Nogo-A expression in the intact and injured nervous system

The expression of Nogo-A mRNA and protein in the nervous system of adult rats and cultured neurons was studied by in situ hybridisation and immunohistochemistry. Nogo-A mRNA was expressed by many cells in unoperated animals, including spinal motor, DRG, and sympathetic neurons, retinal ganglion cells, and neocortical, hippocampal, and Purkinje neurons. Nogo-A protein was strongly expressed by presumptive oligodendrocytes, but not by NG2+glia and was abundant in motor, DRG, and sympathetic neurons, retinal ganglion cells, and many Purkinje cells, but was difficult to detect in dentate gyrus neurons and some neocortical neurons. Cultured fetal mouse neocortical neurons and adult rat DRG neurons strongly expressed Nogo-A in their perikarya, growth cones, and axonal varicosities. All axons in the intact sciatic nerve contained Nogo-A and many but not all regenerating axons were strongly Nogo-A immunopositive after sciatic nerve transection. Ectopic muscle fibres that developed among the regenerating axons were also Nogo-A immunopositive. Following injury to the spinal cord, Nogo-A mRNA was upregulated around the lesion and Nogo-A protein was strongly expressed in injured dorsal column fibres and their sprouts which entered the lesion site. Following optic nerve crush, Nogo-A accumulated in the proximal and distal stumps bordering the lesions.     

Effects of immunosuppression on regrowth of adult rat retinal ganglion cell axons into peripheral nerve allografts

Analysis of the effectiveness of allografts and immunosuppression in the repair of nerve defects in the adult peripheral nervous system (PNS) has a long experimental and clinical history. There is little information, however, on the use of allografts in peripheral nerve (PN) transplantation into the injured central nervous system (CNS). We assessed the ability of PN allografts (from Dark-Agouti rats) to support regeneration of adult rat retinal ganglion cell (RGC) axons in immunosuppressed host Lewis rats. PN allografts were sutured onto intraorbitally transected optic nerves. Three weeks after grafting, regenerating RGC axon numbers were determined using retrograde fluorescent labelling, and total axons within PN grafts were assessed using pan-neurofilament immunohistochemistry. In the absence of immunosuppression, PN allografts contained few axons and there were very few labelled RGC. These degenerate grafts contained many T cells and macrophages. Systemic (intraperitoneal) application of the immunosuppressants cyclosporin-A or FK506 reduced cellular infiltration into allografts and resulted in extensive axonal regrowth from surviving RGCs. The average number of RGCs regenerating axons into immunosuppressed allografts was not significantly different from that seen in PN autografts in rats sham-injected with saline. Many pan-neurofilament-positive axons, a proportion of which were myelinated, were seen in immunosuppressed allografts, particularly in proximal regions of the grafts toward the optic nerve-PN interface. This study demonstrates that PN allografts can support axonal regrowth in immunosuppressed adult hosts, and points to possible clinical use in CNS repair.     

Embryonic Optic Nerve Tissue Fails to Support Neurite Outgrowth by Central and Peripheral Neurons In Vitro

The failure of axon regeneration in the injured mammalian central nervous system has been ascribed, in part, to the inhibitory effects of myelin proteins. To investigate the influence of myelination on neurite growth and regeneration by both central nervous system and peripheral nervous system neurons, isolated rat neonatal retinal ganglion cells and adult and neonatal dorsal root ganglion neurons were cultured on cryostat sections of both immature unmyelinated and mature fully myelinated adult rat optic nerve. In agreement with earlier studies using neonatal peripheral neurons, the adult optic nerve failed to support neurite outgrowth from any of the neurons tested. A new finding was that tissue sections from unmyelinated optic nerve (aged embryonic days 18 and 20, and postnatal days 1–3), also failed to support the growth of neurites from neonatal retinal ganglion cells and both neonatal and adult dorsal root ganglion neurons. Neonatal retinal ganglion cells also failed to extend neurites on sections of pre-degenerated sciatic nerve, a tissue shown in our previous work to be a good substratum for supporting neurite growth for both neonatal and adult DRG neurons. These results suggest that cells in the immature optic nerve either express widely acting axon growth inhibitory molecules unrelated to previously described myelin proteins, or do not synthesize appropriate axon growth promoting molecules. They also reveal that, for axon regeneration, central nervous system and peripheral sensory neurons require distinct substratum interactions.     

Retinal axon regeneration in peripheral nerve, tectal, and muscle grafts in adult rats.

This study examined whether prior regenerative growth through peripheral nerve (PN) bridging grafts influenced the specificity with which lesioned adult rat retinal ganglion cell (RGC) axons grew into co-grafts of developing target tissue (fetal superior colliculus). Growth into nontarget (muscle) tissue was also examined. Autologous PN was grafted onto the transected optic nerve. After 14 days, the distal ends of the PNs were placed next to, or inserted into, embryonic tectal tissue or into autologous muscle grafts placed in frontal cortex cavities. Host retinal projections were examined 3-8 months later using anterograde and retrograde tracing techniques. In rats in which there was good apposition between PN and tectal tissue, small numbers of RGC axons were observed growing into the tectal grafts (maximum distance of 180 microm). No evidence of specific innervation of appropriate target regions within tectal grafts was detected, even though such regions (identified by acetylcholinesterase histochemistry) were often located close to the PN grafts. In rats with PN/muscle co-grafts, the extent of retinal axon outgrowth was greater (up to 465 microm from the PN tip) and labelled profiles that resembled motor endplates were seen contacting muscle fibres. Previous studies have shown that spontaneously regenerating RGC axons consistently and selectively innervate appropriate target areas in fetal tectal tissue grafted directly into optic tract lesion cavities. Together, the data suggest that exposure to a PN environment may have reduced the extent of adult retinal axon growth into fetal tectal transplants and affected the way regenerating axons responded to specific developmental cues expressed by target cells in the co-grafted tissue.     

Erythropoietin is both neuroprotective and neuroregenerative following optic nerve transection

The cytokine hormone erythropoietin (EPO) is neuroprotective in models of brain injury and disease, and protects retinal ganglion cells (RGC) from cell death after axotomy. Here, we assessed EPO's neuroprotective properties in vivo by examining RGC survival and axon regeneration at 4 weeks following intraorbital optic nerve transection in adult rat. EPO was administered as a single intravitreal injection at the time of transection (5, 10, 25, 50 units, PBS control). Intravitreal EPO (5, 10 units) significantly increased RGC somata and axon survival between the eye and transection site. Twenty five units did not improve survival of RGC somata but did increase axon survival between the eye and transection site. In addition, a small proportion of axons penetrated the transection site and regenerated up to 1 mm into the distal nerve. In a second series, intravitreal EPO (25 units) doubled the number of RGC axons regenerating along a length of peripheral nerve grafted onto the retrobulbar optic nerve. Our in vivo evidence of both neuroregeneration and neuroprotection, taken together with the natural occurrence of EPO within the body and its ability to cross the blood-brain barrier, suggests that it offers promise as a therapeutic agent for central nerve repair.     

Adult Retinofugal Axons Regenerating Through Peripheral Nerve Grafts Can Restore the Light-induced Pupilloconstriction Reflex

To investigate the ability of mature regenerating retinal axons to form functional connections within central targets, severed axons were guided into the primary visual centres which subserve the pupillary constriction reflex in response to light. The ocular stump of the transected optic nerve of adult rats was connected by means of an autologous peripheral nerve graft with the pretectal region which contains the relay nucleus of pupillary constriction, the olivary pretectal nucleus. This nucleus is efferently connected with preganglionic neurons in the oculomotor nuclear complex which innervates parasympathetically the muscle constrictors of the iris. Six to sixteen weeks after optic nerve transection and peripheral nerve transplantation, brisk responses were observed in the pupils upon illumination of the transplanted eye. Recovery of the pupil responses indicated that retinal neurons used the peripheral nerve 'bridge' to access the pretectum, in which they established synaptic contacts in sufficient density and with appropriate specificity to reconstitute the function of the traumatically interrupted neuronal circuitry.     

Thrombospondin expression in nerve regeneration I. comparison of sciatic nerve crush, transection, and long-term denervation

Patterns of expression of the extracellular matrix molecule thrombospondin (TSP) were examined during peripheral nerve regeneration following sciatic nerve crush or transection. In noninjured nerve, was present in the axoplasm, Schwann cells, endoneurium, and perineurium of the adult mouse sciatic nerve. Following nerve crush or nerve transection, levels of TSP rapidly increased distal to the trauma site. Elevated levels of TSP were present distal to regenerating axons, while expression gradually returned to normal proximal to the regenerating axons. When reinnervation was blocked, TSP levels remained high in the endoneurium in excess of 30 days, but TSP was absent by 60 days. Following reanastomosis of the proximal and distal segments after 60 days of denervation, TSP was re-expressed in the distal nerve stump. These results indicate that TSP, which is involved in neuronal migrations in the embryo and neurite outgrowth in vitro, appears to play a role in axonal regeneration in the adult peripheral nervous system.     

Chronic infusion of CDNF prevents 6-OHDA-induced deficits in a rat model of Parkinson's disease

The visual system is widely used as a model in which to study neurotrauma of the central nervous system and to assess the effects of experimental therapies. Adult mammalian retinal ganglion cell axons do not normally regenerate their axons for long distances following injury. Trauma to the visual system, particularly damage to the optic nerve or central visual tracts, causes loss of electrical communication between the retina and visual processing areas in the brain. After optic nerve crush or transection, axons degenerate and retinal ganglion cells (RGCs) are lost over a period of days. To promote and maintain axonal growth and connectivity, strategies must be developed to limit RGC death and provide regenerating axons with permissive substrates and a sustainable growth milieu that will ultimately provide long term visual function. This review explores the role olfactory glia can play in this repair. We describe the isolation of these cells from the olfactory system, transplantation to the brain, gene therapy and the possible benefits that these cells may have over other cellular therapies to initiate repair, in particular the stimulation of axonal regeneration in visual pathways. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.     

Presence of growth inhibitors in fish optic nerve myelin: Postinjury changes

This study shows that the fish optic nerve, which is able to regenerate after injury, contains myelin-associated growth inhibitors similar to the growth inhibitors present in mammalian central nervous system (CNS) myelin. The ability of nerves to regenerate was previously correlated with the ability of sections from these nerves to support neuronal attachment to and axonal growth in vitro. Thus neuroblastoma cells or embryonic neurons became attached to and grew axons on sections of rat sciatic nerve or fish optic nerve, which are spontaneously regenerating systems, but not on sections of rat optic nerve, a nongenerating system. Failure of the latter to support axonal growth has been attributed, at least in part, to growth inhibitors. Recently it was shown that adult neurons, which differ in their growth requirement from embryonic neurons, are unable to extend neurites on sections of normal sciatic nerve but are able to extend neurites on sections of sciatic nerve that was injured prior to its excision. We found a similar in the fish optic nerve, i.e. that the nerve is normally not permissive to growth of adult retinal axons but becomes growth permissive after injury. The nonpermissiveness of the normal fish optic nerve was found to correlate with the presence of myelin-associated growth-inhibitory molecules. This inhibitory activity of fish myelin was neutralized by IN-1 antibodies, known to neutralize rat myelin growth inhibitors apparently similar or even identical to those of rat, but possibly present in lower amounts than in the rat. Results are discussed with respect to the possibility that fish optic nerve, like the rat sciatic regeneration of adult neurons. Such changes might include elimination or neutralization of growth inhibitors. © 1994 Wiley-Liss, Inc.     

Precrushed sciatic nerve grafts enhance the survival and axonal regrowth of retinal ganglion cells in adult rats.

We have examined the influence of normal and precrushed ("conditioned") sciatic nerve grafts on the survival and axonal growth of retinal ganglion cells (RGCs) in adult rats. Normal sciatic nerves (group A) or sciatic nerves which had been crushed 1 week before transplantation (group B, conditioned grafts) were used as grafts. The nerves were removed and sutured to the proximal stump of intraorbitally axotomized optic nerves. Neuronal survival and axon growth were determined by counting the numbers of surviving, DiI-prelabled RGCs, cresyl violet-stained RGCs and the numbers of axons which had grown into the grafts 3 and 6 months after transplantation. Counting of axons was performed by combined use of light and electron microscopy. We observed that the use of conditioned grafts (group B) significantly enhanced RGC survival and axonal regrowth as compared to normal grafts 3 months after transplantation. Six months after grafting, RGC survival (as determined in DiI-stained retinae) and axonal growth were not significantly different in both groups. These results suggest that the functional status of a peripheral nerve used for grafting in the CNS influences neuronal viability and axonal reelongation especially during the first 3 months after grafting. Very long-term RGC survival, however, may be determined by functional reconnection of regenerating RGC axons rather than by the graft itself.     

Glial cells in degenerating and regenerating optic nerve of the adult rat

The glial cell reaction both in degenerating and regenerating adult rat optic nerve was studied by immunohistochemistry and electron microscopy. Degeneration in the optic nerve was achieved by complete transection, and the retinal stump was then analyzed. The regeneration was observed by autotransplantation of a sciatic nerve segment to the transected retinal stump. In both cases, optic nerve axons were labeled anterogradely with rhodamine, followed by immunohistochemical staining. Glial fibrillary acidic protein-positive astrocytes covered the transected end of degenerating optic nerve, whereas in the regenerating optic nerve they enwrapped axonal bundles emerging from the optic nerve stump and migrated together into the transitional zone intervening between the retinal stump and graft. In electron microscopy, direct attachment of astrocyte and Schwann cell was found within the transitional zone, whereby these cells were holding axons between them. Decrease of 04 immunoreactivity, which labels oligodendrocytes, was apparent in the transected end of retinal stump during the regeneration. The ED1 -positivity, which labels microglia/macrophages, was found in cells accumulated in the transitional zone of degenerating optic nerve, whereas during regeneration, ED1-immunoreactive cells were also distributed in the retinal stump. These results suggest that astrocytes, usually considered to interfere with optic nerve regeneration, change their characteristics in the presence of peripheral nerve graft and guide the regenerating axons in cooperation with Schwann cells. The response of oligodendrocytes and microglia/macrophages may also be modulated by peripheral nerve.     

Calcitonin gene-related peptide (CGRP) in the regenerating rat sciatic nerve

Calcitonin-gene related peptide (CGRP) is a neuromodulatory peptide present in motoneurons and a subpopulation of sensory neurons of the adult peripheral nervous system. Here we have investigated the changes in axonal transport of CGRP and CGRP receptor expression in the injured and regenerating rat sciatic nerve using CGRP-immunocytochemistry, radioimmunoassay and quantitative in situ receptor autoradiography techniques. Axotomy led to a gradual and prolonged, 2.5- to 3.5-fold increase in specific CGRP binding to the distal part of the crushed sciatic nerve, beginning 4-6 days after axotomy. An even stronger, up to 30-fold increase was observed after 30-42 day denervation in the distal part of the transected sciatic nerve, where neurite reinnervation was prevented by retroversion and ligation of the proximal nerve stump. Reconnection of the proximal and distal nerve stumps 21 days after transection did not lead to a major reduction in specific CGRP binding but prevented a further increase that occurred between 21 and 42 days after transection without reconnection. In contrast, the anterograde axonal transport of CGRP decreased after axotomy to 40-50% of the control values 6-8 days after nerve crush but recovered towards normal levels during successful regeneration. Interestingly, the retrograde axonal transport of CGRP appeared to amount to only 10-20% of the anterograde transport, suggesting that the peptide may be released by the regenerating neurites into the endoneurium of the injured peripheral nerve. In view of the persistent upregulation in endoneural CGRP binding after axotomy these data indicate that axonal CGRP could play a regulatory role in mediating axonal-endoneural cell interaction during peripheral nerve regeneration.     

Growth of optic tract axons in nerve grafts in hamsters

Segments of peripheral nerve, transplanted to the brain or spinal cord, recently have been shown to support regeneration of axons from a variety of central neurons. However, long-tract axons, injured at considerable distances from their cell bodies, have proven refractory to such regenerative support. This report presents evidence for successful, although similarly limited, growth of retinal ganglion cell axons into peripheral nerve grafts placed in the optic tract of adult hamsters. The demonstration of such growth allows the possibility that the primary visual pathways may serve as an advantageous model system in which to study the mechanism of graft-effected regeneration of long-tract axons in the adult mammalian central nervous system.     

Regeneration of axons from the adult rat optic nerve: influence of fetal brain grafts, laminin, and artificial basement membrane

After transection of the optic nerve of adult rats, most of the axons in the proximal stump die and the surviving ones are unable to regenerate into the distal optic nerve. Since the fetal brain has an inherent capacity to regenerate axons, we investigated whether fetal (E16) target regions of optic axons (thalamus and tectum) transplanted to the completely transected optic nerve of adult rats would promote axon regeneration. In control operated rats, axon growth beyond the site of transection was restricted to a few fibers that grew irregularly within the connective tissue scar. By contrast, in grafted animals directed outgrowth of optic axons toward the transplant started at 6 days postoperation (p.o.) and reached its maximum 15 days p.o. and later, when numerous single optic fibers and small axon fascicles had grown toward and into the graft, where they formed arborizations and terminal varicosities. Regenerating optic axons were further advanced than GFAP-positive strands of astroglia that emanated from the proximal optic nerve stump. Laminin immunoreactivity appeared at 6 days p.o. in the zone of reactive astroglia in the terminal part of the optic nerve stump. Later it showed a distribution complementary to the pattern of GFAP immunoreactivity, which it seemd to circumscribe. There was no unequivocal codistribution of laminin immunoreactivity with regenerating axons. In further experiments, target regions from different ontogenetic stages (E14 to neonate and adult) and nontarget regions (E16, cerebral cortex or spinal cord) were grafted to the optic nerve stump. With the exception of the adult grafts, all transplants had effects on axon regeneration comparable to those of E16 target regions. In order to test the effects of extracellular matrix molecules on axon regeneration, a basement membrane gel reconstituted from individual components of the Engelbreth-Holm-Sarcoma (EHS) sarcoma was implanted between proximal and distal optic nerve stumps. No axons were induced to regenerate by this matrix. Likewise, laminin adsorbed to nitrocellulose paper and implanted at the lesion site did not stimulate axon growth from the proximal optic nerve stump. These results indicate that fetal brain is able to induce and direct regrowth of axons from the optic nerve toward the graft across a substrate that is not composed of astroglia or basement membrane components like laminin. The directed growth of axons in the absence of a preformed substrate implies a chemotactic growth response along a concentration gradient mediated by neurotropic molecules released from the graft.     

Predegenerated peripheral nerve grafts rescue retinal ganglion cells from axotomy-induced death

The inability of axons to grow across damaged central nervous system tissue is a well-known consequence of injury to the brain and spinal cord of adult mammals. Our previous studies showed that predegenerated peripheral nerve grafts facilitate neurite outgrowth from the injured hippocampus and that this effect was particularly distinct when 7-, 28-, and 35-day-predegenerated nerve grafts were used. The purpose of the present study was to use the above method to induce and support the regrowth of injured nerve fibers as well as the survival of retinal ganglion cells (RGCs). Adult Sprague-Dawley rats were assigned to three groups. In the experimental groups transected optic nerve was grafted with peripheral nerve (predegenerated for 7 days (PD) or nonpredegenerated). In the control group, the optic nerve was totally transected. RGCs and growing fibers labeled with fluorescent tracers were examined. They were counted and the results were subjected to statistical analysis. Retinal ganglion cells survived in the groups treated with predegenerated as well as nonpredegenerated grafts; however, the number of surviving retinal ganglion cells was significantly higher in the first one. In both groups the regrowth of the transected optic nerve was observed but the distance covered by regenerating fibers was longer in the PD group. No fibers inside grafts and no labeled cells in retinas were present in the control animals. On the basis of the obtained results we can state that the predegeneration of grafts enhance their neurotrophic influence upon the injured retinal ganglion cells.     

Different effects of astrocytes and Schwann cells on regenerating retinal axons

Following a crush injury of the optic nerve in adult rats, the axons of retinal ganglion cells, stimulated to regenerate by a lens injury and growing within the optic nerve, are associated predominantly with astrocytes: they remain of small diameter (0.1-0.5 microm) and unmyelinated for > or = 2 months after the operation. In contrast, when the optic nerve is cut and a segment of a peripheral nerve is grafted to the ocular stump of the optic nerve, the regenerating retinal axons are associated predominantly with Schwann cells: they are of larger diameter than in the previous experiment and include unmyelinated axons (0.2-2.5 microm) and myelinated axons (mean diameter 2.3 microm). Thus, the grafted peripheral nerve, and presumably its Schwann cells, stimulate enlargement of the regenerating retinal axons leading to partial myelination, whereas the injured optic nerve itself, and presumably its astrocytes, does not. The result points to a marked difference of peripheral (Schwann cells) and central (astrocytes) glia in their effect on regenerating retinal axons.     

The cell recognition molecule CHL1 is strongly upregulated by injured and regenerating thalamic neurons

Close homologue of L1 (CHL1) is a cell recognition molecule known to promote axonal growth in vitro. We have investigated the expression of CHL1 mRNA by regenerating central nervous system (CNS) neurons, by using in situ hybridisation 3 days to 10 weeks following the implantation of living and freeze-killed peripheral nerve autografts into the thalamus of adult rats. At all survival times after implantation of living grafts, neurons of the thalamic reticular nucleus (TRN), close to the graft tip and up to 1 mm away from it, displayed strong signal for CHL1 mRNA, even though TRN neurons show very low levels of CHL1 mRNA expression in unoperated animals. When the cell bodies of regenerating neurons were identified by retrograde labelling from the distal portion of the grafts, 4-6 weeks after operation, most of the labelled cells were found in the TRN and could be shown to haveupregulated CHL1 mRNA. In addition, some neurons in dorsal thalamic nuclei near the graft tip transiently upregulated CHL1 mRNA during the first 3 weeks after graft implantation, and glial cells showing CHL1 mRNA expression were present at the brain/graft interface 3 days to 2 weeks after operation. Freeze-killed grafts, into which axons do not regenerate, caused a transient upregulation of CHL1 in very few TRN neurons near the graft tip and in glial cells at the brain/graft interface but did not produce prolonged CHL1 mRNA expression. CHL1 can therefore be added to the list of molecules (including GAP-43, L1, and c-jun) strongly expressed by CNS neurons that regenerate their axons into nerve grafts, but not by those neurons that fail to regenerate their axons.     

Functional activity of rat brainstem neurons regenerating axons along peripheral nerve grafts

To investigate activation and discharge patterns of central nervous system neurons that regenerate lengthy axons along peripheral nerve grafts we inserted a 4 cm long autologous segment of sciatic nerve into the dorsolateral medulla oblongata of adult rats. Two to 6 months after grafting, the distribution of the cells of origin of the regenerating axons in many nuclei of the brainstem was documented by retrograde horseradish peroxidase labelling from the cut end of the grafts. Functional properties of neurons regenerating axons into the grafts were studied by recording from single regenerated fibers teased from the grafts. Conduction velocities of graft fibers ranged from < 1m/s to 25m/s (30°C). Spontaneous centrifugal impulse traffic in the grafts included units firing in bursts synchronously with the respiratory cycle. Activity in other units was either elicited or inhibited by natural or electrical stimulation of the periphery. Most units recorded in the grafts were neither spontaneously active nor responsive to stimulation of primary afferents. We conclude that: (1) there are central nervous system neurons projecting into the grafts that respond to both excitatory and inhibitory transsynaptic influences; (2) at least some of the spontaneous and induced activity recorded from axons in the grafts resembles that known for normal nerve cells in the regions of the brainstem from which axonal growth arises; and (3) it is possible that many central neurons regenerating axons into peripheral nerve grafts have significantly reduced or altered synaptic inputs.     

Studies on the control of myelinogenesis. 3. Signalling of oligodendrocyte myelination by regenerating peripheral axons

Continuing from earlier work which demonstrated the peripheral axonal regulation of Schwann cell myelination, this study has investigated the possibility that a peripheral axon can stimulate oligodendrocyte myelination. To test this hypothesis, regenerating PNS axons were allowed to interact with uncommitted oligodendrocytes by transecting a rat peroneal nerve and inserting a segment of the autologous optic nerve between the cut ends. Grafts were maintained for 4–28 weeks and then examined by light and electron microscopy. A few regenerating peripheral myelinated nerve fibers penetrated the optic nerve graft. Some axons penetrated the outer margin of the graft, were myelinated by Schwann cells, and surrounded by astrocyte processes bordered by basal lamina. More centrally in the optic nerve graft, regenerating peripheral axons displayed myelin of CNS type. The outer myelin lamella abutted directly on the plasmalemma surface of surrounding astrocytic processes and was expanded focally to form a glial tongue. These observations demonstrate the experimental induction of central myelination by regenerating peripheral axons and suggest the existence of a common neuronal mechanism to stimulate myelin formation by both the Schwann cell and the oligodendrocyte.     

Peripheral nerve regeneration through optic nerve grafts

Summary Grafts of optic nerve were placed end-toend with the proximal stumps of severed common peroneal nerves in inbred mice. It was found that fraying the proximal end of adult optic nerve grafts to disrupt the glia limitans increased their chances of being penetrated by regenerating peripheral nerve fibres. Suturing grafts to the proximal stump also enhanced their penetration by axons. The maximum distance to which the axons grew through the CNS tissue remained about 1.5 mm from 2–12 weeks after grafting. Schwann cells were seldom identified in the grafts. Varicose and degenerating nerve fibres were often seen within the grafts. Some varicose profiles were shown to be the terminal parts of axons within the grafts. Axons containing clusters of organelles resembling synaptic vesicles became more abundant in the longerterm grafts. Immunohistochemical studies performed on sutured grafts using a polyclonal antiserum to neurofilaments confirmed the impressions given by the electron microscopical observations. Grafts of neonatal optic nerve lacked myelin debris but were not usually penetrated by regenerating peripheral axons within a 6-week period. Sixty minutes after the intravenous injection of horseradish peroxidase, reaction product could be detected in the extracellular spaces around blood vessels in all types of living optic nerve graft. This indicates that blood-borne macromolecules could penetrate the grafts. However the profiles of axons which were found within living optic nerve grafts had no obvious relationship to blood vessels and were usually surrounded by astrocytic processes. These results suggest that living astrocytes, rather than the absence of serum-derived trophic factors or the presence of CNS myelin, constitute the major barrier to the extension of axons and the migration of Schwann cells into CNS tissue.Supported by a grant from the Wellcome Trust