Peripheral nerve explants grafted into the vitreous body of the eye promote the regeneration of retinal ganglion cell axons severed in the optic nerve

Summary We have conducted experiments in the adult rat visual system to assess the relative importance of an absence of trophic factors versus the presence of putative growth inhibitory molecules for the failure of regeneration of CNS axons after injury. The experiments comprised three groups of animals in which all optic nerves were crushed intra-orbitally: an optic nerve crush group had a sham implant-operation on the eye; the other two groups had peripheral nerve tissue introduced into the vitreous body; in an acellular peripheral nerve group, a frozen/thawed teased sciatic nerve segment was grafted, and in a cellular peripheral nerve group, a predegenerate teased segment of sciatic nerve was implanted. The rats were left for 20 days and their optic nerves and retinae prepared for immunohistochemical examination of both the reaction to injury of axons and glia in the nerve and also the viability of Schwann cells in the grafts. Anterograde axon tracing with rhodamine-B provided unequivocal qualitative evidence of regeneration in each group, and retrograde HRP tracing gave a measure of the numbers of axons growing across the lesion by counting HRP filled retinal ganglion cells in retinal whole mounts after HRP injection into the optic nerve distal to the lesion. No fibres crossed the lesion in the optic nerve crush group and dense scar tissue was formed in the wound site. GAP-43-positive and rhodamine-B filled axons in the acellular peripheral nerve and cellular peripheral nerve groups traversed the lesion and grew distally. There were greater numbers of regenerating fibres in the cellular peripheral nerve compared to the acellular peripheral nerve group. In the former, 0.6–10% of the retinal ganglion cell population regenerated axons at least 3–4 mm into the distal segment. In both the acellular peripheral nerve and cellular peripheral nerve groups, no basal lamina was deposited in the wound. Thus, although astrocyte processes were stacked around the lesion edge, a glia limitans was not formed. These observations suggest that regenerating fibres may interfere with scarring. Viable Schwann cells were found in the vitreal grafts in the cellular peripheral nerve group only, supporting the proposition that Schwann cell derived trophic molecules secreted into the vitreous stimulated retinal ganglion cell axon growth in the severed optic nerve. The regenerative response of acellular peripheral nerve-transplanted animals was probably promoted by residual amounts of these molecules present in the transplants after freezing and thawing. In the optic nerves of all groups the astrocyte, microglia and macrophage reactions were similar. Moreover, oligodendrocytes and myelin debris were also uniformly distributed throughout all nerves. Our results suggest either that none of the above elements inhibit CNS regeneration after perineuronal neurotrophin delivery, or that the latter, in addition to mobilising and maintaining regeneration, also down regulates the expression of axonal growth cone-located receptors, which normally mediate growth arrest by engaging putative growth inhibitory molecules of the CNS neuropil.     

Neovascularization occurs in response to crush lesions of adult frog optic nerves

Summary The capacity of the adult frog optic nerve to regenerate following a crush lesion is well established and is in contrast to the lack of regeneration of mammalian optic nerves after similar lesions. One factor which may contribute to the enhanced regenerative capacity of amphibian optic nerves is the rapid removal of cellular debris from the nerve after injury. In this study the morphology of normal and crushed frog optic nerves has been compared. Although the intraorbital region of the normal adult frog optic nerve is avascular, new intraparenchymal blood vessels appear central to the crush site 24 h after the nerve lesion. The appearance of these blood vessels is coincident with the appearance of granulocytes and macrophages in the nerve. Successful regeneration of the adult frog optic nerve may depend on this neovascularization to facilitate the rapid removal of cellular debris and to supply regenerating axons with trophic substances.     

Microenvironmental changes during axonal regrowth in the optic nerve of the myelin deficient rat. Immunocytochemical and ultrastructural observations

Summary Lesion-induced regenerative sprouting of CNS axons is accompanied by reactions of the supporting glia and vascular and connective tissue which may influence the extent of regeneration. In a previous report, it was shown that after crush injury, the amyelinated optic nerve of the myelin deficient (md) mutant rat contains greater numbers of regrowing axons proximal to the site of crush than that of normally myelinated littermates. The present study was designed to compare the response of the microenvironment, i.e. glial cells and vascular and connective tissue, in md and normally myelinated optic nerves 2, 4 and 6 days after crush injury. In unoperated normal optic nerves monoclonal antibodies to the HNK-1 carbohydrate labelled astrocytic processes at the ultrastructural level whereas in unoperated md mutants HNK-1 staining was restricted to axonal surfaces. Immunoreactivity with monoclonal antibodies to stage-specific embryonic antigen-1 (SSEA-1) was confined to astrocytic surfaces in both md and wildtype animals. After axotomy of md optic nerves regrowing axons were more numerous in the proximal site of the crush and extended further into the lesion than in wildtype animals. In both md and wildtype rats regrowing axons were HNK-1-positive. In md rats strong reaction with antibodies to laminin and fibronectin was only seen in 6-day-old lesions of md rats whereas immunoreactivity was less distinct in operated littermate controls. Immunolabelling was obviously associated with blood vessels, since crush lesions in both md and wildtype rats were Schwann cell-free as assessed by electron microscopy and immunocytochemistry. In both operated md and normal littermates crush lesions contained degenerating astrocytes as well as reactive astrocytes in which the intermediate filaments of the perikarya failed to stain immunocytochemically for GFAP, vimentin, desmin, and a common determinant of intermediate filaments. In contrast, reactive astrocytes in the lesion site of normally myelinated rats expressed the SSEA-1 antigen intracytoplasmically whereas in md mutants astrocytes were completely SSEA-1-negative. Infiltration of crush lesions by macrophages was less extensive in md rats than in normal littermates. However the overall content of macrophages in the peritoneal cavity was also reduced. The present study demonstrates that (1) md optic nerves lack HNK-1-reactive astrocytes; (2) in the axotomized wildtype optic nerve impaired axonal regrowth may be associated with distinct immuno-phenotypes of the supporting glial cells, i.e. SSEA-1-positive astrocytes; (3) laminin and fibronectin seem not to be essential for improved axonal regrowth in md rats.     

Regrowth of PNS axons through grafts of the optic nerve of the Browman-Wyse (BW) mutant rat

Summary We have examined the behaviourin vivo of regenerating PNS axons in the presence of grafts of optic nerve taken from the Browman-Wyse mutant rat. Browman-Wyse optic nerves are unusual because a 2–4 mm length of the proximal (retinal) end of the nerve lacks oligodendrocytes and CNS myelin and therefore retinal ganglion cell axons lying within the proximal segment are unmyelinated and ensheathed by processes of astrocyte cytoplasm. Schwann cells may also be present within some proximal segments. Distally, Browman-Wyse optic nerves are morphologically and immunohistochemically indistinguishable from control optic nerves.When we grafted intact Browman-Wyse optic nerves or triplets consisting of proximal, junctional and distal segments of Browman-Wyse optic nerve between the stumps of freshly transected sciatic nerves, we found that regenerating axons avoided all the grafts which did not contain Schwann cells, i.e., proximal segments which contained only astrocytes; regions of Schwann cell-bearing proximal segments which did not contain Schwann cells; junctional and distal segments (which contained astrocytes, oligodendrocytes and CNS myelin debris). However, axons did enter and grow through proximal segments which contained Schwann cells in addition to astrocytes. Schwann cells were seen within grafts even after mitomycin C pretreatment of sciatic proximal nerve stumps had delayed outgrowth of Schwann cells from the host nerves; we therefore conclude that the Schwann cells which became associated with regenerating axons within the grafts of Browman-Wyse optic nerve were derived from an endogenous population. Our findings indicate that astrocytes may be capable of supporting axonal regeneration in the presence of Schwann cells.     

Optic nerve regeneration after intravitreal peripheral nerve implants: trajectories of axons regrowing through the optic chiasm into the optic tracts

We have studied axon regeneration through the optic chiasm of adult rats 30 days after prechiasmatic intracranial optic nerve crush and serial intravitreal sciatic nerve grafting on day 0 and 14 post-lesion. The experiments comprised three groups of treated rats and three groups of controls. All treated animals received intravitreal grafts either into the left eye after both left sided (unilateral) and bilateral optic nerve transection, or into both eyes after bilateral optic nerve transection. Control eyes were all sham grafted on day 0 and 14 post-lesion, and the optic nerves either unlesioned, or crushed unilaterally or bilaterally. No regeneration through the chiasm was seen in any of the lesioned control optic nerves. In all experimental groups, large numbers of axons regenerated across the optic nerve lesions ipsilateral to the grafted eyes, traversed the short distal segment of the optic nerve and invaded the chiasm without deflection. Regeneration was correlated with the absence of the mesodermal components in the scar. In all cases, axon regrowth through the chiasm appeared to establish a major crossed and a minor uncrossed projection into both optic tracts, with some aberrant growth into the contralateral optic nerve. Axons preferentially regenerated within the degenerating trajectories from their own eye, through fragmented myelin and axonal debris, and reactive astrocytes, oligodendrocytes, microglia and macrophages. In bilaterally lesioned animals, no regeneration was detected in the optic nerve of the unimplanted eye. Although astrocytes became reactive and their processes proliferated, the architecture of their intrafascicular processes was little perturbed after optic nerve transection within either the distal optic nerve segment or the chiasm. The re-establishment of a comparatively normal pattern of passage through the chiasm by regenerating axons in the adult might therefore be organised by this relatively immutable scaffold of astrocyte processes. Binocular interactions between regenerating axons from both nerves (after bilateral optic nerve transection and intravitreal grafting), and between regenerating axons and the intact transchiasmatic projections from the unlesioned eye (after unilateral optic nerve lesions and after ipsilateral grafting) may not be important in establishing the divergent trajectories, since regenerating axons behave similarly in the presence and absence of an intact projection from the other eye.     

Myelination of regenerated axons in goldfish optic nerve by Schwann cells

Summary This study uses immunohistochemistry and EM to examine the site of injury in goldfish optic nerve during axonal regeneration. Within seven days of nerve crush axons begin to regrow and a network of GFAP⁺ reactive astrocytes appears in the nerve on either side of the injury. However, the damaged area remains GFAP^–. By 42 days after nerve crush, the sheaths of new axons acquire myelin marker 6D2, and the crush area becomes populated by a mass of longitudinally-orientated S-100⁺ cells. Ultrastructurally, the predominant cells in the crush area bear a strong resemblance to peripheral nerve Schwann cells; they display a one-to-one association with myelinated axons, have a basal lamina and are surrounded by collagen fibres. It is proposed that these cells are Schwann cells which enter the optic nerve as a result of crush, where they become confined to the astrocyte-free crush area.     

Electron microscopic study of the interaction of axons and glia at the site of anastomosis between the optic nerve and cellular or acellular sciatic nerve grafts

Summary The interactions between retinal ganglion cell (RGC) axons and glia at the site of optic nerve section and at the junctional zone between optic nerve and cellular or acellular peripheral nerve (PN) grafts have been studied electron microscopically. After transection, RGC axons, accompanied by processes of astrocyte cytoplasm, grew out from the proximal optic nerve stump into the scar tissue that developed between proximal and distal stumps. However, axons failed to cross the scar, and none entered the distal stump. By 3 days post lesion (DPL), bundles of RGC axons, accompanied by astrocytes and oligodendrocytes, grew out from the proximal optic nerve stump into the junctional zone between optic nerve and either type of PN graft. The bundles of RGC axons and growth cones that grew towards acellular PN grafts degenerated within 10–20 DPL; by 30 DPL a small number of axons persisted within the end of the proximal optic nerve stump. No axons were seen within the acellular PN grafts. These results suggest that reactive axonal sprouting, axon outgrowth and glial migration from the proximal optic nerve stump are events that occur during an acute response to injury, and that they are independent of the presence of Schwann cells. However, it would appear that few axons entered either scar or junctional zone unless accompanied by glia. There was little evidence that axon outgrowth was laminin-dependent.The bundles that grew towards cellular PN grafts encountered cells that we have identified as Schwann cells within the junctional zone: the axons in these bundles survived and entered the cellular grafts. Schwann cells migrated into the junctional zone from the cellular PN graft. It is probable that Schwann cells facilitated RGC axon entry into the graft directly by both cell contact and the secretion of neuronotrophic factors, and indirectly by modifying the CNS glia in the junctional zone.     

Quantification of the mononuclear phagocyte response to Wallerian degeneration of the optic nerve

Summary We investigated the numbers, origin and phenotype of mononuclear phagocytes (macrophages/microglia) responding to Wallerian degeneration of the mouse optic nerve in order to compare it with the response to Wallerian degeneration in the PNS, already described. We found macrophage/microglial numbers elevated nearly four fold in the distal segments of crushed optic nerves and their projection areas in the contralateral superior colliculus 1 week after unilateral optic nerve crush. This relative increase in mononuclear phagocyte numbers compared well with the four-to five-fold increases reported in the distal segments of transected saphenous or sciatic nerves. Moreover, maximum numbers are reached at 3, 5 and 7 days in the saphenous, sciatic and optic nerves respectively, suggesting that the very slow clearance of axonal debris and myelin in CNS undergoing Wallerian degeneration is not simply due to a slow or small mononuclear phagocyte response. The apparent delay in the response in the CNS occurs because the mononuclear phagocytes respond to the Wallerian degeneration of axons, which is slightly slower in the CNS than the PNS, rather than to events associated with the crush itself, such as the abolition of normal electrical activity in the distal segment. This was demonstrated by the protracted time course of the mononuclear phagocyte response in the distal segment following optic nerve crush in mice carrying theWld ^smutation which dramatically slows the rate at which the axons undergo Wallerian degeneration. By³H-Thymidine labelling or by blocking microglial proliferation by X-irradiation of the head prior to optic nerve crush, we showed that the majority of macrophages/microglia initiating the response to Wallerian degeneration were of local, CNS origin but these cells rapidly (from 3 days post crush) upregulate endocytic and phagocytic functional markers although they do not resemble rounded myelin-phagocytosing macrophages observed in degenerating peripheral nerves. We speculate that the poor clearance of myelin in CNS fibre tracts undergoing Wallerian degeneration compared to the PNS, in the face of a mononuclear phagocyte response which is similar in relative magnitude and time course, is because Schwann cells in degenerating peripheral nerves promptly modify their myelin sheaths such that they can be recognized and phagocytosed by macrophages, whilst in the CNS oligodendrocytes do not.     

Lesion-associated expression of transforming growth factor-beta-2 in the rat nervous system: evidence for down-regulating the phagocytic activity of microglia and macrophages

The mechanisms that control the phagocytic activities of microglia and macrophages during disorders of the nervous system are largely unknown. In the present investigation, we assessed the functional role of transforming growth factor (TGF)beta2 in vitro and studied TGFbeta-2mRNA and protein expression in two CNS lesion paradigms in vivo characterized by fundamental differences in microglia/macrophage behaviour: optic nerve crush exhibiting slow, and focal cerebral ischemia exhibiting rapid phagocytic transformation. Furthermore, we used sciatic nerve crush injury as a PNS lesion paradigm comparable to brain ischemia in its rapid phagocyte response. In normal and degenerating optic nerves, astrocytes strongly and continuously expressed TGF-beta2 immunoreactivity. In contrast, TGF-beta2 was downregulated in Schwann cells of degenerating sciatic nerves, and was not expressed by reactive astrocytes in the vicinity of focal ischemic brain lesions during the acute phagocytic phase. In line with its differential lesion-associated expression pattern, exogenous TGF-beta2 suppressed spontaneous myelin phagocytosis by microglia/macrophages in a mouse ex vivo assay of CNS and PNS Wallerian degeneration. In conclusion, we have identified TGF-beta2 as a nervous system intrinsic cytokine that could account for the differential regulation of phagocytic activities of microglia and macrophages during injury.     

Differential recruitment of CD8+ macrophages during Wallerian degeneration in the peripheral and central nervous system

The strong macrophage response occurring during Wallerian degeneration in the peripheral but not central nervous system has been implicated in tissue remodeling and growth factor production as key requirements for successful axonal regeneration. We have previously identified a population of CD8+ phagocytes in ischemic brain lesions that differed in its recruitment pattern from CD4+ macrophages/microglia found in other lesion paradigms. In the present study we show that crush injury to the sciatic nerve induced strong infiltration by CD8+ macrophages both at the crush site and into the degenerating distal nerve stump. At the crush site, CD8+ macrophages appeared within 24 hours whereas infiltration of the distal nerve parenchyma was delayed to the second week. CD8+ macrophages were ED1+ and CD11b+ but always MHC class II-. Most CD8+ macrophages coexpressed CD4 while a significant number of CD4+/CD8-macrophages was also present. Expression of the resident tissue macrophage marker ED2 was largely restricted to the CD4+/CD8- population. Following intraorbital crush injury to the optic nerve, infiltration of CD8+ macrophages was strictly confined to the crush site. Taken together, our study demonstrates considerable spatiotemporal diversity of CD8+ macrophage responses to axotomy in the peripheral and central nervous system that may have implications for the different extent of axonal regeneration observed in both systems.     

Regeneration of axons in the optic nerve of the adult Browman-Wyse (BW) mutant rat

Summary We have studied the regeneration of axons in the optic nerves of the BW rat in which both oligodendrocytes and CNS myelin are absent from a variable length of the proximal (retinal) end of the nerve. In the optic nerves of some of these animals, Schwann cells are present. Axons failed to regenerate in the exclusively astrocytic environment of the unmyelinated segment of BW optic nerves but readily regrew in the presence of Schwann cells even across the junctional zone and into the myelin debris filled distal segment. In the latter animals, the essential condition for regeneration was that the lesion was sited in a region of the nerve in which Schwann cells were resident. Regenerating fibres appeared to be sequestered within Schwann cell tubes although fibres traversed the neuropil intervening between the ends of discontinuous bundles of Schwann cell tubes, in both the proximal unmyelinated and myelin debris laden distal segments of the BW optic nerve. Regenerating axons never grew beyond the distal point of termination of the tubes. These observations demonstrate that central myelin is not an absolute requirement for regenerative failure, and that important contributing factors might include inhibition of astrocytes and/or absence of trophic factors. Regeneration presumably occurs in the BW optic nerve because trophic molecules are provided by resident Schwann cells, even in the presence of central myelin, oligodendrocytes and astrocytes. All the above experimental BW animals also have Schwann cells in their retinae which myelinate retinal ganglion cell axons in the fibre layer. Control animals comprised normal Long Evans Hooded rats, BW rats in which both retina and optic nerve were normal, and BW rats with Schwann cells in the retina but with normal, i.e. CNS myelinated, optic nerves. Regeneration was not observed in any of the control groups, demonstrating that, although the presence of Schwann cells in the retina may enhance the survival of retinal ganglion cells after crush, concomitant regrowth of axons cut in the optic nerve does not take place.     

Regeneration in theXenopus tadpole optic nerve is preceded by a massive macrophage/microglial response

Summary Changes in the optic nerve following a crush lesion and during axonal regeneration have been studied inXenopus tadpoles, using ultrastructural and immunohistological methods. Degeneration of both unmyelinated and myelinated axons is very rapid and leads to the formation, within 5 days, of a nerve which consists largely of degeneration debris and cells. Immunohistological analysis with monoclonal antibody 5F4 shows that there is a rapid and extensive microglial/macrophage response to crush of the nerve. Regenerating axons have begun to enter the distal stump by 5 days and grow along the outer part of the nerve in close approximation to the astrocytic glia limitans. Between 5 and 10 days after nerve crush, regenerating axons reach and pass the chiasma. Macrophages are seen in the nerve at the site of the lesion within 1 h, and the response peaks between 3–5 days, just before axonal regeneration gets under way.     

Intrastriatal grafts of rat colonic smooth muscle lacking myenteric ganglia stimulate axonal sprouting and regeneration

Grafts of living or freeze-killed freshly dissected colonic smooth muscle from young inbred Fischer rats were implanted into the corpus striatum of adult Fischer rats. Sections of brain were examined electron microscopically 3 and 6 wk after implantation. At both times, living grafts were vascularised and contained healthy differentiated smooth muscle cells, fibroblasts, interstitial cells of Cajal and some macrophages. Large bundles of small nonmyelinated axons, identified as CNS axonal sprouts, could be observed in the brain at and near the interface between the living smooth muscle and the CNS tissue. Bundles of regenerating CNS axons, often associated with astrocyte processes, had grown into the grafts. Some axons within the grafts had matured, enlarged and become myelinated by oligodendrocyte processes or Schwann cells. In some cases, smooth muscle cells were observed in close and intricate association with axons. In contrast to the living grafts, grafts of freeze-killed smooth muscle, examined 3 and 6 wk after implantation, contained macrophages, fibroblasts, collagen and large amounts of cellular debris, but no living muscle cells, astrocytes or Schwann cells. The striatal neuropil around freeze-killed grafts did not contain large bundles of CNS axonal sprouts and bundles of axons were not observed within the freeze-killed graft. This study demonstrates that cells from the smooth muscle layers of the colon, in the absence of myenteric ganglia, can stimulate a vigorous regenerative response from CNS axons when implanted into the corpus striatum of adult rats.     

Schwann cells in the regenerating fish optic nerve: evidence that CNS axons, not the glia, determine when myelin formation begins

Fish optic nerve fibres quickly regenerate after injury, but the onset of remyelination is delayed until they reach the brain. This recapitulates the timetable of CNS myelinogenesis during development in vertebrate animals generally, and we have used the regenerating fish optic nerve to obtain evidence that it is the axons, not the myelinating glial cells, that determine when myelin formation begins. In fish, the site of an optic nerve injury becomes remyelinated by ectopic Schwann cells of unknown origin. We allowed these cells to become established and then used them as reporters to indicate the time course of pro-myelin signalling during a further round of axonal outgrowth following a second upstream lesion. Unlike in the mammalian PNS, the ectopic Schwann cells failed to respond to axotomy and to the initial outgrowth of new optic axons. They only began to divide after the axons had reached the brain. Shortly afterwards, small numbers of Schwann cells began to leave the dividing pool and form myelin sheaths. More followed gradually, so that by 3 months remyelination was almost completed and few dividing cells were left. Moreover, remyelination occurred synchronously throughout the optic nerve, with the same time course in the pre-existing Schwann cells, the new ones that colonised the second injury, and the CNS oligodendrocytes elsewhere. The optic axons are the only common structures that could synchronise myelin formation in these disparate glial populations. The responses of the ectopic Schwann cells suggest that they are controlled by the regenerating optic axons in two consecutive steps. First, they begin to proliferate when the growing axons reach the brain. Second, they leave the cell cycle to differentiate individually at widely different times during the ensuing 2 months, during the critical period when the initial rough pattern of axon terminals in the optic tectum becomes refined into an accurate map. We suggest that each axon signals individually for myelin ensheathment once it completes this process.     

Nerve injury, axonal degeneration and neural regeneration: basic insights

Axotomy or crush of a peripheral nerve leads to degeneration of the distal nerve stump referred to as Wallerian degeneration (WD). During WD a microenvironment is created that allows successful regrowth of nerve fibres from the proximal nerve segment. Schwann cells respond to loss of axons by extrusion of their myelin sheaths, downregulation of myelin genes, dedifferentiation and proliferation. They finally aline in tubes (Büngner bands) and express surface molecules that guide regenerating fibres. Hematogenous macrophages are rapidly recruited to the distal stump and remove the vast majority of myelin debris. Molecular changes in the distal stump include upregulation of neurotrophins, neural cell adhesion molecules, cytokines and other soluble factors and their corresponding receptors. Axonal injury not only induces muscle weakness and loss of sensation but also leads to adaptive responses and neuropathic pain. Regrowth of nerve fibres occurs with high specificity with formerly motor fibres preferentially reinnervating muscle. This involves recognition molecules of the L2/HNK-1 family. Nerve regeneration occurs at a rate of 3-4 mm/day after crush and 2-3 mm/day after sectioning a nerve. Nerve regeneration can be fostered pharmacologically. Upon reestablishment of axonal contact Schwann cells remyelinate nerve sprouts and downregulate surface molecules characteristic for precursor/premyelinating or nonmyelinating Schwann cells. At present it is unclear whether axonal regeneration after nerve injury is impeded in neuropathies.     

Myelin repair by Schwann cells in the regenerating goldfish visual pathway: regional patterns revealed by X-irradiation

Summary In the regenerating goldfish optic nerves, Schwann cells of unknown origin reliably infiltrate the lesion site forming a band of peripheral-type myelinating tissue by 1–2 months, sharply demarcated from the adjacent new CNS myelin. To investigate this effect, we have interfered with cell proliferation by locally X-irradiating the fish visual pathway 24 h after the lesion. As assayed by immunohistochemistry and EM, irradiation retards until 6 months formation of new myelin by Schwann cells at the lesion site, and virtually abolishes oligodendrocyte myelination distally, but has little or no effect on nerve fibre regrowth. Optic nerve astrocyte processes normally fail to re-infiltrate the lesion, but re-occupy it after irradiation, suggesting that they are normally excluded by early cell proliferation at this site. Moreover, scattered myelinating Schwann cells also appear in the oligodendrocyte-depleted distal optic nerve after irradiation, although only as far as the optic tract. Optic nerve reticular astrocytes differ in various ways from radial glia elsewhere in the fish CNS, and our observations suggest that they may be more permissive to Schwann cell invasion of CNS tissue.     

Microglia in tadpoles ofXenopus laevis: Normal distribution and the response to optic nerve injury

Summary We have studied the distribution of microglia in normalXenopus tadpoles and after an optic nerve lesion, using a monoclonal antibody (5F4) raised againstXenopus retinas of which the optic nerves had been cut 10 days previously. The antibody 5F4 selectively recognizes macrophages and microglia inXenopus. In normal animals microglia are sparsely but widely distributed throughout the retina, optic nerve, diencephalon and mesencephalon (other regions were not examined). After crush or cut of an optic nerve, or eye removal, there occurs an extensive microglial response along the affected optic pathway. Within 18 h an increase in the number of microglial cells in the optic tract and tectum can be detected. This response increases to peak at around 5 days after the lesion. At this time the nerve distal to the lesion contains many microglial cells; the entire optic tract is outlined by microglia, extended along the degenerating fibres; and the affected tectum shows a heavy concentration of microglia. This microglial response thereafter decreases and has mostly gone by 34 days. We conclude that the microglial response to optic nerve injury inXenopus tadpoles starts early, peaks just before the regenerating optic nerve axons enter the brain, and is much diminished by the time the retinotectal projection is re-established. The timing is such that the microglial response could play a major role in facilitating regeneration.     

The response of regenerating peripheral neuntes to a grafted optic nerve

Summary Optic nerves, both viable (fresh or pre-degenerate) or non-viable (frozen-thawed) were grafted between the proximal and distal stumps of freshly transected sciatic nerves, using either 10/0 sutures or strips of nitrocellulose paper. The majority of regenerating peripheral neuntes, always in association with Schwann cells, avoided the viable optic nerve grafts, growing along the outside of the grafts in well vascularized minifascicles until they gained the distal stumps. A very small number of axons entered the grafts and grew, for distances typically less than 2mm, between layers of astrocyte processes. The number of axons entering was not increased by using predegenerate grafts or by blocking Schwann cell proliferation in the proximal stumps by pre-treating the latter with mitomycin C. There was no evidence of a continuous cellular-acellular partition between graft and host during the outgrowth phase of the neurites: it was concluded that axons failed to enter the grafts as a result of inhibitory interactions between Schwann cells and astrocytes. When grafts were rendered acellular, all structured debris, including recognizable components of the extracellular matrix, was rapidly removed and the space thus vacated was invaded by minifascicles of Schwann cells and regenerating neurites. Glial fibrillary acidic protein-positive astrocytes and carbonic anhydrase II-positive oligodendrocytes persisted within viable grafts for 17 months; they did not migrate into the surrounding nerve.     

Regeneration of adult rat CNS axons into peripheral nerve autografts: ultrastructural studies of the early stages of axonal sprouting and regenerative axonal growth

Summary If one end of a segment of peripheral nerve is inserted into the brain or spinal cord, neuronal perikarya in the vicinity of the graft tip can be labelled with retrogradely transported tracers applied to the distal end of the graft several weeks later, showing that CNS axons can regenerate into and along such grafts. We have used transmission EM to examine some of the cellular responses that underlie this regenerative phenomenon, particularly its early stages. Segments of autologous peroneal or tibial nerve were inserted vertically into the thalamus of anaesthetized adult albino rats. The distal end of the graft was left beneath the scalp. Between five days and two months later the animals were killed and the brains prepared for ultrastructural study. Semi-thin and thin sections through the graft and surrounding brain were examined at two levels 6–7 mm apart in all animals: close to the tip of the graft in the thalamus (proximal graft) and at the top of the cerebral cortex (distal graft). In another series of animals with similar grafts, horseradish peroxidase was applied to the distal end of the graft 24–48 h before death. Examination by LM of appropriately processed serial coronal sections of the brains from these animals confirmed that up to several hundred neurons were retrogradely labelled in the thalamus, particularly in the thalamic reticular nucleus.Between five and 14 days after grafting, large numbers of tiny (0.05–0.20 m diameter) nonmyelinated axonal profiles, considered to be axonal sprouts, were observed by EM within the narrow zone of abnormal thalamic parenchyma bordering the graft. The sprouts were much more numerous (commonly in large fascicles), smoother surfaced, and more rounded than nonmyelinated axons further from the graft or in corresponding areas on the contralateral side of animals with implants or in normal animals. At longer post-graft survival times, the number of such axons in the parenchyma around the graft declined.At five days, some axonal sprouts had entered the junctional zone between the brain and the graft. By eight days there were many sprouts in the junctional zone and some had penetrated the proximal graft to lie between its basal lamina-enclosed columns of Schwann cells, macrophages and myelin debris. Within the brain, sprouts were in contact predominantly with other sprouts but also with all types of glial cell. Within the junctional zone and graft many sprouts showed no consistent, close associations with other cell processes, although some were in contact or adjacent to processes of astrocytes, Schwann cells or macrophages. There was no evidence to suggest that axonal sprouts grew along astrocytic extensions to reach the junctional zone and graft. At eight days many axons in the junctional zone and graft were in contact with Schwann cell processes. Such axons, particularly those in intimate contact with the Schwann cell, were larger than those which had not established contact. By 14 days, most axons in the proximal graft were surrounded by Schwann cell processes, predominantly in basal lamina-enclosed columns. Some axons were associated with astrocyte processes, either in basal lamina-enclosed columns containing only astrocyte processes and axons or in columns containing a mixture of astrocyte and Schwann cell processes. The astrocyte processes involved in such bundles were concentrated at the periphery of the proximal graft, were not seen in the distal graft and probably represent long finger-like extensions of the astrocytes which rapidly form a glia limitans at the interface between brain and graft. This glia limitans was partially constructed at five days, almost complete at 14 days and subsequently became progressively thicker and more complex.At one month the proximal graft had acquired many of the features of a regenerating peripheral nerve and axons were present in large numbers in the distal graft. However the axon-Schwann cell relationships were immature in many of the Schwann cell columns both proximally and distally at one month, and virtually no myelination was apparent. At two months there were numerous myelinated fibres both proximally and distally although there were larger numbers of nonmyelinated axons, many in immature relationship with associated Schwann cells. Thus the graft appears to offer not only support for axonal elongation but also for a substantial degree of maturation of at least some of the regenerating axons, although (as will be reported elsewhere), the regenerated nerve fibres began to regress after two months.     

Glial cells in transected optic nerves of immature rats. II. An immunohistochemical study

Summary The glial response to Wallerian degeneration was studied in optic nerves 21 days after unilateral enucleation (PED21) of immature rats, 21 days old (P21), using immunohistochemical labelling. Nerves from normal P21 and P42 nerves were also studied for comparison. At PED21, there was a virtual loss of axons apart from a few solitary fibres of unknown origin. The nerve comprised a homogeneous glial scar tissue formed by dense astrocyte processes, oriented parallel to the long axis of the nerve along the tracks of degenerated axons. Astrocytes were almost perfectly co-labelled by antibodies to glial fibrillary acid protein and vimentin in both normal and transected nerves. However, there was a small population of VIM⁺GFAP^– cells in normal P21 and P42 nerves, and we discuss the possibility that they correspond to O-2A progenitor cells describedin vitro. Significantly, double immunofluorescence labelling in transected nerves revealed a distinct population of hypertrophic astrocytes which were GFAP⁺VIM^–. These cells represented a novel morphological and antigenic subtype of reactive astrocyte. It was also noted that the number of oligodendrocytes in transected nerves did not appear to be less than in normal nerves, on the basis of double immunofluorescence staining for carbonic anhydrase II, myelin oligodendrocyte glycoprotein, myelin basic protein, glial fibrillary acid protein and ED-1 (for macrophages), although it was not excluded that a small proportion may have been microglia. A further prominent feature of transected nerves was that they contained a substantial amount of myelin debris, notwithstanding that OX-42 and ED1 immunostaining showed that there were abundant microglia and macrophages, sufficient for the rapid and almost complete removal of axonal debris. In conclusion, glial cells in the immature P21 rat optic nerve reacted to Wallerian degeneration in a way equivalent to the adult CNS, i.e. astrocytes underwent pronounced reactive changes and formed a dense glial scar, oligodendrocytes persisted and were not dependent on axons for their continued survival, and there was ineffective phagocytosis of myelin possibly due to incomplete activation of microglia/macrophages.