesion of the fimbria-fornix results in loss of innervation
arising from the magnocellular cholinergic neurons of the basal
forebrain.
Concurrent with the degeneration of the cholinergic
20
terminals is a substantial rise in levels of nerve growth factor (NGF)
protein, but not mRNA,
within the denervated hippocampus within 10 days
of axotomy (Larkfors et al, 1987; Korshing et al., 1986).
Since only
the protein and not the message increased, it has ,been suggested that
this increase is due to the loss of cholinergic efferents which
retrogradely transported the NGF protein away from the hippocampus.
With the subsequent collateral sprouting of cholinergic fibers within
the denervated field, the NGF protein levels return to normal (Milner
and Loy, 1980).
As a result of the axotomy the cholinergic neurons of
the basal forebrain degenerate.
However, chronic ventricular infusion
of NGF (Hefti, 1986; Williams et al., 1986) or transplantation of NGF
secreting tissues (Springer et al., 1988) will result in the survival of
the axotomized neurons.
These data indicate the reliance of specific
central neurons in the adult brain on NGF for survival and plasticity.
Although the mechanism underlying NGF activity within a target
tissue remains enigmatic, it is known that the ability of a target
tissue to utilize NGF is reliant upon the presence of high affinity
nerve growth factor receptor (NGFR)(Springer, 1988).
of the receptor exist:
At least two forms
a high affinity form which is absolutely
required for the internalization and utilization of NGF by the receiving
cell, and a low affinity form which binds NGF but does not internalize
the resulting NGF-NGFR complex, a step that is essential for NGF
activity (Bernd and Greene, 1984; Hosang and Shooter, 1987; Johnson et
al., 1988).
Autoradiographic mapping of I125 NGF binding has
demonstrated high affinity NGF receptors in the basal forebrain (Bernd
et al., 1988), the caudate putamen, and most sympathetic and primary
sensory neurons (Richardson et al., 1986).
Immunocytochemical analysis
and immunoprecipitation assays have demonstrated NGF receptors in the
olfactory tubercle, medial septal nucleus (Taniuchi et al., 1986), the
vertical and horizontal limbs of the diagonal band of Broca, the basal
nucleus of the forebrain (Kiss et al., 1988), Purkinje cells of the
21
cerebellum (Taniuchi et al., 1886; Pioro and Cuello, 1988), brainstem,
cortex, hippocampus (Taniuchi et al., 1986), suprachiasmatic nucleus
(Sofroniew et al., 1989), and posterior pituitary (Yan and Johnson,
1990) of the rat. Hence, there is a growing body of evidence implicating
NGF activity and NGFR expression throughout the adult CNS.
A growing number of non-neuronal tissues and cell types, not
believed to be NGF responsive, have been shown to express the low
affinity form of NGF receptor and/or the NGF protein during development,
following injury, or in cell culture.
These include Schwann cells
(Taniuchi et al., 1986; Johnson et al., 1988; Assouline and Pantazis,
1989; Matsuoka et al., 1991), cultured astrocytes (Furakawa et al.,
1986; Yoshida and Gage, 1991), fibroblasts (Furukawa et al., 1985;
Yoshida and Gage, 1991), and activated microglia (Yoshida and Gage,
1991) .
Fibroblasts will secrete NGF but have not been shown to express
NGF receptor in rat or human (Assouline and Pantazis, 1989).
CNS
astrocytes also express NGF message (Lu et al., 1991) and protein, but
not the NGF receptor,
(Assouline and Panatazis, 1989) in culture
(Yoshida and Gage, 1991), during development and following axotomy of
the optic nerve of the adult rat (Lu et al., 1991).
In vitro studies
have demonstrated the expression of NGF to be highest during stages of
rapid glial growth (Yoshida and Gage, 1991).
Non-neuronal cells express only the low affinity form of .the NGF
receptor (Johnson et al., 1988) and are not capable of internalizing or
utilizing the NGF protein.
The expression of NGF receptors and
secretion, of NGF protein by Schwann cells is developmentally regulated
(Johnson et al., 1988).
development.
Binding of NGF occurred from days E6-E16 of
After this stage the receptor expression diminished to
undetectable levels (Johnson et al., 1988).
However, following
peripheral axotomy a reexpression of low affinity NGF receptor by the
ensheathing Schwann cells (10-50 fold) appeared within 3 days and peaked
at 5-7 days (Taniuchi et al., 1986; Johnson et al., 1988).
The
22
reexpression was confined to the region distal to the lesion.
No
reexpression was detected proximal to the lesion where axon-Schwann cell
contact remained, indicating a contact mediated suppression of NGF
receptor expression by Schwann cells (Johnson et al., 1988).
The
presentation of bound NGF on the surface of the Schwann cell (presumably
secreted by the same Schwann cell) may play a role in the cell's
contribution to axonal regeneration following axotomy by providing an
NGF-Iined path for the regenerating axon to follow (Johnson et al.,
1988).
Once the regenerating axon reached its NGF secreting target
tissue, NGF secretion and expression of NGF receptors were shut off.
Within the posterior pituitary of the neonatal rat, NGF receptor
expression (detected immunocytochemically) continues to intensify in
expression throughout the first year of life (Yan et al., 1990).
Following transection of the pituitary stalk, the NGF receptor
immunoreactivity rose dramatically within 3 days and peaked at
approximately 15 days following stalk transection (Yan et al., 1990).
This correlates closely to the time when axon degeneration was occurring
and phagocytosis of the axonal debris was at a maximum.
Dehydration,
lactation, castration in male rats, pregnancy and colchicine injection
all produced alterations in glial and/or neurosecretory activity in the
neural lobe.
However, none of these manipulations resulted in an
increase in NGF receptor immunoreactivity.
These data would suggest
that the increased glial activity associated with phagocytosis of the
axonal debris was responsible for the increase in NGFR immunoreactivity.
The cell type within the neural lobe thought to be responsible for
phagocytosis is the perivascular microglial cell (Zambrano and Robertis,
1979; Watt and Paden, 1991).
In summary, the occurrence of NGF and NGF receptors has been
repeatedly demonstrated in both neuronal and non^neuronal cells of the
peripheral and central nervous systems.
Within the adult CNS the role
of NGF and NGF receptors remains unresolved.
Indications of up
23
regulation of NGF protein and receptor following axotomy reflect a
potential for CNS repair similar, but more limited in extent, to that
observed in the peripheral nervous system.
Justification, Rational and Specific Objectives
Role of Glia in Axonal Sprouting.
The proliferative astroglial response to penetrating injury often
leads to inflammation and the development of scar tissue which may act
as a barrier to axonal regeneration.
However, there is accumulating
evidence to suggest that astroglia and microglia may also play a
supportive role in axonal sprouting, typically under conditions in which
inflammation, tissue insult and disruption of the blood-brain barrier do
not occur (Gall et al., 1979; Gage et al., 1988).
The principle
advantage of our model of collateral sprouting (Watt and Paden, 1991) is
that the use of a unilateral hypothalamic knife cut, instead of an
infundibular stalk section, hypophysectomy or neural lobectomy, permits
the sprouting response of uninjured neurons to be studied in the intact
NL without the complications arising from hemorrhage or scar formation.
The NL in turn offers a highly simplified tissue, of CNS origin but
lying outside the blood-brain barrier, in which to investigate problems
such as the role of glial and neuronal activation in the sprouting
response of central neurons.
At least two distinct populations of glial cells reside within the
N L , pituicytes and perivascular cells.
Immunocytochemical application
of an antibody directed against glial fibrillary acidic protein (GFAP),
a known astrocyte-specific marker, indicates pituicytes are GFAPpositive and thus an astroglial-like cell (Velasko et al., 1979; Suess
and Pliska, 1981; Salm et al., 1982).
Pituicytes are characterized by
highly asymmetric and extensive processes in close juxtaposition to
neurosecretory axons and have a more velate appearance compared to
astrocytes (Salm et al., 1982).
Although the functional role of
24
pituicytes remains unresolved, they are known to ensheathe axonal
endings within the NL when these axons are not in contact with the
external basement membrane of the perivascular space.
However, upon
stimulation of the anti-diuretic system the pituicytes will rapidly
withdraw from the axon terminals allowing terminal/basement membrane
contact for neurosecretion.
Additionally, pituicytes will cover the
basement membrane left exposed upon axon terminal retraction (Wittkowski
and Brinkman, 1974).
We believe the intimate relationship between
pituicytes and axons may be crucial in the sprouting response.
A
similar contiguous relationship between pituicytes and axons has been
reported in the developing NL (Galabov and Scheibler, 1978; Dellman and
Sikora, 1981) and during regenerative sprouting in the proximal
infundibulum (Kiernan, 1971).
NL perivascular cells are thought to be related to CNS microglia
(Fugita and Hartman, 1961; Zambrano and de Robertis, 1968).
Microglia,
in turn are thought to be of mesenchymal origin and thus are not related
to neuronal or neuroglial elements which arise from the germinal matrix
(Dickson and Mattiace, 1989).
Antibodies which recognize mesodermally-
derived elements such as macrophages will also recognize microglia
(Perry et al., 1985; Guilian et al., 1976; Esiri and McGee, 1986; Hickey
and Kimura, 1986).
Likewise antibodies directed against major
histocompatibility complex (MHC) class II, commonly detected on immune
cells, will also recognize microglia (Dickson and Matiace, 1989).
However, microglia can be distinguished from tissue macrophages and
blood monocytes on the basis of histochemical, morphologic and mitogenic
analyses (Giulian et al., 1986).
Our data indicate a functional
relationship between perivascular cells of the NL and activated
macrophages. We have observed that perivascular cells are solely
responsible for phagocytosis of degenerating neurosecretory axons
following knife cut (Watt and Paden, 1991).
This activity is apparent
at 5 days following knife cut and is characterized by an apparent
25
increase in the number of perivascular cells within the N L , an increase
in the number of electron dense cytoplasmic lysosomal granules, highly
dilated rough endoplasmic reticulum (RER) filled with a dense amorphous
material and the presence in some perivascular cells of phagocytic
debris (Watt and Paden, 1991).
Likewise, Zambrano and De Robertis
(1968) have shown perivascular cells to be exclusively involved in the
phagocytosis of degenerating neurosecretory axons within the NL
following bilateral electrolytic destruction of the PVN.
Neuroglia and microglia may play an important role in neural
repair following injury.
Hypertrophy of astrocytes occurs surrounding
sites of brain injury and neuronal death (Jacobson, 1978) as does
proliferation of astrocytes in proximity to the site of an injury
(Cavanagh, 1970; Latov et al., 1979).
Microglia proliferate at a very
high rate within 2-5 days following axotomy of the facial nerve
(Kreutzberg and Barron, 1978) and within the outer molecular layer of
the hippocampus following partial deafferentation (Gall et al, 1979).
In this instance the proliferation of microglial cells preceded the
increase in numbers of astrocytes.
This sequential pattern is supported
by evidence suggesting microglial cells secrete factors involved in the
proliferation of astrocytes (Giulian and Baker, 1985).
Furthermore,
production of "glia-promoting factors" can be elicited by brain injury
(Giulian et al, 1976).
The possibility remains that the perivascular
cells of the neurosecretory system are in fact a form of microglial
cell.
If so, it would seem likely that these cells would express
immunologic cell surface markers as do microglia and macrophages (Perry
et al, 1985; Esiri and McGee, 1986; Hikey and Kimura, 1988; Dickson and
Mattiace, 1989).
Likewise, immunopositive staining of pituicytes with
GFAP indicates that these cells are at least related to, and possibly
share a common ontogeny with, brain astrocytes.
If so, the relationship
between these cell types in the neural lobe may closely parallel that
observed between the microglia and astrocytes of the brain, particularly
26
in response to brain injury or neuronal death.
My working hypothesis is that the degenerative debris resulting
from axonal degradation stimulates the activation, proliferation and
phagocytic response of perivascular cells.
This phase is followed by
the activation, hypertrophy and possible proliferation of resident
pituicytes, possibly as a result of one or more factors released by the
activated perivascular cell population.
The focus of this investigation
was on the hypertrophy and hyperplasia of the glial population.
Neuronal Activation in Collateral Sprouting.
Cellular hypertrophy has been repeatedly demonstrated to correlate
with the onset of compensatory sprouting of intact afferents. to a
partially denervated site in a variety of CNS systems (Goldschmitt and
Steward, 1980; Headon et al., 1985; Hendrickson and Dineen, 1982;
Pearson, et al., 1987a; Pearson, et al., 1987b).
Likewise, hypertrophy
of SON neurons has been demonstrated to occur concomitant with the
reformation of neurosecretory nerve terminals in the proximal
infundibulum following hypophysectomy (Raisman, 1987).
'
We have
demonstrated that hypertrophy of magnocellular neurons in the
contralateral SON coincided with the increase in axon number observed at
30 and 90 days following partial denervation of the NL (Watt and Paden,
1991).
These observations are consistent with the conclusion that
compensatory sprouting of intact neurosecretory efferents was occurring
at this time.
Cellular hypertrophy in the magnocellular neurosecretory
system has also been associated with heightened secretory activity
induced by dehydration or salt loading (Enestrom, 1967; Morris and
Dyball, 1974; Peterson, 1966).
However, these acute effects occur
rapidly, and the somata return to normal size soon after cessation of
the osmotic stimulation (Enestrom, 1967; Morris and Dyball, 1974).
contrast, the prolonged delay observed in our study between partial
denervation of the NL and the increase in cell size suggests the
In
27
cellular enlargement is not the result of increased vasopressin
synthesis or secretion.
We believe the increase in SON and PVN cell size observed may
reflect the increased cellular metabolic activity required to maintain
an enlarged terminal arbor.
Furthermore, this enhanced activational
state may be a critical factor in determining the extent of the
regenerative response.
Herman and Gash (1987) demonstrated that
vasopressinergic, vs oxytocinergic, cellular activity following NL
ablation could be selectively inhibited via chronic vasopressin
infusion.
Results of their study suggest inhibition of vasopressinergic
cell activity inhibits axonal regeneration and ultimately results in
cell death, with no effect on the activational state, cell survival, or
regenerative capacity of the axotomized oxytocinergic neurons.
These
results indicate the critical importance of heightened cellular activity
to
regenerative capacity (although, the specific form of activity,
e.g., gene expression, synthetic, or electrophysiological, remains
unclear).
An advantage of the MNS as a model system for the study of
sprouting is the possibility of selectively activating and suppressing
vasopressinergic or oxytocinergic cell activity using different
hormones,
(e.g., vasopressin and oxytocin), and different physiological
states (e.g., pregnancy, lactation, dehydration).
Other investigators
have demonstrated a difference in the temporal pattern of vasopressin
and oxytocin immunoreactivity in the median eminence following
hypophysectomy relative to the onset of sprouting of vasopressin axons
as a function of age in both rats (Ibata et al., 1983; Kawamoto and
Kawashima, 1987) and mice (Kawamoto, 1985).
These findings suggest that
there may well be differences in the ability of oxytocin vs vasopressin
cells to undergo compensatory sprouting.
The basis of such differences
and their relation to cell activity remain unknown.
I proposed to
explore this problem by determining the temporal pattern of oxytocin vs
vasopressin neuronal hypertrophy in the contralateral SON and its
28
possible correlation with the onset of compensatory sprouting by
oxytocin and vasopressin axons in the partially denervated N L .
Further
exploration of any differences in vasopressin vs oxytocin neuronal
activation and sprouting may provide new information regarding factors
that affect the neuronal propensity for compensatory sprouting.
Specific Objectives.
A novel model of compensatory collateral
sprouting of intact magnocellular vasopressinergic efferents following
partial denervation of the rat NL has been reported (Watt and Paden,
1991). In addition to the sprouting response, further analysis revealed
an increase in the relative area of the NL occupied by glial cells.
This increase corresponded to the period of degeneration, activation of
the vasopressinergic neurons and onset of sprouting.
My objectives are
divided into two principle areas of investigation: I) to further
investigate the role of non-neuronal elements in the facilitation,
initiation, and/or maintenance of the compensatory sprouting response
and, 2) to measure the neuronal response to partial denervation and
possible correlations with the sprouting response.
These data may provide clues regarding the mechanisms regulating
the glial and neuronal response to denervation and ultimately to improve
our understanding of the glial role in central nervous system (CNS)
repair.
The specific aims of the proposed research and hypotheses to be
tested are as follows:
Non-Neuronal Contribution to MNS Response to Injury
I) To determine
if the observed increase in relative area of partially denervated NL
occupied by glia is due to cellular hypertrophy of resident pituicytes
and/or perivascular cells or to a proliferative response resulting in an
increase in the absolute number of cells in either glial population.
2) To determine the vascular response to partial denervation of the NL
and its possible correlations with degeneration and sprouting of
neurosecretory axons.
29
3) To investigate the expression and possible upregulation of NGF
receptors on glial cells in the N L .
Neuronal Activity
I) To investigate the possible occurrence of
regenerative sprouting of neurosecretory axons within the hypothalamus.
2) To investigate the possible alterations in drinking volume, urine
excretion volume and urine osmolality as an indirect means of
determining whether the sprouting response may result from a lesioninduced deficit in plasma vasopressin levels.
3) To determine if a differential response in cellular
activation/hypertrophy exists between oxytocin- and vasopressinimmunoreactive cells in the SON.
30
METHODS
Animals
Male Holtzman rats (bred from stocks originally obtained from
Charles River) were 35 to 40 days of age at the time stereotaxic surgery
was performed.
The sprouting efficacy of transected neurosecretory
axons has been shown to decrease with age (Kawamoto and Kawashima, 1985,
1987) and this reduced propensity has been observed as early as 23 days
post-parturition (Kawamoto and Kawashima, 1987).
Therefore, young rats
between the ages of 35-40 days post-parturition were utilized in an
effort to minimize any age-related impairment of the neurosecretory
system response to partial denervation.
Animals were either
individually or group housed under a 12L:12D light cycle and had ad lib
access to lab chow and tap water throughout the investigations.
Surgery
Prior to surgery each animal received Sodium-Pentobarbital,
(50mg/kg intraperitoneal (IP)) to induce a surgical plane of anesthesia.
Animals received methoxyflurane to prolong or deepen the plane of
anesthesia, as necessary.
Each animal received a preanesthetic
injection of atropine sulfate, 0.5mg subcutaneous (SC), to reduce mucous
secretions.
Following anesthesia, the heads were shaved, washed with
100% ethanol (ETOH) and positioned in the stereotaxic apparatus with the
incisor bar set at 3 mm below the horizontal position.
Incisions were
then made in a caudal to rostral direction and the dorsal surface of the
cranium exposed, cleared of fascia and dried.
The tip of the
stereotaxic knife blade (see appendix for materials and design), was
positioned at bregma and the coordinates recorded.
coordinates, AP -3mm to +5mm, ML -0.5mm,
The lesion
(Paxinos and Watson, 1986) were
then marked on the surface of the cranium with bregma serving as
stereotaxic zero.
A burr hole was then made through the skull using a
31
Dremel moto-flex drill and a #3 carbide burr, with care taken to ensure
that the hole had clean, smooth edges to reduce the possibility of scalp
irritation after surgery.
The dura mater was incised using a sterile,
27 gauge hypodermic needle.
Any excessive bleeding was interrupted by
a.pplying direct pressure with a Kimwipe pad directly to the wound site.
Once the incision was completed and cleared of debris, the stereotaxic
knife blade was then repositioned at -2mm A P , and lowered through the
brain to the ventral surface of the cranial cavity.
When the knife
blade contacted the skull surface, the blade flexed slightly.
The blade
was then retracted Imm and the knife slowly brought forward to the
rostral extent of the lesion.
The blade was then retracted and any
blood and/or debris was cleared from the site.
first filling the burr hole with Gelfoam.
The wound was closed by
The surface of the cranium
was then gently wiped clean and nitrofurazone topical antibiotic applied
liberally, and the scalp sutured.
The animal was then removed from the
stereotaxic apparatus and replaced in his home cage for recovery.
All
sham controls were prepared in a like manner except that the knife blade
was lowered only 4 mm from the dorsal surface of the skull.
This lesion
depth transected the cortex but did not impact the hypothalamus, thus
sparing the PVN and SON nuclei from damage.
Tissue Preparation
Tissue was collected from groups of experimental animals
sacrificed between 12 hours and 150 days PS as well as from age-matched
sham-operated and intact controls.
Animals to be used for morphometric
analysis of immunocytochemically-labeled vasopressin and oxytocin
neurons of the SON were perfused intracardially with 4% paraformaldehyde
in .IM phosphate buffer under deep ether anesthesia, then decapitated
and the brain and NL removed.
The brain and attached NL were then
submerged and postfixed in the perfusion medium overnight, then
dehydrated, cleared in xylene and embedded in paraffin.
Animals to be
32
used for Bromodeoxyuridine (Brdu) substitution analysis were first
placed under light ether anesthesia and then given a I ml IP injection
of 20.0 mg/ml Brdu in .007M NaOH vehicle.
Animals were allowed to
survive for three hours following injection and then were perfused
intracardially with saline solution for three minutes under deep ether
anesthesia.
The brain and attached NL were removed and immersion fixed
with Carnoys' acidified alcohol fixative overnight.
The tissue was then
dehydrated for 3 hours in absolute ethanol, cleared for three hours in
xylene and embedded in paraffin.
Animals involved in the capillary morphometry and glial population
estimates were anesthetized with ether and then perfused intracardially
with 4% paraformaldehyde plus 2% glutaraldehyde in .IM phosphate buffer.
The NL was then removed and postfixed overnight in the perfusion medium,
then dehydrated, cleared in xylene and embedded in paraffin. The NL was
postfixed 4 hours in 2% osmium tetroxide, dehydrated in ethanol and
propylene oxide and then embedded with Spurr's epoxy resins in flatmount Beem capsule molds with the rostral end of the NL positioned at
the tip of the Beem capsule for orientation.
Tissues to be utilized for immunohistochemical analysis of the p75
low affinity nerve growth factor receptor (NGFR) were first perfused
intracardially under deep ether anesthesia with cold saline for two
minutes and then perfused for 20 minutes with Nakane's periodate-lysineparaformaldehyde (PLP) fixative.
paraformaldehyde,
The PLP fixative containing 2%
.075M lysine and .OlM sodium periodate in .IM PO4 was
prepared immediately before use.
Following perfusion, the brain and NL
were removed intact and postfixed overnight in PLP, cryoprotected in 30%
sucrose/PBS for 24-48 hours at 4°C and then snap frozen in molds filled
with OCT freezing compound using isopentane chilled to -160 degrees
centigrade in liquid nitrogen.
The tissues were then stored at -70
degrees centigrade until sectioned.
Once sectioned and mounted on
gelatin-coated slides, the sections were stored at room temperature.
33
Tissue Microtomy
Paraffin microtomy was performed on a Leitz Wetzlab microtome.
For the investigation of SON neuronal hypertrophy ten micron serial
sections were collected from each specimen block, float-mounted singly
on gelatin coated slides and dried overnight on a slide warmer prior to
use.
Those sections utilized for Brdu analysis were sectioned at six
microns and mounted in a similar manner.
NGFR analysis were
All fresh frozen tissues for
sectioned at ten microns using a Reichert-Jung
Frigocut 2800 cryostat and thaw mounted on gelatin-coated slides.
For capillary morphometry and glial nuclear counts one micron
semithin sections of Spurr's embedded NL were sectioned using a Reichert
Nr 318 435/E ultramicrotome using glass knives and collected from each
of four subject animals per lesion group and three animals per agematched control group.
Sections were mounted on gelatin-coated
microscope slides by placing each section on a drop of distilled water
and then drying briefly on a slide warming tray.
All sections on a
given slide were then coated with a liberal volume of 1% basic toluidine
blue and warmed for approximately 5-7 minutes before being rinsed in
distilled water.
The staining intensity was then adjusted by brief
immersion of the slide in 95% and 100% ethanol and coverslipping with
Permount.
Immunohistochemistrv
In a previous investigation we were able to demonstrate
hypertrophy of SON and PVN neurons and their respective nuclei which
coincided with the collateral sprouting response of vasopressinergic
neurosecretory axons (Watt and Paden, 1991).
However, the Nissl stain
utilized to visualize these neurons did not allow for a direct
comparison between the vasopressin- and oxytocin-immunoreactive neurons
within either nuclei.
Therefore, the possibility of a differential
response between vasopressin and oxytocin immunoreactive neurons within
34
the SON was investigated by selectively immuneIabeling each of the two
neuronal populations for subsequent morphometric analysis.
Both
peroxidase and silver-intensified immunogold preparations were utilized
in an effort to produce the most sensitive and accurate estimation of
neuronal size, as described in detail below.
Immunohistochemical application of anti-vasopressin-neurophysin
(gift of Dr. Alan Robinson) and anti-synthetic oxytocin (Accurate) was
performed on paraffin-embedded coronal sections from each of three
experimental groups (6-8 animals/group) and four intact control groups
(4 animals/group).
The intact groups were included to control for any
age-related increase in neuronal and/or nuclear size.
The number of
sections prepared for each animal was solely dependent on the number
required to measure a minimum of 25 cell bodies and their respective
nuclei in each animal.
and nuclei measured.
Most animals, however, had 35 to 50 cell bodies
Sections were cleared of all traces of paraffin
and then rehydrated through decreasing concentrations of ethanol leading
to phosphate buffered saline (PBS).
All sections were then incubated in
either rabbit anti-vasopressin-neurophysin (1:1000) or rabbit anti­
synthetic oxytocin (1:2000) in blocking serum composed of bovine serum
albumin and normal goat serum, pH 7.4, overnight at room temperature in
a plastic hydration chamber.
Sections were then rinsed in PBS for two
successive ten minute increments and exposed to the secondary antibody,
biotinylated goat anti-rabbit IgG (1:200)(Cappel) for one hour at room
temperature in a hydration chamber.
Sections were again rinsed twice in
PBS then incubated in avidin-biotin complex (ABC)(Vector) for one hour
as before.
Finally, all sections were rinsed in PBS and then developed
in 3,3' diaminobenzidine (DAB)(Sigma) solution containing 300 jul of I
mg/ml glucose oxidase and 600 jul of 1% cobalt chloride per 100 ml of DAB
solution.
Silver-enhanced immunohistochemistry was performed as follows.
Sections were cleared of paraffin in xylene and then rehydrated through
35
successive decreasing concentrations of ethanol to tris-phosphate
buffered saline (TPBS) pH 7.4.
All sections were then incubated for one
hour in TPBS blocking serum (GSTPBS) containing 3.0% normal goat serum.
The GSTPBS was then wicked away using Kimwipes and the primary antisera,
rabbit anti vasopressin-neurophysin (1:40,000)(gift of Dr. Alan .
Robinson), was applied and left overnight at room temperature in a
plastic hydration chamber.
All sections were then washed for 60 minutes
in GSTPBS and the secondary antibody, goat anti-rabbit IgG conjugated to
a I nm gold particle (BioCell), was applied and incubated at room
temperature for 60 minutes.
All sections were then washed in GSTPBS for
60 minutes and then washed for 10 additional minutes in sterile water
(pyrogen-free for injection)(Kendall McGraw).
Sections were washed in
.2M citrate buffer, pH 3.85, for 10 minutes to slow the rate of silver
deposition.
Sections were then placed in a silver development solution
which was prepared by combining 15 mis of 5.6% hydroquinone in sterile
water, 15 ml of 0.7% silver lactate in sterile water, 10 ml of 2.OM
sodium citrate buffer (pH 8.5) and 60 ml sterile water.
Sections were
then developed under a sodium vapor lamp for 8-16 minutes or until the
reaction was judged to be complete.
The development reaction was then
halted by replacing the silver solution with a 5% acetic acid stop bath
for one minute, and then fixing the sections in Kodak fixer for 10
minutes.
All sections were then washed in a continuous flow of tap
water for 30 minutes, counterstained in a 1% working solution of cresyl
violet (Aldrich) and coverslipped in Permount.
Low affinity (p75) nerve growth factor receptor (NGFR) expression
in the NL was measured by applying the mouse monoclonal antibody 192-IgG
(gift of Dr. E.M. Johnson, Jr.) to tissue sections selected from lesion
and sham operated animals sacrificed at post-surgery days 0, 2, 5, 7,
10, 35 and 90.
A minimum of 8 sections of NL were selected from each of
4-6 animals within each group.
Tissue sections were selected from
approximately the same depths through the NL across all animals in all
36
groups to ensure that a reasonably equivalent comparison was made
between animals and between experimental groups.
The large number of
sections stained and the nature of the analysis (see below) required
that sections be prepared in batches of 18.
In order to ensure that
variations in staining intensity not bias the analysis, each batch
contained sections selected from animals representing both lesion and
sham preparations from several time points.
Sections were washed for 30
minutes in Tris-buffered saline and then incubated overnight with
primary antibody 192-IgG (1:100) in TBS blocking serum (TBS/NHS), pH
7.6, containing 1% normal horse serum.
All incubations were conducted
at room temperature in a plastic hydration chamber.
Following the
primary antibody, the sections were washed for a minimum of two 10
minute intervals in TBS and then incubated with the secondary antibody,
biotinylated horse anti-mouse (1:500)(Vector) in TBS/NHS, pH 7.6.
Sections were then washed in TBS for two 10 minute intervals and
incubated in ABC for 60 minutes.
Again, sections were washed in TBS for
two successive 10 minute intervals and then developed in DAB in darkness
for 60 minutes.
All sections were then washed for a minimum of ten
minutes in TBS, dehydrated through increasing concentrations of ethanol,
cleared in xylene and coverslipped in Permount.
Immunocytochemical analysis of Brdu substitution was performed to
determine if the apparent increase in the number of pituicyte nuclei was
due to cell mitosis.
Cell counts indicated that this increase occurred
as early as 24 hours following the knife cut.
Therefore, animals
injected with Brdu were sacrificed at .5, I, 2, 3, 4, 6, 7, 10 and 30
days PS.
Sham controls were sacrificed at I, 2 and 10 days PS.
As
before, tissue sections were selected from approximately the same depths
through the NL across all animals in all groups to ensure that a
reasonably equivalent comparison was made between animals and between
experimental groups.
Selected sections were cleared of all traces of
paraffin in xylene and then rehydrated through decreasing concentrations
37
of ethanol leading to PBS.
Because the anti-Brdu antibody will
recognize only single stranded Brdu substituted DNA, denaturation was
performed by a 60 minute incubation in ZN HCL.
The primary antisera,
mouse anti Brdu (1:100)(Becton-Dickenson), diluted in blocking serum
(PBS containing 1% bovine serum albumin (BSA) and 1% normal goat serum)
was then applied and left overnight.
All incubations were performed at
room temperature in a plastic hydration chamber.
Sections were then
washed in two successive 10 minute PBS rinses and then exposed in
succession to the secondary antibody, biotinylated goat anti-mouse
(1:200) in blocking serum for 60 mins., two 10 minute PBS rinses, ABC
for 60 minutes, two 10 minute PBS rinses, then .025% DAB containing .03%
glucose oxidase solution (Sigma) with .03% cobalt enhancement for 15-20
minutes in darkness.
Each section was then counterstained in Gill's
Hematoxylin, dehydrated through a series of increasing concentrations of
ethanol, cleared in xylene and coverslipped in Permount.
Once having determined that Brdu substitution had occurred, it was
necessary to identify the proliferative cell type(s ).
This was
accomplished by utilizing both dual-label immunofluorescence and dual­
label peroxidase immunocytochemistry.
Immunofluorescent preparations
were subsequently analyzed utilizing confocal microscopy.
For dual fluorescence immunocytochemistry, selected sections were
cleared of all traces of paraffin in xylene and then rehydrated through
decreasing concentrations of ethanol leading to PBS.
Sections were then
incubated in 2N HCL for 60 minutes, followed by three 10 minute PBS
washes.
The primary antisera, both mouse anti-Brdu (1:100)(Becton-
Dickenson), and rabbit anti-glial fibrillary acidic protein
(GFAP)(1:250)(Dako) were diluted in blocking buffer (containing 1% (BSA)
and 1% normal mouse serum), was then applied and left overnight.
All
incubations were performed at room temperature in a plastic hydration
chamber.
Sections were then washed in two successive 10 minute PBS
rinses and exposed in succession to: biotinylated goat anti-mouse
38
antibodies (b-GAM) for 60 minutes, two 10 minute PBS washes, an avidinphycoerythrin (1:200)(Avidin-PE)(Accurate-Phycoprobe) and fluorescein
isothyocyanate-goat anti-rabbit (1:500)(FITC-GAR)(Hyclone) solution for
60 minutes, and two 10 minute PBS washes.
Slides were then coverslipped
in PBS:glycerol with Img/ml O-phenylenediamine.
For dual label peroxidase immunocytochemistry the sections were
cleared, rehydrated and denatured as described above and then exposed to
the following sequence: anti-Brdu (1:100) overnight at room temp., 2x10
min PBS washes, b-GAM (1:200) for 60 minutes at room temp., 2x10 min PBS
washes, ABC for 60 minutes, room temp., 2x10 min PBS washes, development
in DAB with cobalt enhancement for 15-20 minutes, PBS wash for a minimum
of 30 minutes, the second primary antibody (e.g. anti GFAP overnight at
room temp.), 2x10 min PBS washes, secondary antibody b-GAR (1:200) for
60 minutes at room temp., 2x10 min PBS washes, ABC for 60 minutes at
room temp., 2x10 min PBS washes, DAB without cobalt enhancement for a
15-30 min development period, followed by a PBS wash, dehydration,
clearing and coverslipping in Permount.
This paradigm resulted in a
highly compartmentalized and very black Brdu nuclear staining against a
ruddy brown cytoplasmic GFAP staining, allowing the two antibody
staining patterns to be easily discerned and compared.
Image Analysis and Morphometric Measurements
192-IaG Proportional Area Analysis
Quantification of 192-IgG immunopositive proportional area was
performed on an MCID image analysis system (Imaging Research Inc.).
Microscope illumination was stabilized with a SOLA CVS transformer to
prevent fluctuations in illumination intensities from biasing
measurements of the 192-IgG immunopositive area.
Caution was taken when
reaccessing the MCID program that illumination intensities and
microscope apperature settings were precisely the same as for all
previous sessions.
Calibrations were checked prior to each sampling
39
session against a known sample.
Images were projected from an Olympus
BH-2 light microscope using a IOx objective through a Sierra Scientific
CCD high resolution camera for relay to the MCID program and displayed
on an Electrohome monitor for image editing and analysis.
The IOX objective was the lowest magnification objective that
could be utilized without compromising the clarity and resolution of the
immunostained region under analysis.
However, this magnification
required that each coronal section of the NL be divided into multiple
sample frames for analysis.
These data were then compiled to determine
the proportional area of NL occupied by 192-IgG immunopositive cells
within a given animal and within a given experimental group.
All data
were downloaded directly into a spreadsheet program (Quatro Pro) for
statistical analysis.
SON Cellular Morphometry
Morphometric measures of somata and their respective nuclear were
collected from the SON contralateral to the knife cut.
The purpose of
these measures was to investigate the possibility of a differential
response between the vasopressin-neurophysin and oxytocin immunoreactive
neuronal populations.
Previous measures had shown a close correlation
between somata and cell nuclear hypertrophy (Watt and Paden, 1992).
Therefore, nuclear measures were again collected as a means of providing
evidence of enhanced neuronal activity and verification of the somata
measures.
The criteria for cell selection was that a distinct cell boundary,
a clearly defined nuclear envelope and a prominent nucleolus all be
present.
Measures were collected by first projecting a microscopic
image of the cells of the contralateral SON onto a ZIDAS digitizing pad
via an Olympus drawing tube.
First, the periphery of the cell body was
traced using a stylus interfaced with the digitizing pad.
The nucleus
was then measured and the cross sectional area of each measure was then
40
automatically printed.
All measures collected were then loaded into a
spreadsheet program (Quatro Pro) for analysis.
Mean cross sectional
area of cell bodies and nuclei were then calculated for each animal and
each experimental time point.
Glial Morphometry
Morphometric measures of pituicyte nuclei numbers and cross
sectional area were collected from one micron thick sections of Spurr'sembedded N L .
Pituicyte nuclei have previously been reported to undergo
a mitotic and hypertrophic response during periods of increased
neurosecretory activity induced by dehydration (Krsulovic and Bruckner,
1969).
The purpose of these measures was to determine if pituicyte
nuclei may also enlarge during degeneration or axonal sprouting in the
partially denervated N L .
Such a response would indicate heightened
pituicyte activity and provide indirect evidence of pituicyte
involvement in the response to partial denervation.
Measures were
collected by first projecting a microscopic image of a coronal section
of the NL onto a ZIDAS digitizing pad via a drawing tube.
Sample fields
were selected in each of three NL coronal sections per animal by
carefully aligning a grid containing five evenly spaced squares along
the long axis of the tissue section.
The grid was placed on the ZIDAS
and observed overlying the section via the drawing tube.
Each square
corresponded to one .02 mm2 sample field as viewed through a IOOX oil
objective.
At IOX each respective sample field was centered within the
viewing field and the IOOX objective then engaged.
A blank paper was
then placed on the ZIDAS overlying the grid and each nuclear profile
counted was checked off with either a black (pituicyte) or red
(perivascular cell) felt tip pen, thereby ensuring that no nuclei would
be counted more than once.
This also served to provide a permanent
record of all cell counts.
The cross sectional area of each NL coronal
section sampled was then measured and the mean number of nuclei per unit
41
area then determined for each section, animal and experimental group.
Measures of pituicyte nuclear cross sectional area were collected
by tracing the boundary of each pituicyte nuclear envelope within the
sample field that was clearly defined and contained a prominent
nucleolus.
All measures were then loaded into a spreadsheet (Quatro
Pro) for analysis.
The luminal surface of every.blood vessel displaying
a clearly defined endothelial wall within each of the five sample fields
was traced in a like manner.
Again, these data were then loaded into a
spreadsheet for analysis.
Metabolic Analysis
A total of 12 lesion animals and 6 sham control animals were
individually housed in Nalgene metabolic cages throughout the 100 day
experimental period.
Daily drinking and urine excretion volumes were
determined directly from graduated drinking and urine collection tubes,
respectively.
Urine samples were collected at a consistent time every
day (10:00 am) by pulling off a 2.0 ml sample from a consistent sample
depth and the sample was briefly centrifuged at low speed to remove any
precipitation or food contaminants prior to analysis.
A 10.0 ul sample
was then applied to a vapor pressure osmometer (Wescor) to determine the
relative urine osmolality for each animal every day.
Baseline data on
urine excretion volume, daily drinking volume and urine osmolality were
established over a seven day period prior to surgery.
This period
ensured the subject animal's adjustment to the metabolic cage as well as
the daily sample collection and cage cleaning routine.
Statistical Analysis
One way analysis of variance was performed using the MSUSTAT AVlW
program (developed by Dr. R.D. Lund).
ANOVA was followed by testing the
significance of differences in group means using the MSUSTAT program
COMPARE and the Neuman-Keuls and Least Significance Difference test.
>
42
RESULTS
Post-Mortem Observations within the Hypothalamus
The efficacy of the hypothalamic knife cut was determined by
examining brain sections stained with cresyl violet which were selected
from the most rostral to the most caudal aspect of the lesion tract in
every animal.
Only those animals which sustained a complete unilateral
transection of the hypothalamo-neurohypophysial tract were included in
any subsequent investigation.
The knife cut was often accompanied by a
marked edema and cerebral hemorrhage resulting in varying degrees of
cavitation surrounding the knife path.
This cavitation extended from
the dorsal surface to varying depths of the brain in most lesion
animals, particularly at early post-lesion times, and to a lesser extent
in sham controls.
The cavitation typically compressed the ipsilateral
PVN, resulting in complete neuronal death.
PVN neurons spared.
Rarely were any ipsilateral
No apparent displacement of the contralateral PVN
or ipsilateral SON was ever observed.
Within the cavity of the lesion
tract was an extensive invasion of macrophages, monocytes and other
blood born elements which persisted throughout the first 10 days
following surgery.
Within the parenchyma surrounding the knife cut an
extensive gliosis was consistently observed which persisted for the
first two weeks post-lesion.
A marked hypertrophy of astrocytes was
apparent throughout the ipsilateral hemisphere and particularly in close
proximity to the lesion tract.
No such astrocyte hypertrophy was
notable in the contralateral hemisphere except within the corpus
callosum.
Neovascular growth was observed surrounding the knife tract in all
animals as early as 10 days following the knife cut with new vessels
developing in close juxtaposition to the lesion wall.
These vessels
ranged in size from small arterioles to vessels with diameters greater
than any observed within the hypothalamus of the normal animal.
In a
43
serendipitous observation it was noted that by 90 days following the
knife cut much of the neovasculature surrounding the lesion tract was
selectively immunostained, using silver-enhanced immunogold labeling,
with antibodies against the neuropeptide oxytocin-neurophysin.
However,
when adjacent sections were prepared in an identical fashion with
antibodies against vasopressin-neurophysin, no immunostaining was ever
observed (Fig. I).
This observation was made in animals of both the 30
and 90 day PS groups across literally dozens of like preparations.
Immunostaining was never observed in any tissues prior to 30 days PS,
Figure I. Oxytocinergic Perivascular Plexis Within the Hypothalamus.
A. Silver-enhanced immunogold localization of oxytocin-neurophysin
immunoreactive plexis surrounding the neovasculature in the 90 day
animal (solid arrows). B. An adjacent section stained with vasopressinneurophysin in an identical fashion (solid arrows). A linear scar forms
the boundary of the lesion tract (asterisk). Magnification, 200X.
44
nor were individual axons or fiber bundles observed coursing toward
these apparent neurosecretory plexuses. However, fibers were observed
projecting between individual plexuses in close juxtaposition to the
cavity wall.
Perivascular plexuses were observed in animals regardless
of the extent of neuronal loss in the ipsilateral SON.
The possibility
of non-specific staining seems remote given that the internal surface of
the lesion cavity and tears within the tissue show no immunoreactivity.
Likewise, many vessel profiles were observed throughout the brain which
showed negative immunoreactivity.
The specificity of staining for oxytocin vs vasopressinneurophysin peptides, the appearance of immunopositive plexuses at only
30 and 90 days PS and the absence of non-specific staining suggest that
the plexuses form through extensive outgrowth of hypothalamic
neurosecretory axons.
Lesion-Induced Changes in Urine Osmolality. Excretion, and Daily Water
Intake.
The principle objective of this experiment was to determine if the
stimulus for the compensatory axonal sprouting observed was due to a
response by the neurosecretory system to recover from a functional
deficit in circulating plasma VP levels or to a growth response due in
part to lesion-induced stimuli.
The first step in exploring these
possibilities was to determine if, in fact, the antidiuretic system had
been compromised by the lesion.
The possibility of lesion-induced changes in circulating plasma VP
levels was explored indirectly by measuring urine osmolality as well as
daily drinking and urine excretion volumes.
Daily measurements of urine
osmolality revealed an initial transient hypoosmolality, followed within
48 hours by a chronic and significant (P<.05, t-test, t=l.76, n=79 )
hyperosmolality which persisted throughout the 90 day post-surgical
survival period (Fig. 2).
The occurrence of the transient
hypoosmolality would suggest a substantial decrease in plasma VP levels
45
immediately following surgery which occurred in spite of a concomitant
decrease in both water intake and urine excretion volumes.
The
transient rebound in urine osmolality which occurred from day 2 to day 5
PS may reflect either a compensatory over-responsiveness resulting in
heightened secretion of plasma vasopressin or release of VP stores from
degenerating terminals.
The urine osmolality then stabilized at a level
significantly above that of sham control values for the remainder of the
experimental period.
A chronic and significant (P<.05, t-test, t=1.78, n=79 ) decrease
in both daily water intake (Fig. 3) and urine excretion volumes (Fig. 4)
URINE OSMOLALITY
2750
♦
LESION
D
SHAM CONTROL
2500
M 2250
2000
1750
1500
1250
0
5§ 10 15 20 25 30 35 40 45 50 55 60 65 70 75 50 55 90
I
DAYS PO ST-LESION
Figure 2. Effects of unilateral hypothalamic lesion on urine
osmolality.
Urine osmolality decreased markedly within the first 24
nours foiiowing surgery and then immediately increased to a
signifacantly (P<.05) hyperosmotic state which persisted throughout the
experimental period.
46
began immediately following surgery and persisted throughout the postsurgical period, paralleling the increased urine osmolality.
These data
suggest that the lesion did not result in a functional deficit in
circulating VP levels.
Hypertrophy of SON Somata and Nuclei Following Partial Denervation.
Cellular hypertrophy within the magnocellular neurosecretory
system occurs following experimental induction of heightened metabolic
activity via salt loading (Paterson and Leblond, 1977) or dehydration
and lactation (Watson, 1965) and is an accepted indicator of heightened
cellular activity (Vogels et al., 1990).
DAILY DRINKING VOLUME
Figure 3. Effects of unilateral hypothalamic lesion on daily drinking
volume. Daily water intake was significantly (P<.OS) reduced within 24
hours of the surgical intervention and remained depressed throughout the
experimental period.
47
Furthermore, cellular hypertrophy following partial denervation of a
terminal field has been correlated with axonal sprouting contralateral
to the site of injury (Goldschmidt and Steward, 1980).
In a previous
investigation (Watt and Paden, 1991) an enlargement of both
magnocellular somata and their respective cell nuclei was demonstrated
in the contralateral SON and PVN following partial denervation of the
NL.
Furthermore, these data indicated the hypertrophy occurred during
the sprouting phase of the post-lesion response.
However, the
hrstochemical staining paradigm used did not distinguish between
oxytocin and vasopressin magnocellular neurons, the two cell types in
the nucleus (Fig. 5).
URINE EXCRETION VOLUME
35 H
♦
LESION
D
SHAM CONTROL
25 -
15 10 -
0
5 1 10 15 20 25 30 35 40 45 50 55 60 65 70 75 SO S5 90
5
DAYS PO ST-LESICN
48
These distinct cell populations display differences in the temporal
pattern of axonal outgrowth and penetration of the NL terminal field
during development.
It seemed reasonable then to explore the
possibility of a differential hypertrophic response between the
vasopressin and oxytocin immunoreactive neurons within the contralateral
SON following partial denervation of the N L .
A highly significant
enlargement of vasopressin immunoreactive neurons (F=12.6, P<.00001, one
way ANOVA, df=6/29) and nuclei (F=8.67, P<.00001, one way ANOVA,
df=6/30) occurred following surgery when compared to controls.
Figure 5. Photomicrograph Demonstrating Silver-Enhanced Oxytocinergic
SON Neurons. Demonstration of the silver-enhanced immunogold labeling
of oxytocinergic magnocellular neurons in the SON utilizing anti
synthetic-oxytocin antibodies (solid arrowheads). The non-immunolabeled
cells are vasopressinergic magnocellular neurons (arrows). Note the
numerous immunolabeled fibers within and dorsal to the nucleus.
(OC)
Optic chiasm, (BV) blood vessel. Magnification, IOOX
49
By 10 days PS there was a 12% increase (one way ANOVA, Neuman-Keuls post
hoc comparison, not statistically significant (NS)) in the cross
sectional area of vasopressin-immunoreactive somata over age-matched
control values (Fig. 6).
By 30 days PS the difference had increased to
23.7% (P<.01, Neuman-Keuls) followed by a return to a 12% enlargement
above age-matched intact control values by 90 days post lesion (NS).
Within the control groups a continued growth of the vasopressin somata
was evident throughout the experimental period.
A significant 27%
(P<.01, Neuman-Keuls) increase in somata size was apparent between the
lesion day 0 and 90 day PS control.
Hypertrophy
of V a s o p r e s s i n
N e ur o ns
Figure 6. Hypertrophy of Vasopressinergic Neurons.
The cross-sectional
areas of at least 25 vasopressin magnocellular neurons were measured in
the contralateral SON nucleus of each animal. Age-matched sham controls
were included at 10, 30 and 90 days post-surgery.
Somata were
hypertrophic by 10 days, had reached significantly larger areas than
controls by 30 days and remained enlarged throughout the post-surgical
period.
Note the continued enlargement of somata with age in control
animals.
50
This continued cellular growth beyond the first 35 days post­
parturition demonstrates the importance of including age-matched
controls in these experiments.
The nuclei of these same VP-IR neurons
reflected a similar, albeit not identical, pattern (Fig. 6).
At 10 days
PS there is a non-significant but readily apparent 12.7% decrease in the
lesion group nuclear cross-sectional area when compared to age-matched
control values.
However, by 30 days PS a significant (P<.01, Neuman-
Keuls) increase in cross sectional area became evident with nuclear area
20% above age-matched control values.
By 90 days PS the nuclear
enlargement was no longer significantly different from
Hypertrophy
of V a s o p r e s s i n
Cell
Nuclei
Figure 7. Hypertrophy of Nuclei in Vasopressinergic Neurons.
The
cross-sectional areas of at least 25 vasopressin magnocellular cell
nuclei were measured in the contralateral SON of each animal. Agematched sham controls were included at 10, 30 and 90 days post-surgery.
A significant hypertrophy of somata occurred by 30 days (PC.01), which
then returned to values no different from age-matched controls by 90
days PS.
51
age-matched control values.
Thus, VP cellular nuclei demonstrate a more
temporally restricted response than observed in VP somata.
A continuing
age related enlargement in nuclear cross sectional area was also
apparent in the control groups reflected by a significant 28% (P<.01,
Neuman-Keuls) increase between day 0 and 90 days PS.
A highly significant enlargement of OT-immunoreactive somata
(F=9.24, P<.00001, one way ANOVA, df=6/28) and cell nuclei (F=6.6,
P < .0002, one way ANOVA, df= 6/29) was also observed (Fig. 7).
By 10
days PS a robust enlargement of OT somata was evident with mean cell
Hypertrophy
0
of Oxyto ci n
10
30
DAYS POST-SURGERY
Neurons
90
Figure 8. Hypertrophy of Oxytocinergic Neurons.
The cross-sectional
areas of at least 25 oxcytocin magnocellular neurons were measured in
the contralateral SON nucleus of each animal. Age-matched sham controls
were included at 10, 30 and 90 days post-surgery. A significant
hypertrophy of somata occurred by 30 days (P<.01), which then returned
to values no different from age-matched controls by 90 days PS.
52
cross sectional area 24.7% above the age-matched control value.
This
difference was maintained throughout the experimental period, with
experimental values 17% and 9.8% above age-matched control values at 30
and 90 days post-lesion, respectively.
Average nuclear cross sectional
areas were also markedly enlarged (NS)
by 10 days PS reaching 19% above
age-matched control levels (Fig. 8).
This difference was maintained at
17.4% at 30 days PS but had disappeared by 90 days PS.
Enlargement of
somata and cell nuclei was also evident in the control tissues
reflecting a continuing growth process in these cells throughout the
experimental period.
Hypertrophy
of
Oxytoci n Cell
Nuclei
Figure 9. Hypertrophy of Nuclei in Oxytocinergic Neurons.
The crosssectional areas of at least 25 oxytocinergic magnocellular cell nuclei
were measured in the contralateral SON of each animal. Age-matched sham
controls were included at 10, 30 and 90 days post-surgery. A marked
enlargement was apparent by 10 days PS persisting at 30 days, and
disappearing by 90 days PS.
53
Although both the vasopressin and oxytocin immunoreactive neurons
undergo a lesion-induced hypertrophy there are indications that the
oxytocinergic neurons and their respective nuclei may respond more
rapidly and/or more vigorously than their vasopressinergic counterparts.
Morphometric Assessment of the Neural Lobe Vascular Plexus.
Vascular endothelial cells show little proliferative activity in
the mature animal (D'Amore and Thompson, 1987) indicating a low rate of
collateral vessel growth under normal conditions.
However, under
certain conditions these normally quiescent cells may exibit
considerable growth.
Formation of the corpus luteum, chronically
excersized muscle, psoriasis, atherosclerosis, arthritis, tumor
vascularization and wound healing are all conditions which promote or
require vascular growth.
In the neurosecretory system an extensive
collateral vessel ingrowth has been demonstrated in the relatively
avascular proximal infundibulum following hypophysectomy (Raisman, 1973;
Adams, et al, 1969; Beck, et al, 1969).
Furthermore, the neovascular
sprouting precedes that of the regenerative sprouting of the
neurosecretory axons.
It has been suggested that this vascular growth
may act as a stimulus for the subsequent regenerative axonal sprouting
(Raisman, 1973).
Given this possible link between neovascular sprouting
and neurosecretory axonal sprouting, a morphometric investigation of the
vascular network within the partially denervated NL was undertaken in an
effort to further elucidate the mechanisms involved in the initiation or
maintanance of compensatory sprouting in the N L .
Figure 10 illustrates the mean number of vessel profiles/mm2
within the NL of lesion and age-matched intact control animals.
Although development of the NL vascular plexus is thought to reach
maturity by 20 days of age in the mouse (Eurenius, 1977) and 30 days in
the rat (Galabov and Schiebler, 1983) these data, indicate a continued
increase in capillary profiles until at least 45 days of age (PS 10).
54
The number of vessel proflies/unit area then shows a downward trend
reflecting an age-related reduction in vessel numbers or an increase in
NL area continuing to at least 90 days PS.
In contrast, the lesion
groups show a slight increase in vessel density by 2 days PS which
continues for at least 90 days reaching levels significantly higher
(P<.01, Neuman-Keuls) than age-matched control values at 90 days (Fig.
10).
It seems likely that these data reflect the maintenance of the
early postnatal vessel density as opposed to a lesion-induced collateral
sprouting of NL vessels.
The difference observed in vessel densities
between 90 day lesion (Fig. 11a) and age-matched controls
Density of Vessel Profiles
(PC.OI)
IN T A C T
1200
N=4
LESIO N
5
5
N=4
960-
LU
CC
<
z>
O
720-
</)
LU
E
O
cr
a.
480-
240-
0
0
2
5
10
DAYS POST-LESION
30
90
Figure 10. Effects of lesion on blood vessel density in the N L . A
gradual increase in vessel density occurred throughout the post-surgical
period.
There was a concomitant age-related decline in the vessel
density in control animals which resulted in a significant difference in
vessel density by 90 days PS.
55
(Fig. 11b) was readily apparent.
In order to further explore this
possibility the size of the vessels within each experimental group was
sorted by cross-sectional area into I of 10 bins (Table I).
The
rational for this approach was that an increase due to endothelial
proliferation would be evident as a relative increase in the number of
small vessels, i.e. capillaries, as opposed to the larger arterioles and
venuoles.
Table I.
Size Distribution Bins of NL Vascular Morphometry
Eouivalent Circle
Vessel Area
Range (jum2)
Bin #
.933-1.33
1.34-5.34
5.35-9.34
9.35-13.4
13.5-33.4
33.5-53.4
53.5-93.4
93.5-134
134-540
541-941
I
2
3
4
5
6
7
8
9
10
Mean (pm2)
1.13
3.33
7.33
11.35
23.37
43.38
73.38
113.39
340.00
741.00
diameter(pm) perimeter(pm)
3.77
6.47
9.60
11.94
17.14
23.35
30.37
37.75
65.36
96.50
1.20
2.06
3.05
3.80
5.45
7.43
9.67
12.02
20.81
30.72
Examination of the PS 2 lesion group indicates little difference
in the number of small vessels, when compared to day 35 intact controls.
However, there is a marked increase in the number of larger vessels
(bins 6-9) which may be indicative of an acute vasodilatory response
(Fig. 12).
By PS 5 these differences in distribution of vessel size are
still apparent although to a less pronounced degree, consistent with
indications of an early, acute vasodilatory response.
By 10 days PS a marked increase in the number a smaller vessels
becomes apparent.
By 30 days the continued increase in vessel numbers
now extends to include all size distribution bins (data not shown).
This trend is best illustrated at 90 days PS where increased vessels
were observed in all size categories (Fig. 13).
In summary, the
increase in large diameter vessels at 2 and 5 days PS with no comparable
increase in the number of small vessels indicates a vasodilatory
56
response.
This is followed at 10 days PS by a slight increase in the
number of small vessels which continues at 30 and 90 days PS.
By 90
days PS however, a shift to increased numbers of large vessels is
apparent as well.
By 125 days of age (PS 90) a distinct pattern becomes evident.
Vessel numbers drop off markedly from earlier age groups in bins 3-5
while remaining essentially equal in bins 7-10.
These data indicate an
age-related reduction in the number of small vessels with no equivalent
reduction in the largest vessels measured (Fig. 11).
These data indicate either growth of new collateral vessels or the
maintenance of the early postnatal vascular plexus over a period when,
in the normal animal, a decline in the number of small vessels is
apparent.
Figure 11. Photomicrograph of NL Vasculature.
Higher density of blood
vessels is maintained following unilateral knife cut. A. Neural lobe
of the lesion animal at 90 days PS. Note the large number of small
vessels perforating the N L . B. Neural lobe of age-matched intact
control tissue demonstrating the relative absence of blood vessels.
Magnification, 1000X.
57
The proportional area of the sample fields occupied by vessel
lumen was calculated to determine the net effect of the change in vessel
numbers on proportional vascular area (Fig. 14).
These data indicate a
significant (P<.01, Neuman-Keuls) increase in the proportional area of
the NL occupied by vascular elements within 48 hours PS when compared to
lesion day 0 controls.
By 5 days PS the response had waned with no
further differences between lesion and control animals observed.
However, from 30-90 days PS an upward trend in vessel number/unit area
became apparent in all size distribution bins (see Fig. 11).
Why then
would this increase not be evident in the proportional area analysis?
SIZE DISTRIBUTION OF NEURAL LOBE VASCULATURE
O
i
I
1
2
I
3
I
4
I
0
I
6
B IN
7
i
8
l
9
l
I
1
0
#
Figure 12. Size Distribution of PS 2 Neural Lobe Vasculature.
Increased vessel profiles were observed in the largest bin size
categories reflecting a vasodialation response 2 days following partial
denervation.
58
As a means of reapproaching this apparent discrepency the proportional
areas were again plotted but excluding bins 8, 9, and 10 (Fig. 15).
The
largest vessels, presumably arterioles and venuoles, by their very size
could mask any increase in proportional area resulting from an increase
in the size of the capillary plexus.
These results demonstrate a
pattern essentially identical to those displayed in Figure 10.
The
increase in proportional area observed at 2 days PS has returned to
levels more representative of the vessel density observed at the same
time, while by 90 days PS the proportional area of the lesion group is
significantly higher (P<.05, Neuman-Keuls) than intact controls.
Figure 13. Size Distribution of PS 90 Neural Lobe Vasculature.
Increased vessel profiles were observed in all size categories at 90
days PS reflecting the absence of an age-related decline in vessel
density apparent in control tissues.
59
These values now appear to more accurately reflect the observed increase
in vessel density. Although these data do not necessarily reflect a
growth response per se they do indicate that the principle alteration of
the NL vasculature was in the capillary plexus as opposed to the larger
arterioles and venuoles.
Low Affinity (o75) Nerve Growth Factor Receptor Immunoreactivitv in N L .
Monoclonal antibody (Mab) IgG-192 has been shown to bind
specifically to low affinity nerve growth factor receptors (NGFR) in
intact N L , infundibulum and median eminence but not anterior lobe.
Proportional
1.00
<
UJ
Area
of
Capillary
Plexus
INTACT
(P<.05)
LESION
N =4
0.80
GC
<
-J
<
O
0.60
N=3
U- CU
O
LU
Z
—
O
0.40
E
O
CL
O
CC
CL
0.20
0.00
0
Figure 14. Mean Proportional Area of NL Vasculature. An early, acute
increase in the mean proportional area of NL vasculature is evident at 2
days PS (P<.01). No further differences were observed between lesion
animals and controls.
60
An up-regulation of NGFR densities has also been demonstrated in
neural lobe at 7 and 15 days following infundibular stalk section with
the immunopositive cells found predominately, but not always, in the
vicinity of capillaries (Yan and Johnson, 1990).
However, the identity of the cells demonstrating this increased
expression of NGF receptors has not yet been conclusively determined.
Armed with these data, I sought to explore the possible upregulation of
NGFR-immunoreactivity (NGFR-IR) in the NL and its possible correlation
with degeneration and sprouting.
Pr o p o rt i o n a l A re a of Total V a s c u la t u r e
INTACT
0.60
oc
<
—J
g
(PC.01)
N=4
<
Ul
LESION
N=4
0.48
0.36
0.24
OC
2
O
OC
O-
0.12
0.00
5
10
DAYS POST-LESION
Figure 15. Mean Proportional Area of NL Capillary Plexus.
Proportional
area analysis of the capillary plexus demonstrates no acute vasodilation
at 2 days PS. An age-related decline in capillary area was observed in
all control tissues.
In contrast, the lesion animals show an increase
in the proportional area occupied by capillaries which correlates
closely with the increased density of the capillary plexus noted
earlier.
61
Figure 16 illustrates the results of the proportional area
analysis of NGFR-IR in the N L .
A highly significant increase (F=9.8,
P<.0002, one way Anova, df=11/59) in the proportional area of NL
occupied by NGFR-IR elements occurred following partial denervation.
Two days following surgery no noticable difference existed between
lesion animals and age-matched sham controls.
By 5 days PS a
significant 70% (Pc.05, least significant difference test (LSD))
increase in the proportional area of NL occupied by NGFR-IR elements had
occurred in lesion animals when compared to sham controls (Fig. 17).
N G F R P ropo rtional A rea A nalysis
0.20
0.16
<
UJ
CE
<
-I
<
Z
O
t—
CC
O
CL
O
0.12
0.08
CE
CL
0.04
0.00
2
5
7
10
35
DAYS POST DENERVATION
90
Figure 16. 192-IgG Immunoreactivity in the N L . A significant increase
in the proportional area of NL immunoreactive for nerve growth factor
receptor was evident within 5 days PS. This trend continued reaching
peak values by 10 days PS then returned to normal levels by 30 days PS.
An age-related increase in NGFR proportional area is evident as well.
62
Figure 17. Photomicrograph of NGFR immunoreactivity patterns in the NL
at 5 days PS. A) Immunocytochemical application of 192-IgG in 5 day
post-lesion animals demonstrates a highly immunoreactive staining
pattern surrounding blood vessels and extending outward in long finger­
like projections (solid arrows), presumably within the perivascular
space. Numerous 192-IgG negative glial cell nuclei are evident
throughout the section (solid arrowhead). B) Age-matched sham control
tissue demonstrating a reduced intensity 192-IgG immunoreactivity and
proportional area. Magnification, 650X
63
This difference was also significant at 7 days PS (P<.01, LSD) and at 10
days following surgery.
By 35 days following surgery the proportional
area of NGFR-IR elements had fallen below those observed at 10 days PS
and had returned to levels no longer different from age-matched control
values.
By 90 days PS NGFR-IR levels had increased over those observed
at 35 days PS but were not significantly different from age-matched sham,
control (Fig. 18).
The observed increase in NGFR-IR which was apparent
in. control tissue reflects a previously reported age-related increase in
NGFR-IR in the NL of rats (Yan and Johnson, 1990).
What is important to
note here is the significant increase in NGFR-IR which occurs between 5
and 10 days following surgery, a time when perivascular cell activation
and phagocytic activity is at a maximum (Watt and Paden, 1991).
These
data suggest that a heightened expression of the p75 low affinity NGF
receptor coincides with these cellular events.
The possible expression of NGFR antigens by microglia was further
explored immunocytochemically utilizing known microglial markers Mab OX42 (iC3b receptor; Serotec) and OX-I (leucocyte common antigen;
Serotec). OX-I antigens are thought to be upregulated in activated
microglial cells (Perry and Gorden, 1987).
staining patterns observed.
Figure 19 illustrates the
Mab OX-42 stains microglial cells in close
proximity to blood vessels and putative parenchymal microglial cells in
a highly specific and localized manner.
Individual OX-42 positive
microglial cells and their characteristic reticular processes could
easily be discerned at the light level.
The distribution of these
immunoIabeled cells was essentially homogeneous with no apparent
difference in the staining pattern or relative immunoreactivity between
the parenchymal and perivascular compartments.
A very similar pattern
was characteristic of the OX-I immunostaining reflecting no differences
in the distribution of labeled cells as a result of the activation of a
subpopulation of microglial cells.
64
Figure 18. Photomicrograph of NGFR immunoreactivity patterns in the NL
at 90 days PS. A. 90 days post-lesion 192-IgG staining intensity (solid
arrowheads) is reduced from levels observed at 5 days PS and are no
different from age matched sham controls. However the staining pattern
and the proportional area of staining are similar to that observed at 10
days PS reflecting an age-related increase in nerve growth factor
receptor expression.
B. Age-matched sham control tissue.
Magnification, 650X.
65
Figure 19. Photomicrograph of microglial cells in the NL at 5 days PS.
A. OX-42 immunoreactive microglial cells at 5 days PS are distributed
randomly throughout the tissue. Both putative parenchymal (solid
arrows) and perivascular (solid arrowhead) microglia are apparent. B .
OX-I microglial staining pattern at 5 days PS demonstrates a similar
pattern to, but a higher staining intensity than, OX-42 labeled cells
(solid arrows).
C. NGFR immunoreactive microglia are located
predominately surrounding and extending out from blood vessels as long
velate projections (solid arrows). Magnification, 650X.
66
The NGFR staining pattern showed similarities as well as some
differences compared to OX-42 and OX-I staining pattens in the
‘
distribution of the NGFR-IR cells.
Distinctly labeled microglial cells
appear to be located within the NL parenchyma.
However, the majority of
the NGFR-immunoreactive sites have a more reticular and somewhat velate
appearance.
This is particularly evident surrounding the blood vessels
and the 192-IgG positive cells appear to be localized primarily within
the perivascular space.
This staining pattern becomes more evident with
time and is particularly well illustrated at 90 days PS.
The OX-42 and
OX-I staining patterns show no maturational change.
Morphometric Assessment of Glial Cell Numbers
Astrocyte hypertrophy and proliferation have been reported to
occur both within and surrounding sites of brain injury in a variety of
experimental paradigms (Sjost^rand, 1966; Rose et al., 1976; Latov et
al., 1979; Nathaniel and Nathaniel, 1981).
Perivascular and parenchymal
microglia have also been implicated in the extensive gliosis which
accompanies brain injury (Streit and Kreutzberg, 1988; Streit et al.,
1989).
We too have reported an injury-related increase in the area of
the NL occupied by glial elements which reached peak levels by five days
post-surgery before gradually returning to normal a month later (Watt
and Paden, 1991).
However ,■ the specific glial cell populations
contributing were not determined.
Therefore an investigation was
undertaken to determine whether glial proliferation and/or cellular
hypertrophy might account for the observed increase in glial area.
The initial experiment involved determining the relative density
of pituicyte and microglial cells by counting glial nuclei in one micron
coronal sections of toluidine blue stained plastic-embedded NL at
varying times following knife cut (Table 2).
As illustrated in Figure
20, a significant increase (F=IO.7, PC.OOOl, one way ANOVA, df=9/23) in
pituicyte nuclear density was observed within 48 hours of denervation
67
which then remained above normal for 30 continuous days.
By 2 days P S ’
pituicyte nuclei have increased a significant 39% above intact control
values (P<.05, Neuman-Keuls).
This increase was reduced slightly by 5
days PS reaching a value of 36% above normal (P<.05, Neuman-Keuls).
The
pituicyte numbers continue to decrease with no significant difference
observed between PS 30 lesion animals and controls.
Table 2.
Exp.
Group
Results of Glial Cell Counts - Lesion Group Means*
*.
Pituicytes
Lesion Animals:
418.0 ±
2 dps
309.0 ±
5 dps
10 dps 395.0 ±
30 dps 239.7 ±
91.6 ±
90 dps
150 dps 197.3 ±
11.5
26.1
21.2
23.8
18.4
15.7
NL
Area mm
Microglia
293.0
225.0
257.1
179.4
93.25
125.2
Intact Control Animals:
315.0 ± 23.7 293.0
0 dps
10 dps 210.0 ± 12.9 154.2
30 dps 190.2 ± 13.6 121.6
91.7
90 dps 102.7 ± 8.8
±
±
±
±
±
±
Pits/mm
18.2
24.9
18.0
8.9
16.2
6.7
.34
.26
.37
.26
.12
.39
1229.4
1188.0
1067.5
921.9
763.2
505.7
+ 34.9
± 9.5
± 14.5
± 12.6
.37
.23
.26
.41
851.3
913.4
731.6
250.4
±
±
±
±
±
±
±
±
±
±
Micro/mm
49.1
39.2
17.1
68.4
54.5
32.3
69.2
46.2
67.3
46.1
861.8
865.4
694.8
690.0
777.1
321.0
800.8
669.0
467.5
764.0
±
±
±
±
±
±
38.0
130.4
13.0
40.9
57.3
46.0
± 36.1
± 37.8
± 43.4
±47.3
Four animals were included in all lesion groups.
Three coronal sections were analyzed per animal.
Three animals were included in all control groups except 0 dps which
included four.
*Mean ± sem
Days post-surgery (dps) or equivvalent age for sham controls.
We have previously reported shrinkage of the NL at 10 days
following surgery with a return to normal size by 30 days PS (Watt and
Paden, 1991).
Such a reduction in NL cross-sectional area would almost
certainly result in an apparent increase of cell density in the absence
of glial death.
However, the substantial elevation in pituicyte nuclei
observed at 30 days PS provides strong evidence that the increase in
cell density noted here reflects a true increase in relative pituicyte
cell numbers.
Further analysis of pituicyte contribution to the observed gliosis
was investigated by measuring the cross-sectional area of pituicyte
68
nuclei.
Having previously demonstrated a strong correlation between
increased neuronal size and cell nuclear hypertrophy in the SON it
seemed plausible that a similar response might be observed in the event
of
pituicyte cellular hypertrophy.
Contrary to expectations, a
significant 25% reduction in nuclear cross-sectional area was apparent
by 2 days PS (F=9.11, P<.01, one way ANOVA, df=7/22) when compared to
day O intact controls (Fig. 21).
By 5 days PS the nuclear size had
enlarged to levels slightly below, but not significantly different from,
that of control values.
No further differences were observed through
the remainder of the investigative period.
P it ui cy t e
Density
WMA
LESION
1200
Lobe
INTACT CNTL
(P<.05)
(Re.05)
N -4
1500
5
5
iu
cr
<
3
O
in Ne ur al
N -4
-
N -4
N -4
900-
%
LU
600-
O
=>
Q-
300"
0
5
10
30
DAYS POST-SURGERY
90
150
Figure 20. Pituicyte density in N L . Pituicyte density in the NL
increases significantly 5 days following partial denervation of the NL
(P<.05) when compared to day 0 control values. Pituicyte density
remains high through the first 30 days PS then returns to normal levels.
69
The relative density of microglial cell nuclei was also examined
in the same Spurr's embedded, coronal NL sections described above.
These data indicate a moderately significant lesion-induced increase in
microglial density (F=3, Pc.016, one way ANOVA, df=9/23)(Fig. 22).
Two days following the lesion microglial nuclear counts revealed a
non-significant 7% increase in cell density over lesion day O intact
controls.
These values continue to climb reaching a substantial 23%
increase over lesion day O control values by 5 days PS.
By 10 days PS
microglial numbers have returned to values similar to age-matched
controls.
By 30 days PS microglial numbers in lesion brain have
Nuclei C r o s s - S e c t i o n a l A r e a
###
LESION
INTACT CNTL
0.75
N -4
0.60
0.45
0.30
0.15
0.00
0
2
5
10
30
DAYS POST-SURGERY
90
150
Figure 21. Pituicyte nuclei cross-sectional area. Pituicyte nuclei
cross-sectional area decreases significantly (P<.01) over the first 48
hours following surgery when compared to day 0 intact controls then
returns to normal size by 5 days PS. These data may reflect the
difference in size between immature daughter cells and their nuclei and
the larger more mature pituicyte nuclei following mitotic activity.
70
remained essentially unchanged from values obtained at 10 days PS.
By 90 days PS microglial numbers in control tissues have returned
to values more typical of normal N L .
Note that microglial numbers drop
remarkably between 90 and 150 days PS reaching values significantly
lower than observed at 2, 5 and 90 days PS.
differences exist between groups.
No other significant
Having established that an increase
in the number of pituicytes and microglia had occurred we next chose to
determine whether or not this increase stemmed from cellular
proliferation.
Counts of Brdu immunopositive nuclei revealed that in
the normal animal .4% of the NL cell population is actively synthesizing
Mi cr ogl ia
Density
Figure 22. Microglial density in the neural lobe following surgery.
An increase in microglial density occurs within the first 5 days PS then
returns to normal values by PS 10. Note the age-related decrease in
microglial density at PS 150.
71
DNA at any given time (Table 3).
Within 24 hours of partial denervation
this estimate increases to 1.2% of the cell population, an increase of
65%.
By 2 days PS a significant 1.6% of the cell population is Brdu
positive reflecting the peak level of mitotic activity observed in the
NL (P<.05, Neuman-Keuls).
A subsequent decline in Brdu positive nuclei
occurs by 3 days PS dropping to .84% of the cell population.
This trend
continues with Brdu cell numbers returning to normal values by 6 days
following surgery.
Thus, the mitotic activity observed within the NL
occurs within a period of time when degeneration and phagocytic activity
within the NL are at peak levels.
Table 3. Results of Glial Population/Proliferation Estimates.
Exp.(n)
Sham:
I dps(7)
Lesion:
I dp s (6)
2 dp s (4)
3 dp s (3)
4 dps(5)
6 dps(2)
7 dps(4)
Brdu
cells\section
%Brdu
2008+115
8.3+.69
0.4
2046+156
2089+71.8
1887+108.4
1829+86.2
2196+229
1983+137
24+1.7
33.4+4.2
16+3.7
14±3.8
9.2+2.1
4±.5
1.2
1.6
0.84
0.76
0.41
0.20
Estim. cell
numbers\sect.
% increase
over control
65.5
76.1
51.8
40.7
9.7
— 2--- -— ;-- TT-,— =-7
Three .02mm" sample fields were measured/section.
Six sections were measured/animal.
( ) denotes the number of animals in the indicated group,
(dps) days post-surgery
Immunocytochemical markers were next employed to demonstrate the
specific cell type(s ) undergoing the mitotic activity.
Dual peroxidase
immunocytochemistry using anti-GFAP, an astrocyte-specific immunolabel,
and anti-Brdu showed that in all sections and all animals examined the
Brdu-positive nuclei did not co-localize with GFAP immunoreactivity.
A
second attempt was made by utilizing the superior resolution achievable
with the confocal microscope and dual fluorescence immunocytochemistry.
Again, the GFAP and Brdu labels were clearly not localized to the same
cell type (Fig. 23a) indicating the pituicytes are not actively
dividing.
Next, a dual fluorescence label pairing anti-GSA Lectin, a
72
marker for microglia and macrophages, and anti-Brdu was also examined
with confocal microscopy.
These data showed a definite colocalization
of both antibodies to the same cell type (Fig. 23b).
However, these
data cannot differentiate between blood-born monocytes, macrophages and
brain-derived microglia, leaving any conclusions regarding the cell
type(s) undergoing mitosis unresolved.
Figure 23. Confocal Photomicrographs of GFAP/Brdu and GSA-Lectin/Brdu
labels in the NL at 5 days PS. A. GFAP-immunoreactive pituicytes
(arrows) at 5 days PS are clearly not Brdu immunopositive (solid
arrows). B. GSA-Lectin immunopositve microglial cells (arrows) show
intense Brdu label in the cell nuclei (solid arrowheads).
Magnification, 450X.
73
DISCUSSION
Observations of cavitation, astrocytic hypertrophy surrounding the
wound, gliosis, the influx of massive numbers of monocytes and
macrophages within the wound cavity and neovascular growth surrounding
the knife track are consistent with the reports of others (Rio-Hortega,
1932; Danilova and Polenov, 1977; Antunes et al., 1979; Ling, 1981;
Yoshida et al., 1986; Dellman et al., 1987a).
In this investigation
numerous newly formed vessels were apparent as early as 10 days PS, the
earliest time investigated in hypothalamic tissue, indicating the rapid
onset of this growth response.
No regional heterogeneity in vessel
growth was observed with new vessels forming at all levels of the lesion
tract.
The stimulus for the gliosis and neovascularization may be due
in part to the mononuclear phagocytes observed within the wound cavity
and penetrating the surrounding brain parenchyma (Banda et al., 1982;
Knighton et al., 1983; Guilian, et al., 1988).
Outside the CNS these
cells are recognized as potent secretors of interleukin-I (!LI)(Guilian,
et al., 1988) and have been demonstrated to induce rapid and extensive
astrocytic hypertrophy and neovascular proliferation following injection
into the rat brain parenchyma (Guilian, et al., 1988).
Both types of
mononuclear phagocytes present here, e.g., blood-born macrophage and
brain microglia, have been shown to secrete IL-I (Guilian and Baker,
1985, 1986; Giulian, 1987) as well as other factors (Banda et al., 1982;
Knighton et al., 1983) which act to condition the wound fluid and
surrounding tissues resulting in the subsequent gliosis and
neovascularization.
The regenerative response by neurosecretory axons observed in the
hypothalamus following hypothalamic knife cut is consistent with the
reports of others (Danilova and Polenov, 1977; Antunes et al., 1979;
Dellman et al., 1987a) but differs in several ways.
Immunocytochemical
indications of regeneration by neurosecretory axons were observed within
the hypothalamus and ventral thalamus and were specifically localized to
74
vessels in close proximity to the lesion tract.
Perivascular plexuses
were located on both the medial and lateral boundaries of the knife
track and extended dorsal to the paraventricular nucleus.
In all cases
complete loss of the ipsilateral PVN had occurred, while the ipsilateral
SON had remained intact.
A differential growth response was observed
between the oxytocin and vasopressin producing neurons.
These data
clearly indicate a regenerative sprouting response of oxytocinergic
neurons and the absence of growth stemming from vasopressinergic
neurons.
In our preparations the occurrence of these plexuses was never
observed prior to 8 weeks following the knife cut.
a much more rapid response.
Others have reported
Dellman et al (1987a) observed neurophysin-
immunoreactive axons within the perivascular space of vessels
surrounding a knife tract by 5 days after transection of the HNT tract
in the rat.
Antunes et al., (1979) reported the appearance of
neurophysin-immunoreactive perivascular plexus in the rhesus monkey no
earlier than 8 weeks following hypothalamic lesion.
The magnitude of
this response observed by Antunes (1979) was quite remarkable.
The
neurosecretory plexuses were found surrounding vessels throughout the
hypothalamus as well as the ventral thalamic regions.
Formation of the
most dorsal plexuses would require growth over distances of several
millimeters from the ipsilateral SON.
Our observations of plexuses both
proximal and distal to the ipsilateral SON raises questions regarding
the origin of the axons involved.
One cannot exclude the possibility
that the plexuses located on the distal side of the lesion tract,
particularly those found in the ventral thalamus, originated from the
contralateral SON and/or PVN nuclei.
However, the lack of clearly
defined immunoreactive axons coursing toward the plexus from either
nuclei precludes any attempt to determine the ^ource of origin of these
presumptive neurosecretory terminals.
Having established the remarkable propensity of the MNS to undergo
compensatory axonal growth following injury, we next undertook an
75
investigation of the stimulus responsible for illiciting the growth
response.
Therefore, chronic measurements of urine osmolality, urine
excretion volume, and water intake were undertaken with the expectation
that any initial deficit in VP secretion following partial denervation
of the NL might be reversed by subsequent compensatory sprouting of VP
axons.
However, the complete absence of any lesion-induced deficit,
other than a very brief post-surgical depression in urine osmolality,
makes it impossible to correlate the observed sprouting with functional
recovery.
The absence of a functional deficit in VP secretion was not
the result of an incomplete unilateral lesion, since all lesions were
confirmed post-mortem prior to data analysis.
In addition, animals
receiving bilateral hypothalamic lesions using the same stereotaxic
coordinates exhibited lowered urine osmolality as well as heightened
water intake and urine excretion characteristic of diabetes insipidus
(unpublished observations).
Rather it appears that the reserve capacity
of the VP system is such that destruction of almost half of the
magnocellular neurosecretory terminals is insufficient to compromise its
normal function.
This conclusion is consistent with the report of Jones
and Pickering (1972) who demonstrated that the normal daily requirement
of VP in water balanced rats comprises only 5% of the total
neurohypophysial content.
Thus, in retrospect, it is not surprising
that unilateral transection of the hypothalamo-neurohypophysial tract
failed to compromise normal VP secretion.
No attempts were made to
determine whether any functional deficit in VP release might become
apparent following partial denervation of the NL if subjects were
subjected to hyperosmotic stress or water deprivation.
The decrease in urine excretion volumes and daily drinking volume
was also an unexpected observation.
One possible explanation for these
events is that the lesion may have induced an increase in tonic VP
secretion by disrupting the contralateral connection between the PVN and
SON nuclei.
The PVN nuclei are characterized by reciprocal synaptic
76
connections between oxytocinergic neurons of the PVL and PVM
subdivisions (Silverman et al., 1980).
The SON nuclei have been
reported to have polysynaptic reciprocal connections as well (Takano et
al., 1990).
Although the precise function of these reciprocal
connections remains enigmatic, they may be involved in coordinating
secretory activity between their respective nuclei.
Hence, their
disruption could result in the disinhibition of VP secretion,
resulting
in reduced daily water intake and a concomitant decrease in urine
excretion volume.
The chronic urine hyperosmolality and decreased urine excretion
volume observed following the lesion indicates that a functional deficit
in circulating VP is not required to induce compensatory sprouting of
magnocellular neurosecretory axons.
Herman et al. (1986) have
hypothesized that activation of damaged neurons may play a role in
determining their regenerative success following injury.
These authors
have demonstrated that continuous infusion of VP will selectively impair
vasopressinergic cell survival following neurohypophysectomy (Herman et
al, 1987), an effect which may be attributed to negative feedback of
peripheral VP on vasopressinergic cell activity (Herman et al, 1987;
Hubert et al, 1989).
Likewise, sprouting of intact parvocellular
vasopressinergic axons into the zona externa of the median eminence
following contralateral lesion of the PVN was only apparent when
hyperactivity of these neurons was induced by adrenalectomy (Silverman
and Zimmerman, 1982).
The precise mechanisms underlying the VP
hypersecretory activity observed in the present experiments remain
unresolved.
However, the reduced water intake may have led to increased
neurosecretory activity by intact vasopressinergic neurons, as reflected
in increased ^irine osmolality.
Thus, increased neuronal activity may be
associated with the sprouting response, whereas deficits in circulating
hormone levels play no role in the compensatory sprouting response
previously reported by Watt and Paden (1991).
77
Neuronal activity was further investigated by examining the
neuronal hypertrophy which occurs in the SON following partial
denervation of the NL (Watt and Paden, 1991).
Each neuronal population
was specifically immunolabeled allowing a direct and highly selective
comparison to be made of the cross sectional area of oxytocin and
vasopressin somata and their respective cell nuclei.
The results
confirm and extend our previously reported observations of cellular
hypertrophy accompanying the compensatory sprouting response (Watt and
Paden, 1991).
A highly significant cellular enlargement was observed in
both populations of neurons following surgery with each showing a
similar temporal pattern of response.
By 10 days PS both the
oxytocinergic and vasopressinergic somata had enlarged above age-matched
controls.
This was an unexpected observation as previous investigations
gave no indications of a cellular response prior to 30 days PS (Watt and
Paden, 1991).
the two groups.
However, the dynamics of this response differed between
The greater enlargement of the oxytocin vs vasopressin
neurons suggests either an earlier onset of hypertrophy and/or sprouting
of oxytocin neurons than VP neurons or that a heightened level of
activity may be necessary to support the growth of new terminals within
the hypothalamus as well as the N L .
In a previous investigation (Watt and Paden, 1991) we had argued
that the neuronal hypertrophy observed was a reflection of the
heightened cellular activity necessary to support the ongoing axonal
sprouting and continued maintenance of the enlarged terminal arbor (Watt
and Paden, 1991). This interpretation was based on the tight correlation
observed between the onset of cellular enlargement at 30 days PS and the
robust sprouting event first detected at this time.
Our interpretation
was consistent with the reports of others demonstrating a correlation
between the onset of sprouting of intact afferents and hypertrophy of
the host neurons in other CNS systems (Goldschmidt and Steward, 1980;
Headen et al., 1985; Hendrickson and Dineen, 1982; Pearson et al.,
78
1987a, 1987b).
Likewise, in the SON neuronal hypertrophy has been shown
to occur concomitant with the reformation of neurosecretory axon
terminals in the proximal infundibulum following hypophysectomy
(Raisman, 1977).
Hypertrophy of magnocellular neurons has also been
associated with increased neurosecretory activity induced through
osmotic stimulation via dehydration or salt loading (Enestrom, 1967;
Morris and Dyball, 1974; Peterson, 1966; Reinhardt et al., 1969).
For
example, dehydration of 1.5, 3 and 5 days will result in an enlargement
of 25, 28 and 86% in SON neuronal cross-sectional area, respectively
(Reinhardt et al., 1969).
With 12 days of dehydration SON cellular
enlargement reached 180% of normal (Enestrom, 1967).
However, these
effects were notably transient with a rapid return to normal following
cessation of the osmotic stress (Enestrom, 1967; Morris and Dyball, 74).
Watt and Paden (1991) reported a delay of 30 days observed prior to the
detection of a cellular hypertrophy which led to the conclusion that the
hypertrophy was correlated solely with the sprouting response and not
related to a heightened secretory activity.
The present results
necessitate a reevaluation of these conclusions.
That heightened cellular activity can result in enlargement of the
active cell appears indisputable.
In this paradigm two factors may
contribute to such a response; the osmotic stress induced by having
effectively decreased the volume of daily drinking intake with the
subsequent increase in secretory activity, and the heightened synthetic
activity necessary to both provide the materials required for terminal
growth and the continued maintenance of the enlarged terminal arbor.
Although the time of initiation of the sprouting process has not yet
been elucidated the duration of the growth has conclusively been shown
to proceed for at least 90 days post-surgery (Watt and Paden, 1991).
Likewise, the chronic increase in urine osmolality begins within 2 days
and continues throughout the 90 day investigative period.
Hence, either
of these two responses could account for the cellular hypertrophy and
79
each probably contributes substantially to the overall effect.
Determining the relative contribution of each would certainly be the
logical next step in the pursuit of this investigative paradigm.
Non-neuronal components of the magnocellualr neurosecretory system may
also play a role in the initiation and maintanace of the compensatory
sprouting response.
Analysis of the vasculature in the partially
denervated NL illustrated several distinct alterations resulting from
the lesion.
A significant increase in proportional area of the NL
occupied by vessel lumen 48 hours following denervation indicates a
vasodilatory response which then wanes by 5 days PS with no further
change.
This dilatory response presumably corresponds with increased
blood flow in the NL at a time when hypersecretion is thought to occur.
An increase in blood flow and vasodilation of NL vasculature has been
reported to accompany moderate stimulation of hormone release
(Sooriyamoorthy and Livingston, 1972, 1973).
Under more chronic
stimulation endothelial proliferation has also been reported (Paterson
and Leblond, 1977), exemplifying the apparent malleability of the NL
vasculature to enhanced metabolic demand.
The return of luminal area to
normal presumably reflects the stabilization of fluid balance in these
animals although hypersecretory activity is maintained throughout the
experimental period.
An increase in the density of blood vessels also arises as a
result of the lesion and correlates closely with the temporal pattern of
axonal sprouting reported previously (Watt and Paden, 1991).
The
relative increase in vascular density, which presumably corresponds to
an increase in perivascular basement membrane, may provides additional
target area for the sprouting fibers.
However, under these conditions
the partial denervation would result in a surplus of target area.
It
seems more likely that the hypervascularization occurs either as a
result of the heightened secretory activity which remains elevated
throughout the 90 day post-surgical period or as a result of endothelial
80
stimulating factors arising from activated non-neuronal cells within the
NL.
A number of factors may be involved in the induction of collateral
vessel growth.
Hypoxia associated with areas of high metabolic activity
stimulates neovascular growth (Knighton, et al., 1981).
Hypoxia may in
turn stimulate the release of angiogenic factors from macrophages
(Knighton, et al., 1981).
Macrophages are the major cell component of
wound fluid (Greenburg and Hunt, 1978) and act to condition the fluid
with secreted factors (Banda, et al., 1982; Knighton, et al., 1983)
which in turn can induce a cascade of events leading to
neovascularization of the area surrounding the wound site.
Ameboid
microglia, thought to arise from a monocyte line (Perry and Gordon,
1988), are considered brain macrophages and secrete a variety of
stimulatory factors including IL-I (Fagen and Gage, 1990; Giulian et
al., 1986) which may in turn stimulate the proliferation of fibroblasts
(Greenburg and Hunt, 1978; Knighton, et al., 1983).
The activated
fibroblasts may then release angiogenic factors such as acidic and basic
fibroblast growth factors (Y anagisawa-Miwa, et al., 1992) or secrete
extracellular matrix components which may stimulate angiogenesis as well
(Young and Herman, 1985).
Substrates of fibronectin and interstitial
collagen have been shown to induce a rapid growth response of cultured
endothelial cells (Young and Herman, 1985).
In addition, mechanical
factors such as shear stress resulting from intraluminal turbulence or
heightened intraluminal pressure (Yanagisawa-Miwa, et al., 1992) and
vasodilation (D'amore and Thompson, 1987) may promote collateral vessel
outgrowth.
In addition to questions regarding the stimulatory factors
involved in collateral vessel outgrowth the possible role of
hypervascularity in the compensatory sprouting response must also be
explored.
During embryogenesis the NL anlage is penetrated first by
blood vessels then by the neurosecretory fibers.
Maturation of both
81
fibers and capillary plexus then proceeds together indicating a possible
trophic interaction between vessels and neurosecretory fibers during
development (Galabov and Scheibler, 1983).
Likewise., following
hypophysectomy the initial development of a capillary plexus within the
relatively avascular stalk precedes the penetration of regenerating
neurosecretory fibers (Billenstein and Leveque, 1955; Moll, 1958;
Raisman, 1973), leading to the suggestion that capillary growth may act
as a stimulus for the subsequent regenerative sprouting of
neurosecretory fibers (Raisman, 1973).
A similar regenerative pattern
has been observed in the hypothalamus.
Following transection of the
hypothalamo-neurohypophysial tract the proximal segments of the
transected neurosecretory fibers will form new neuroheamal contacts with
the neovasculature surrounding the lesion tract (Danilova and Polenov,
1977; Antunes et al., 1979; Dellman et al., 1987a) and with resident
vessels of the hypothalamus not previously innervated by neurosecretory
fibers (Antunes et al., 1979).
Considerable growth of neurosecretory
axons has also been observed in the ventral leptomeninges and
surrounding pial vessels following disruption of the ventral
hypothalamic surface (Danilova and Polenov, 1977; Antunes et al., 1979;
Dellman et al., 1988).
Stereotaxic placement of lntrahypothalamlc
allographs of sciatic nerve, optic nerve and hypodermal connective
tissue within the HNT has also been demonstrated to result in varying
degrees of penetration by neurosecretory axons (Dellman et al., 1985a,
1985b, 1986a, 1987).
The relative density of invading neurosecretory
axons appears to be dependent upon the degree of vascularization
(Dellman et al., 1987b), with the highest density of neurosecretory
fibers always found in the immediate vicinity of blood vessels (Dellman
et al., 1989).
These observations indicate that the vasculature must
play a prominent role in the attraction and maintenance of the
neurosecretory axonal ingrowth.
Although the data from this study do
not exclude the possible, and likely, contribution of pituicytes to the
82
development and growth of neurosecretory axons in the normal animal,
they do indicate the underlying importance of blood vessels and the
various components of the perivascular network to a regenerative and/or
compensatory sprouting response within the compromised neurosecretory
system.
In addition to the vasculature the pituicyte, parenchymal
microglia and perivascular cells also play a role in the response of t h e ,
MNS to partial denervation.
Therefore a series of investigations were
undertaken to describe specific attributes of the glial response to
denervation.
The first was to investigate the role of nerve growth
factor receptor regulation in the MNS response.
Increased levels of
NGFR immunoreactivity have been reported during the massive degeneration
that occurs in the posterior pituitary following infundibular stalk
transection (Yan et al., 1990).
However, that preparation does not
allow for regeneration of neurosecretory axons.
We chose to explore the
expression of NGFR immunocytochemically in the partially denervated NL
during the degenerative phase and the subsequent sprouting response as a
means of further elucidating the role of NGFR expression and the
contribution of microglial cell activity to the sprouting event.
Results clearly demonstrate an increase in the proportional area of the
NL occupied by NGFR-immunoreactive elements during the first 10 days of
post-denervation response (Fig. 16).
This is a time when degeneration
of the severed axons and phagocytosis o f .the resulting axonal debris is
at a maximum (Watt and Paden, 1991).. Yan et al. (1990) have demonstrated
192-IgG (NGFR) immunoreactivity on the surface of perivascular
microglial cells and what appeared to be microglial cells within the
parenchyma of the NL following stalk transection.
These cells are
thought to be solely responsible for phagocytosis of axonal debris in
the partially denervated NL following bilateral electrolytic destruction
of the paraventricular nuclei (Zambrano and de Robertis, 1988) or
unilateral transection of the hypothalamo-neurohypophysial tract (Watt
83
and Paden, 1990) and during the turnover of neurosecretory axons in the
intact NL (Pow et al., 1989).
Pituicytes have not been conclusively
demonstrated to express the NGF receptor (Yan et al., 1990). The return
of NGFR proportional area to normal values by 30 days PS could indicate
either a downregulation of NGF receptor expression by microglia or a
reduction in the number of microglia expressing this receptor.
This
reduction in NGF receptor immunoreactivity was observed at a time when
phagocytic activity was complete and a robust compensatory sprouting
response was in progress (Watt and Paden, 1991).
These observations
indicate that the principle period of activation of the microglial cells
within the NL is concurrent with phagocytic activity.
The role of NGF
receptor expression in the initiation or maintenance of the sprouting
response remains undetermined.
However, the activity of the NGFR
expressing glia may activate the pituicytes (Giulian and Young, 1986)
thus preparing the NL for the subsequent sprouting event.
Comparisons of 192-IgG, OX-42 and OX-I staining patterns suggest
some differences in the distribution of NGFR immunoreactive microglial
cells when compared to the other microglial labels (Fig. 19).
The NGFR
staining pattern is reticular and velate in appearance and is localized
primarily surrounding blood vessels and extending outward in long
finger-like projections.
Although the limiting basement membrane of the
perivascular space cannot be readily discerned at the light level, the
general pattern of NGFR staining suggests that the majority of NGFR
immunoreactive cells are located within this compartment.
Ultrastructural analysis will be required to confirm these observations.
In contrast, the OX-42 and OX-I staining patterns are indicative of
isolated, stellate cells scattered uniformly throughout the NL
parenchyma and in close proximity to, but not encompassing, blood
vessels.
Nor are these cells joined in the long wispy pattern observed
with NGFR staining.
These observations indicate that the NGFR
immunoreactive cells represent a subpopulation of NL microglial cells
84
comprised predominantly of the perivascular type.
The increase in
staining area which accompanies the post-lesion response may reflect the
expression of NGF receptors on microglial cells that have entered a
phagocytic state.
Ultrastructural analysis of the degenerating NL
demonstrates the presence of degenerating axons within the perivascular
compartment at 10 days PS (unpublished observations).
Alternatively,
the perivascular microglial cells, having become activated by the
degeneration or injury, may then migrate into the parenchymal
compartment to engulf axonal debris (Perez-Figares et ail., 1986).
Yan
et al., (1990) reported the infrequent occurrence of NGFR immunoreactive
microglia located directly within the parenchymal basement membrane
following stalk transection.
These immunoreactive cells were typically
within necrotic areas indicative of phagocytic activity.
The reduction
in relative proportional area immunoreactive for NGF receptors by 30
days PS supports this hypothesis.
The age-related increase in proportional area immunoreactive for
NGFR is in agreement with earlier reports (Yan et al, 1990).
The
similarity in staining intensity and pattern observed between young and
mature animals suggests a similar underlying mechanism involved in both
the lesioned animal and in mature aging rats.
Pow et al (1989) have
reported the involvement o f ,perivascular microglial cells in the
engulfment of otherwise healthy neurosecretory axons in the intact N L .
These authors suggest that growing neurosecretory axons penetrate the
parenchymal basement membrane and are then engulfed by the resident
microglia.
This activity may reflect a continuous remodeling of the
neurosecretory terminal field.
I have also observed an increased
incidence of axonal engulfment by perivascular microglial cells in the
90 day PS animal, supporting the possibility of such remodeling
(unpublished observations).
The turnover of intact neurosecretory
terminals may provide an explanation for the age-related increase in NL
areas occupied by NGFR immunoreactive cells.
85
In summary, an increase in proportional area of NL immunoreactive
for NGF receptor was observed during the first 10 days following partial
denervation and as a result of normal aging.
Comparison of staining
patterns of known microglial markers with NGFR suggests a subpopulation
of microglial cells may be involved in this response.
This in turn may
reflect the phagocytic activity ongoing during the period of heightened
receptor immunoreactivity .
The return of NGFR staining area to normal
levels following this period suggests little direct contribution of NGFR
expression to the sprouting response.
CNS injury either as a result of peripheral axotomy or penetrating
brain injury initiates a cellular response from the non-neuronal
elements surrounding the sites of injury and/or distal degeneration.
This response may include proliferation of glial cells (Gall et al.,
1979: Schwartz et al., 1988; Steit and Kreutzberg 1987; Kreutzberg and
Barron, 1978), invasion of blood born elements including macrophages and
monocytes (Perry et al., 1987), or migration and hypertrophy of
astrocytes (Gall et al., 1979).
Increased glial proliferation has also
been demonstrated in the SON and NL following chronic water deprivation
or NaCL imbibement (Murray, 1968; Krsulovic and Bruckner, 1969; Paterson
and Leblond, 1977), pituicytes have since been shown to be one of the
proliferating cell types (Murugaiyan et al., 1992).
In the present
investigation the proliferative response of the glial population within
the partially denervated NL was studied using counts of cell nuclei on
semithin plastic sections, bromodeoxyuridine substitution and
immunocytochemistry.
Counts of pituicyte nuclei revealed a significant
increase in the density of pituicytes within the NL from 2-30 days
following surgery, thus supporting earlier findings of increased volume
occupied by glial elements (Watt and Paden, 1991).
However, the
substantial increase in pituicyte density observed within 48 hours of
deafferentation is surprising in light of previous reports of delayed
activation of astrocytes within regions of axonal degeneration (Gall et
86
al., 1979; Fagen and Gage, 1990).
In this paradigm all nuclei were
counted in a given coronal section thus eliminating the possibility of
sampling error as a result of a heterogeneous distribution of glia.
One
cannot exclude the possibility of shrinkage in these tissues, yet the
cross-sectional areas Of the sections examined across experimental
groups offer no indication of such an effect.
In, those cases where an
equivalent sample area was compiled between two age-matched groups e.g.,
PS 2 day vs lesion day 0 control and PS 30 day lesion vs PS 30 day
control, the absolute numbers of glia within the lesion groups are
substantially higher than the age-matched control (Table 2).
These data
support indications of an increase in pituicyte numbers, while arguing
against the possibility of a shrinkage-related artifact.
Morphometric analysis of pituicyte nuclear cross-sectional area
was done to determine the contribution of pituicyte cellular hypertrophy
to the post-lesion glial response.
Neuronal hypertrophy within the
magnocellular neurosecretory system has been demonstrated to occur as a
result of heightened metabolic activity (Watson, 1965; Murray, 1968;
Paterson and Leblond, 1977) and is an accepted indicator of heightened
cellular activity (Vogels et al., 1990).
The enlargement of
magnocellular neuronal nuclei during heightened cellular activity has
been demonstrated in this and a previous investigation (Watt and Paden,
1991).
Therefore, the possibility of such a response by activated
pituicytes was investigated.
However, measures of pituicyte nuclei
during the post-lesion period reflect no increase in nuclear size.
On
the contrary, a significant reduction in nuclear size was observed at 2
days PS.
This reduction occurs at a time when cell numbers are rapidly
increasing, and may provide further indirect evidence of pituicyte
proliferation.
Following cell division the nuclei of both parent and
daughter cells may be reduced in size.
The subsequent return to normal
size over the next 3-8 days would indicate reduced pituicyte
proliferation, with subsequent growth and maturation of the post-mitotic
87
cells.
Hence, these data may also reflect the time course over which
the proliferative event may occur.
The absence of nuclear hypertrophy
may indicate the absence of cellular hypertrophy coinciding with the
post-lesion response.
However, to my knowledge the incidence of glial
nuclear enlargement coinciding with cellular enlargement has not
previously been reported for any glial type.
Therefore, these data do
not necessarily reflect the absence of pituicyte cellular hypertrophy
per se.
The occurrence of astrocyte cellular hypertrophy within
deafferented regions (Rose et al., 1976; Visayan et al., 1991) and
surrounding wound sites (Author, unpublished observations) has been
repeatedly observed.
Thus, it remains a distinct possibility that such
an event may be occurring in the partially denervated NL and warrants
further investigation.
Microglial nuclear counts were compiled in the same plastic
coronal sections discribed above.
These data reflect an insignificant
increase in nuclear numbers between 2 and 5 days PS.
By 10 days PS the
numbers of microglial cells have returned to normal values where they
remain for the rest of the investigative period.
The transient
increase in cell numbers observed here coincides with the period of
degeneration and phagocytosis of axonal debris which occurs over the
first 10 days following knife cut (Watt and Paden, 1991).
These data
agree very closely with those of Kreutzberg and Barron (1978) who
reported a rapid proliferation of microglial cells in the facial nucleus
2-5 days following facial nerve axotomy.
activity had ceased.
Thereafter, all proliferative
Proliferation of microglial cells has also been
reported in sites of neuronal degeneration distant from the site of
axotomy (Kreutzberg and Barron, 1978) and surrounding the site of
transection in the optic nerve (Perry et al., 1987).
The microglial
response typically precedes astrocyte activation (Fagen and Gage, 1990)
and may be responsible for the release of factors which stimulate
astroglial activity (Giulian and Young, 1986).
Ameboid microglia are
88
considered brain macrophages and are responsible for the removal of
degenerating tissue in the CNS.
The early, albeit limited, increase in
microglial cells reported here correlates closely with the post-lesion
degeneration observed in the N L .
The return of microglial numbers to
normal levels following this degenerative phase indicates an indirect
role of microglia in the sprouting response.
However, it must be
emphasized that the insignificant increase in microglial density
observed suggests a limited role at best.
Although the glial nuclear counts provide strong indirect evidence
of glial hyperplasia, further investigation of this response was
undertaken using Brdu substitution to specifically label the cells
undergoing mitosis.
All animals were injected at approximately the same
time of day (10-llam) and sacrificed 3 hours later.
This provided
control for circadian differences in the cell cycle from animal to
animal and across groups.
The normal cell cycle for astrocytes is 20
hours with an S phase of 9.4 hours; G2 less than 3 hours; G2+M approx. 5
hours, and G1 lasts 5 hours (Korr et a l ., 1979).
Therefore, the Brdu
substitution period of 3 hours utilized here must be interpreted as a
snapshot of the ongoing proliferative activity and as such, a
conservative estimate of the percent population of glial cells in a
proliferative state.
In the normal animal (35 days post-parturition)
the percent population undergoing DNA synthesis in the NL was found to
be 0.4% of the total glial population (Table 3).
Korr et al., (1979)
has reported a similar ratio of growing glial cells to all other glial
cells of 0.4% in mouse brain.
Within 24 hours of partial denervation
we observed an increase to 1.2% of the total glial population
representing a 65% increase over control values.
At two days PS a peak
value of 1.6% of the glial population was found in a growth state, a 76%
increase over control values.
The growth activity then begins a gradual
decline over the next 4 days, reaching levels below normal (ns) by 7
days PS, the latest time point thus far examined.
89
Brdu immunopositive nuclei were characterized by a black DAB-CoCL2
precipitate localized within the nuclear envelope, thus allowing stained
nuclei to be easily discerned from unlabeled, counterstained nuclei.
The distribution of Brdu nuclei was essentially random throughout the
sections with no apparent segregation within specific compartments e.g.,
perivascular space, surrounding blood vessels or within the parenchyma,
making it difficult to ascertain the specific cell type(s ) that had been
labeled based on nuclear morphology at the light level.
Therefore, dual
label fluorescence lmmunocytochemistry was utilized as a means of
identifying the Brdu immunopositive cell species.
These results
indicate an absence of co-localization of Brdu nuclei with GFAP
immunopositive cell bodies in all sections examined.
However, the
relatively thin 6 micron sections and the limited number of sections
analyzed leave room for the possibility of pituicytes (GFAP+) which are
Brdu positive but were not observed in the course of this investigation.
Ultimately, confirmation of the cell types exhibiting Brdu substitution
will require E.M. immunocytochemical localization or further dual label
immunocytochemical analysis at the light level.
Pituicyte numbers may increase as a result of migration of
pituicytes and/or astrocytes from the infundibular stalk.
Astrocyte
migration into regions of denervation and axonal degeneration have been
reported to occur within the partially denervated hippocampus (Gall et
al., 1979).
The pituicyte is a characteristically motile cell
particularly during periods of heightened neurosecretory activity
(Tweddle and Hatton, 1980; Hatton et al.,1984; Montagnese et al., 1988).
Hence, the pituicyte displays all the attributes necessary for such a
response.
Dual fluorescence labeling of cells with Brdu and GSA-Lectin
confirmed the presence of microglial hyperplasia in the N L .
Some
caution must be exercised in the interpretation of these data however,
as GSA-Lectin will also stain invading monocytes and macrophages.
Given
90
the extensive axonal degeneration and the lack of a blood brain barrier
in the NL the presence of invasive blood-born cells cannot be
discounted.
However, invading leukocytes were never observed in the
course of extensive analysis of high-clarity plastic semithin sections
of NL or during exhaustive ultrastructural analysis completed in a
previous investigation (Watt and Paden, 1991).
Given that such an event
is possible it must be a rare occurrence at best.
Summary
The compensatory collateral sprouting of intact neurosecretory
fibers which follows partial denervation of the NL in the rat provides a
useful model system to investigate the cellular correlates of both
degenerative and sprouting events.
Unilateral transection of the
hypothalamo-neurohypophysial tract results in the loss of over 40% of
the axons within the NL terminal field (Watt and Paden, 1991).
A period
of axonal degeneration and phagocytosis by resident microglial cells
then occurs followed by the .compensatory sprouting of the remaining
intact neurosecretory fibers within the terminal field between 10 and 90
days post-lesion (Watt and Paden, 1991).
The period over which these
events occur can be divided into two principle phases, a degenerative
phase including approximately the first 10 days PS, and a sprouting
phase which follows and includes the first 90 days PS.
During the
degenerative phase lesion-induced activity first becomes evident with
the appearance of Brdu labeled glial nuclei within 24 hours PS, and the
subsequent increase in pituicyte and microglial cell numbers counted in
cross sections of NL within 48 hours PS.
return to normal values by 10 days PS.
Microglial cell numbers then
However, the pituicyte numbers
remain elevated at least 30 days PS, well into the sprouting phase.
The
degenerative phase is further characterized by the metabolic alterations
in urine osmolality, urine excretion volumes and drinking volumes and
the acute vasodilation of the NL vasculature within 48 hours PS.
Between 5 and 10 days PS continued glial activity was indicated by the
91
increase in proportional area of NL immunoreactive for nerve growth
factor receptors.
However,
b y
10 days PS, the approximate end of the
degenerative phase, NGFR proportional area had returned to normal.
By the end of the degenerative phase, as defined by the lack of
axonal debris and phagocytic activity within the NL (unpublished
observations), a marked hypertrophy of the contralateral intact
neurosecretory neurons was observed.
The neuronal enlargement,
indicative of heightened cellular activity, continued through at least
30 days PS before returning to normal by 90 days PS.
The pattern of
neuronal hypertrophy correlated closely with the sprouting phase and may
reflect the heightened cellular activity necessary to sustain the
sprouting response.
The occurrence of hypothalamic perivascular
neurosecretory plexuses by 30 days PS provided further indications of
cellular activity and highlight the propensity of the MNS for structural
plasticity.
Finally, an injury-related increase in the density of blood
vessels in the NL was also observed between 30 and 90 days PS,
coinciding with the sustained increase in pituicyte density through the
sprouting phase.
In conclusion, anatomical and metabolic aspects of the temporal
pattern of cellular activity within the partially denervated NL have
been described.
These data offer indications of the cellular activity
which may underlie the sprouting response but add little to our present
understanding of the mechanisms responsible for initiating and
maintaining such a response.
However, the anatomical simplicity of the
N L , the robust nature of the MNS sprouting response, and the anatomical
data described herein provide an excellent model system for the
continued investigation of factors involved in the CNS response to
injury.
92
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