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Astrocytes display properties that are both inhibitory and beneficial to neural regeneration. Unfortunately, the exact nature of these inhibitory properties as of yet are unknown. Two hypotheses have been offered. One is based on the mechanically inhibitory properties of astrocyte scars. The other is based on the chemically inhibitory properties of a yet unknown substance. Fortunately, a great deal more is known about the beneficial properties of astrocytes. The goal of this paper is threefold: First, to review both the inhibitory and beneficial properties of astrocytes in neural regeneration. Second, to critique the strength of the more prevalent arguments found in the literature that deal with these inhibitory and beneficial properties. Third, to propose an experiment designed to draw out yet another beneficial property of astrocytes.
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University of Colorado at Boulder
THE ROLE OF ASTROCYTES IN NEURAL REGENERATION OF THE CENTRAL NERVOUS SYSTEM
Troy S. Knapp
Introduction to Neuroscience11:00-12:00 M/W/F
Dr. Tim SmockDecember 11, 1995
ABSTRACT
Astrocytes display properties that are both inhibitory and
beneficial to neural regeneration. Unfortunately, the exact
nature of these inhibitory properties as of yet are unknown. Two
hypotheses have been offered. One is based on the mechanically
inhibitory properties of astrocyte scars. The other is based on
the chemically inhibitory properties of a yet unknown substance.
Fortunately, a great deal more is known about the beneficial
properties of astrocytes. The goal of this paper is threefold:
First, to review both the inhibitory and beneficial properties of
astrocytes in neural regeneration. Second, to critique the
strength of the more prevalent arguments found in the literature
that deal with these inhibitory and beneficial properties.
Third, to propose an experiment designed to draw out yet another
beneficial property of astrocytes.
REVIEW OF THE CURRENT LITERATURE
Almost a century ago the German anatomist Rudolf Virchow
recognized that brain cells could be divided into two distinct
categories: (1) Neurons and (2) Neuroglia (Levitan et al, 73)
Neuroglia is by far the most numerous of the cell types, out
numbering neurons by a factor of approximately ten to one
(Bignami et al, 1.) In fact, according to an anonymous
corespondent in Nature about half of the volume of the vertebrate
brain is composed of glial cells (Bignami et al). Until the
1920's neuroglia was believed to be a single functional unit that
served only as a putty providing structural support to adjoining
neurons (Streit et al, 54.) At this time Pio del Rio-Hortega
developed a silver carbonate stain that made possible
differentiation of the three types of glial cells, astrocytes,
oligodendrocytes and microglia (Streit et al). Each type of
glial cell has a specialized function relatively independent of
the other. Here we are most interested in the actions of
astrocytes and their contribution to neural regeneration in the
CNS.
Astrocytes perform a myriad of functions in the CNS,
including, maintaining a stable neuronal microenvironment, uptake
of amino acids, production of growth factors, and protection from
oxygen toxicity. Astrocytes also seem to play an inhibitory role
in the neural regeneration of the CNS. Whether this role is
mechanical or chemical is still a matter of debate.
The main function of astrocytes is to assure the stability
of the neuronal micro-environment (Bignami et el, 31). In both
gray matter and white matter we find that astrocytes are ideally
located for carrying out this task. In gray matter astrocytes
surround neurons and their processes, while in white matter
astrocytes mainly surround the oligodendrocytic product, myelin.
Further, astrocyte processes form a continuous lining on the
surface of the brain and of blood vessels entering the brain from
the leptomeninges. Astrocytes exhibit gap junctions with other
astrocytes in both white and gray matter, forming a functional
syncytium equilibrating changes in concentrations of ions and
small solutes (Bignami et al, 36). These gap junctions
facilitate astrocytes in maintaining the neuronal
microenvironment. Potassium homeostasis is the clearest example
of astrocytes maintaining the neuronal microenvironment. The
amount of potassium [K+] released during neuronal activity, such
as an action potential, is relatively small, though it results in
a significant increase in extracellular [K+]. These excess
levels of [K+] are taken up by the astrocyte and dispersed
through the syncytium (Bignami et al, 36). The effectiveness of
this syncytium is evidenced by an increase in local extracellular
[K+] being redistributed through the syncytium to distant areas
where extracellular [K+] is lower (Brightman et al, 113).
Research indicates that this syncytium may be responsible
for the protection astrocytes seem to receive from some types of
neuronal toxicity. Investigators found, using L-trans-pyrolidine-
2, 4-dicarboxlic acid (trans-PCDA), that astrocytes were
generally neuroprotective under excitotoxic conditions (Dugan et
al, 3). The rational being that the functional syncytium easily
dissipated the toxicity away from the region of highest
concentration.
Astrocytes also mediate toxicity by uptake of amino acid
neurotransmitters. For example, astrocytes show a much grater
uptake capacity for glutamate, one of the main excitatory
neurotransmitters in the brain, then do neurons. Regional
differences do exist in this respect however. This uptake
capacity for glutamate is not surprising as this amino acid, like
many other acidic amino acids, are neurotoxic (excitotoxins)
(Bignami et al, 36).
The glutamate that astrocytes uptake is used for ammonia
detoxification, yet another example of astrocytes maintaining the
neuronal microenvironment. The evidence supporting this is
twofold. First, the brain enzyme responsible for the formation
of glutamine from glutamate and ammonia is exclusively localized
in astrocytes (Murphy et al, 383). Secondly, the swelling and
vacuolation of astrocytic nuclei is the most prominent finding in
patients dying of hepatic coma, believed to be caused by excess
levels of blood ammonia (Murphy et al).
As Bignami discussed the requirements of maintaining the
neuronal microenvironment are so much more stringent in gray
matter than in white matter that one would expect gray matter and
white matter astrocytes to be different in this respect.
Synaptic activity in the integrating zone of neurons found in
gray matter are presumably more sensitive to small changes in the
microenvironment then the conduction of action potentials found
in white matter. Bignami therefore concluded that cerebral white
matter is more comparable to peripheral nerve then to gray matter
in respect to maintaining neuronal microenviroments.
Astrocytes exhibit receptors for several types of growth
factors as well as appear to produce both Nerve Growth Factor
(NGF) as well as Basic Fibroblast Growth Factor (bFGF).* bFGF is
known to promote not only the survival of neuronal cells, but
* More specifically bFGF has a wide range of tissue distribution and the broadest spectrum of biological activities. Due to striking physiochemical properties several different factors are lumped under the term bFGF. They include: Astroglial growth factor , Heparin-binding growth factor class II and tumor angiogenesis factor.
also the proliferation and differentiation of non-neuronal cells
like astrocytes (Enokido et al, 106). Since bFGF is found to
have a positive growth action in both astrocytes and neurons the
question is raised as to which cell is exhibiting an influence on
the other.
Bignami notes that two possibilities exist as to the source
of these neurotrophic factors. They are either produced by the
innervated target or by the glial cells responsible for these
targets (p, 34). Some confusion exists in the literature as to
this point. The uncertainty being what quantity of neurotrophic
factors are produced in astrocytes verses neurons. It is well
established, however, that neurons are the main site of NGF
expression in normal CNS tissue. Though, tissue evidence suggests
that astrocytes may by the source of NGF in damaged CNS tissue
(Bignami, 34). The rational for this being that NGF mRNA levels
have been found to be increased in primary astrocyte cultures
stimulated by several cytokins and bFGF. Furukawa, however,
asserts that healthy astroglial cells are known to synthesize NGF
(p.42).
In addition to the production of NGF and bFGF astrocytes
have been found to contain receptor sites for epidermal growth
factor (EGF) (Huff, 659). The investigators found that binding
of EGF by the astrocytes was saturable, specific and not competed
by NGF or bFGF. They did find, however, a 70% reduction in EGF
binding when the astrocytes were pretreated with bFGF for an 18
hour period. This lead them to the conclusion that bFGF may
serve as an off switch for the EGF mitogenic signal in
astrocytes.
While the exact physiological significance of neurotrophic
synthesis (whether in healthy or damaged CNS tissue) is unclear,
it is clear that astrocytes are one of the sources providing
neurotrophic factors to neurons.
Regardless of the conditions under which astrocytes produce
trophic factors, it has been securely documented that neurons
require a glial environment to grow. Dugan and colleagues found
this by developing "pure" neuronal cultures from mouse neocortex
to study the effect of glial cells on the response of neurons to
injury. They found that neuronal cultures grew best on a glial
base, coming to the conclusion that cortical neurons require a
glial conditioned medium in order to survive (p, 4545). Mark
Noble came to the same conclusion in his study on the
developmental biology of the optic nerve. Noble compared the
growth of optic neurons on a variety of substrates including,
optic astrocytes, schwann cells, skin fibroblasts, and cardiac
myocytes. He found that dissociated neurons plated onto
astrocytes grew as if they preferred the astrocytic surface to
any other surface availalbe (p, 9). Noble saw extensive
crossing over of neuronal processes, and an occasional instance
of processes running in parallel for short distances (p, 9).
Growth factors are not the only astrocyte product that are
beneficial to neuronal survival. Astrocytes have been found to
release pyruvate which has also been determined to have a
positive effect on neuronal survival (Selak et al, 23).
Astrocytes also seem to play a role in protecting CNS
neurons from oxygen toxicity, some of which is thought to be
produced by microglia. These reactive oxygen species are
believed to have the beneficial effect of damaging microbe
membranes, proteins and DNA. Unfortunately, reactive oxygen
species damage healthy cells in the same way (Streit et al, 61).
These oxygen free radicals have been implicated as a potential
cytotoxic mechanism responsible for nigral cell death in
Parkinson's disease (Jovoy-Agid, 92). There are several enzymes
in the CNS that act in concert to defend against this oxygen
toxicity. Fist the superoxide radical is mutated to hydrogen
peroxide by superoxide dismutase (SOD). The hydrogen peroxide is
then decomposed to water or removed by glutathion peroxidase
(GPX) which uses hydrogen peroxide to oxidize the reduced
glutathion (Jovoy-Agid). Through immunocytochemical studies on
human mesencephalon Jovoy-Agid found that GPX is found
exclusively in astrocytic cells, showing that astrocytes are
responsible for mediating oxygen toxicity.
Knowing the functions of astroglial cells we now turn our
attention to their role of neural regeneration in the CNS. A
profound difference exists in the ability of neural regeneration
in the CNS and the peripheral nervous system (PNS). This is
demonstrated by the following, if an efferent dorsal root
ganglion is cut the axon will regenerate normally reaching its
peripheral target allowing functional recovery. However, if a
CNS axon is severed the regenerative attempt will be abortive and
non functional. A regenerating PNS axon will stop at the PNS-CNS
junction. This apparently happens for one of two reasons.
First, the lack of CNS regeneration is due to an active
inhibition on the part of the CNS. Second, the CNS lacks
regenerative factors normally found in the PNS. Astrocytes at
the PNS-CNS junction would share responsibility for the lack of
neural regeneration in either of these two cases.
One of the longest held hypothesis for the lack of neural
regeneration in the CNS is astrocyte scar tissue resulting from
CNS injury. The major mass of astrocytic scar tissue is formed
from bundles of cytoplasmic intermediate filaments which are made
of GFAP, an astrocyte-specific protein (Bignami, 84).
Upregulation of GFAP production is a main factor in the formation
of glial scars. In this case astrocyte proliferation also occurs
but is limited and confined to the area of injury. These
astrocytic scars have been believed to be responsible for
inhibition of neural regeneration (Bignami, 85).
It has been suggested that increased GFAP expression by
astrocytes is the most sensitive indicator of neural damage in
the CNS. Farooque and colleagues found this to be true in rats.
The investigators used immunohistochemistry to detect changes in
the expression of GFAP in spinal tracts after using blocking-
weight techniques to induce spinal cord compression at the level
of the eight and ninth thoracic vertebrae (Farooque et al, 41).
The investigators found that within 24 hours post compression
widespread astrocyte reaction occurred. Even mild compressions
that did not produce any signs of dysfunction induced widespread
astrocytic alterations. Further, the astrocyte response was more
marked in rats with more severe compression leading to more
pronounced neurological deterioration (Farooque).
Recent research has indicated that this astrocyte injury
response is not entirely local. Studies by Janeczko point to the
possibility that astrocytes migrate from peripheral areas to
participate in the process. His evidence for this is that
[3H]thymidine-labeled astrocytes at first scattered over a
relatively wide area later became concentrated around CNS lesions
(p, 236). According to Bignami several publications now suggest
that normal glial cells (astrocytes and oligodendrocytes) have
the ability to migrate in CNS tissue (p, 85).
Astrocytes are found to exhibit scaring in both injuries
that produce dysfunction and in injuries that do not. Yamada and
colleagues have found the formation of astrocyte scar tissue does
not create a major barrier in CNS neuronal regeneration in lower
vertebrates. The researchers investigated axonal regeneration in
the CNS using fine structural and histochemical aspects of the
carp spinal cord, which was completely transected at the level of
the dorsal fin. Fusion of the transected region and regeneration
of axons was apparent at 26 days post lesion. By 115 days post
lesion the rostral and caudal portion of the transected spinal
cord were completely connected by the regeneration nervous tissue
(p, 324). Horseradish peroxidase injected in the spinal cord at
the portion caudal to the transection site was detected in the
cytoplasm of large neurons located in the reticular formation of
the midbrain (p, 325). This demonstrates that long axons
regenerated through the ablation gap, indicating that
regenerating axons in carp spinal cord can pass through the glial
scar bundle formed in the transected portion. Many of these
regenerating axons were found to be in contact with astrocytes,
indicating that glial cells do not play a major role as an
obstacle for the prolongation of axons in the carp spinal cord
(p, 325).
Unfortunately, several observations are not readily
explained by the scar hypothesis. First, extensive axonal growth
may be observed in glial scars, as noted by Yamada and
colleagues. Secondly, a study performed by Chi and Dahl on nerve
grafting found that glial scars formed as the result of damage
done during surgery at the interphase between brain and
peripheral nerve implants do not prevent axons growing from the
brain into the graft (p, 245).
These inconsistencies are best accounted for by a second
hypothesis. A chemical mechanism has been proposed by Liuzzi and
Tedesch as the barrier to CNS neural regeneration. Knowing that
regeneration of a peripheral axon stops at the junction to the
CNS they proposed that astrocytes transmit some sort of 'stop'
signal when contacted by a growing axon (Liuzzi, 4783). This
signal would be similar to the physiological mechanisms that stop
growth when axons reach their destination in development, except
that in the first case the message is delivered by the astrocyte
membrane and in the second case by the post-synaptic membrane
(Bignami, 95). Unfortunately, this signal has not been found or
identified. Oligodendrocytes however seem to exhibit a sort of
stop signal on their surface that act as an axonal repellent
(Bignami et al, 95). This was observed when dorsal root ganglion
(DRG) axons are put in contact with sciatic nerve (a PNS nerve)
and an optic nerve (a CNS nerve) in vitro. The DRG grows inside
the sciatic nerve and avoids the latter optic nerve. This lead
to the isolation of two inhibitory proteins fractions at 250 and
35 kD and to the demonstration that antibodies to these proteins
promote axonal growth in the spinal cord (Schnell et al, 269).
We have found that astrocytes perform a variety of functions
in the CNS. Including maintaining a stable neuronal
microenvironment, uptake of amino acids, production of growth
factors, and protection from oxygen toxicity. Unfortunately,
none of these functions elucidate hints as to why astrocytes
appear to have an inhibitory effect on neural regeneration in the
CNS. Both a mechanical and chemical hypothesis have been
explored. The glial scar hypothesis has several unresolved
contradictions. Therefore, the chemical hypothesis seems more
solid.
CRITIQUE OF THE LITERATURE
Within the reviewed literature there are three areas lacking
clarity. First, the effect of bFGF on astrocytes is unclear.
Second, there is confusion in whether we find greater NGF
production in healthy or damaged astrocytes. Third, though a
physiological mechanism seems to prevent regenerating axons from
crossing the PNS-CNS barrier, none has been found or identified.
As stated before bFGF is known to promote not only the
survival of neuronal cells but also the proliferation and
differentiation of non-neuronal cells like astrocytes (Enokido et
al, 106). Both neurons and astrocytes express receptors for and
produce bFGF. Further, Enokido found that astroglia fibers
increased in number with the addition of bFGF. Because we find
both astroglia and neurons responding to and producing bFGF the
question is raised as to which cell is having an action on the
other. Or, does the possibility exist that they share a mutually
beneficial relationship. It appears that the literature is silent
on any mutually inclusive action bFGF may have on astrocytes and
neurons.
Secondly, uncertainty exists as to the production of NGF in
astrocytes. Bignami has cited tissue evidence suggesting that
astrocytes may be a source of NGF in damaged CNS tissue (Bignami
et al, 34). His rational for this is the finding of NGF mRNA in
primary astrocyte cultures. Furukawa states very clearly that
healthy astrocytes are known to synthesize NGF in cultures. His
evidence for this is that murine astrocytes synthesize and
secrete molecules identical to murine submaxilary gland derived
NGF with respect to molecular weight, isoelectric point,
antigenicity and neurite promoting activity (Furukawa, 62). The
question as to whether healthy or injured astrocytes produce NGF
is of importance. If healthy astrocytes produce NGF then they
could be considered to have a maintenance role in the CNS. If
injured astrocytes produce NGF then they could be considered to
have restorative role. The answer to this question goes to the
very basis of defining the role astrocytes play in the CNS.
Thirdly, Bignami asserts as a reasonable hypothesis that
astrocytes possess on their surface a chemical that transmits a
signal to growing axons that in effect tells them to stop their
regeneration. Bignami puts forth this hypothesis though he is
unable to provide any information as to the nature of this
chemical signal. This chemical hypothesis is found elsewhere in
the literature though in no place is the structure or action of
the chemical signal elucidated. The basis for Bignamis'
hypothesis seems to be that oligodendrocyte appear to possess
this chemically inhibitory property. Bignami states that in
oligodendrocytes these inhibitory proteins, as well as antibodies
for them have been identified (p, 95). Bignami is the only place
in the literature that I have found a statement that the
inhibitory proteins in oligodendrocytes have been identified.
While the chemically inhibitory hypothesis appears reasonable it
seems directly opposed to the other actions of astrocytes. We
find astrocytes playing a variety of beneficial roles in relation
to neurons. For example, the literature is very secure that
astrocytes produce and maintain several types of neurotrophic
factors, as well as protect neurons from oxygen and ammonia
toxicity. In light of these beneficial aspects stating that
astrocytes also possess a strong repellent to neuronal growth is
difficult.
This dilemma goes back to the previous dilemma raised by
Bignami and Furukawa. Bignami felt that injured astrocytes were
the source of astrocytic NGF production, while Furukawa believed
that healthy astrocytes produced this NGF. This lead us to the
question of whether astrocytes play a maintenance or restorative
role in the CNS. The answer to this question in turn will
provide hints as to which hypothesis against neural regeneration
(mechanical or chemical) is correct. If the role of astrocytes
in the CNS is found to be restorative then the chemical
inhibition hypothesis against neuronal regeneration seems out of
place. The reasoning here being that a restorative role would be
in opposition with a chemically inhibitory signal. If the role
is indeed one of maintenance then the chemically inhibitory
hypothesis of neuronal regeneration would appear valid. The
thought here being that a chemically inhibitory signal on
astrocytes provides a balance system against the neurotrophic
factors, while the neurotrophic factors provide a check system
against the chemically inhibitory signal. This arrangement would
create a homeostatic state in which neither influence could exert
its will unchecked. This would be in keeping with a maintenance
role.
Unfortunately, experimental verification of this check and
balance theory of astrocyte chemical message and neurotrophic
factor homeostasis would be difficult to carry out without
knowing the make up or antibody factors of the chemically
inhibitory signal. If NGF production was inhibited in a
controlled environment one would expect the inhibitory chemical
signal on the astrocyte to be free to express its action. This
action would be to inhibit neuronal growth. However, it would be
difficult to determine if any decrease in axonal sprouting or
growth was due to the inhibitory chemical signal, or if it was
due to a lack of NGF.
If antibodies to the inhibitory chemical were known then an
experiment to test this theory could most likely be carried out.
If the inhibitory chemical signal could be inhibited then NGF
would be expected to express its effect unchecked. This could be
easily checked by looking for any increases in axonal growth or
sprouting in a controlled environment. The answers to a great
many questions are resting on the discovery of the structure of
the proposed inhibitory chemical signal on astrocytes.
The most exciting discovery in recent literature is that of
astrocytes having the ability to migrate in the CNS from
peripheral areas to lesion areas as discovered by Janeczko and
verified by Bignami (p, 236: p, 85). In light of the many
beneficial aspects that astrocytes have been shown to exhibit in
neuronal maintenance, and possibly in neural regeneration, this
discovery is even more exciting. Naturally, the question is
raised, if astrocytes migrate to injured areas and exhibit their
beneficial properties can they be placed at the site of injury
and remain viable so as to exhibit their beneficial properties?
PROPOSED EXPERIMENT
In hopes of gaining further understanding of the potentially
beneficial aspects of transplanted astrocytes I propose the
following experiment. In fifteen male Wistar rats of the same
age (approximately 90 days) create a 1mm lesion in the white
matter of the right cerebral hemisphere underlying the cerebral
cortex.* At 30 minutes post lesion inject .25mL of pure cultured
astrocytes (as derived by Dugan and colleagues (p, 4546)) that
have been labeled with [3H]thymidine. At 40 days post lesion
kill the animals and fix the brains in Bouins's fixative and
section at 5 m intervals in the coronal plane. Label 5 of
the coronal sections according to the method developed by
Janeczko and Bignami
* The rational for creating a lesion in the white matter is based on Bignami's hypothesis that cerebral white matter is less sensitive then gray matter to small changes in the microenvironment. Because white matter is more robust to changes in the microenvironment accidental contamination due to the surgery will play less of a role as a confounding variable.
Knapp 1
(Janeczko, p. 237).# Examine a 100 x 100 m area of lesion in
each of the 5 coronal sections. Tally both the number of labeled
and unlabeled astrocytes in these sections as well as the number
of astrocytes in the corresponding contralateral unlesioned area.
Further, estimate the number of labeled astrocytes placed into
the lesion area.
From these numbers perform a Students two tailed t-test to
determine the following: (1) If there is a significant
difference between the number a labeled astrocytes added to the
lesion area verses the number of labeled astrocytes found in the
lesion area after the animal was killed. A significant
difference here would suggest one of two things. First, that the
astrocytes are incapable of growing into the lesion area.
Second, that there is some maximum number of astrocytes that the
area is able to support and that number had been exceeded.
Finding no significant difference would suggest that the labeled
astrocytes are capable of growing into the existing neural
structure. (2) If there is a significant difference between the
total number of astrocytes on the lesion side verses the
unlesioned side. A significant difference may indicate that the
labeled astrocyte culture was able to grow into the existing
structure. No significant difference between the two sides may
indicate that there is some maximum level of astrocytes that a
# The method is as follows: stain the coronal sections immunocytochemically by the peroxidase-antiperoxidase method according to Van Noorden. Then prepare autoradiographs from the immunocytochemically stained sections by the dipping technique using illford K-2 emulsion, expose for 21 days, develop and stain with Harris' hematoxylin and eosin.
Knapp 2
given area can support, based on the assumption that the
unlesioned side is close to this maximum density. The optimal
results would be to find after analysis of the data that no
significant difference was found in the first case while a
significant difference was found in the second case. This would
indicate a probability that astrocytes are capable of being
placed into a damaged neuronal environment and surviving.
Knapp 3
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