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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