Anomalous axonal outgrowth at the retina caused by injury to the optic nerve or tectal ablation in adultXenopus

Summary The retina and optic nerve head have been examined by light and electron microscopy in adultXenopus laeuis after injury to optic nerve fibres. Intraorbital resection, transection or crush of the optic nerve all resulted in the appearance at the retina of a mass of actively growing axons which formed a ring around the intraretinal and adjacent choroidal portions of the optic nerve head. Formation of this heterotopic axon population was first noted at two weeks after nerve injury and fibres persisted for at least six months. The ectopic fibres were seperated from the optic nerve head by astrocytes within the retina or by blood vessels and fibroblasts of the leptomeninges at extraretinal locations. In general, the orientation of the ectopic fibres was perpendicular to the fibres of the optic nerve. Bundles of axons were found between the ring of ectopic fibres and the pigment epithelial layer of the retina or among the blood sinuses of the choroid. Similar ectopic fibres were seen following transection of the optic nerve at the chiasm and after tectal ablation although the onset of these changes was slower than that seen after nerve resection. It is concluded that damage to visual pathways in the frog induces dramatic morphological alterations in the optic nerve and retina far proximal to the site of injury in this regenerating system.     

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.     

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.     

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 and retrograde degeneration of axons in the rat optic nerve

Summary The retinal stump of the rat optic nerve was examined histologically 1–64 weeks after intracranial section of the nerve with or without grafting of autologous peripheral nerve segments. Single unmyelinated axons and bundles of unmyelinated axons appeared in cut optic nerves and were most abundant 2–4 weeks after section. With light and electron microscope radioautography after injection of tritiated amino acids into the globe, it was confirmed that many unmyelinated fibres arose from the optic nerve rather than from nearby peripheral nerves and it was estimated that some axons regenerated as far as 0.5 mm. At or near the end of retinofugal axons, structures resembling growth cones were seen at 2 weeks and vesicle-containing swellings similar to synapses were found at 1–2 months. Outgrowth from optic nerve axons was not obviously enhanced by peripheral nerve grafts although a few retinofugal axons became ensheathed by Schwann cells. Retrograde axonal degeneration was rapid in both cut and grafted optic nerves, the number of nerve fibres near the globe falling to less than 10% of normal after 4 weeks. A few myelinated and unmyelinated fibres were still present 64 weeks after nerve transection. In conclusion, some cut axons in the rat optic nerve display a transient regenerative response before undergoing retrograde degeneration.     

Temporal and spatial patterns of expression of laminin, chondroitin sulphate proteoglycan and HNK-1 immunoreactivity during regeneration in the goldfish optic nerve

Summary Current views suggest that the extracellular environment is critically important for successful axonal regeneration in the CNS. The goldfish optic nerve readily regenerates, indicating the presence of an environment that supports regeneration. An analysis of changes that occur during regeneration, in this model may help identify those molecules that contribute to a favourable environment for axonal regrowth. We examined the distribution and expression of two extracellular matrix molecules, laminin and chondroitin sulphate proteoglycan, and a carbohydrate epitope shared by a family of adhesion molecules (HNK-1), using immunocytochemical detection in sections from the normal adult goldfish optic nerve and in nerves from one hour to five months following optic nerve crush. We also usedin vitro preparations to determine if neurites in retinal explants could express these same molecules.The linear distributions of laminin and chondroitin sulphate proteoglycan immunoreactivity in control optic nerves are co-extensive with the glia limitans, suggesting both are expressed by non-neuronal components surrounding the axon fascicles. Between one and three weeks postoperatively when axons elongate and reach their target, laminin and chondroitin sulphate proteoglycan immunoreactivity increases around the crush site and distally. At six weeks postoperatively the pattern of immunoreactivity has returned to normal. While the temporal pattern of changes in immunoreactivity is similar, the spatial pattern of these two extracellular proteins in the regenerating nerve differs. Chondroitin sulphate proteoglycan immunoreactivity is organized in discrete columns associated with regenerating axons while laminin immunoreactivity is more diffusely distributed. Examination of retinal explants reveals growing neurites express chondroitin sulphate proteoglycan but not laminin. Our results suggest that laminin is only associated with non-neuronal cells, while chondroitin sulphate proteoglycan is associated with axons as well as non-neuronal cells.HNK-1 immunoreactivity is co-extensive with both the glia limitans and axon fascicles and is more extensively distributed in the intact nerve than either laminin or chondroitin sulphate proteoglycan immunoreactivity. In contrast to laminin and chondroitin sulphate proteoglycan, HNK-1 immunoreactivity is substantially decreased at the crush site within one week following optic nerve crush. HNK-1 immunoreactivity reappears through the crush site during the next several weeks, although non-immunoreactive regions, co-extensive with areas predominantly containing non-neuronal cells, persist both proximal and distal to the crush, up to six weeks postoperatively. The pattern suggests that HNK-1 epitope expression by these non-neuronal cells is decreased during axonal regeneration.Our results show that each of these molecules is constitutively expressed with a unique distribution in the normal goldfish optic nerve and each exhibits different patterns of change during regeneration. It thus appears that each may contribute to modifications of the environment that supports axonal regeneration. Both neurons and non-neuronal cells contribute to these changes.     

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.     

Regeneration and remyelination of Xenopus tadpole optic nerve fibres following transection or crush

Optic nerves of stage 54-56 Xenopus laevis tadpoles were either transected or crushed, and subsequent Wallerian degeneration, regeneration, and remyelination were examined. After 4 days, normal myelinated fibres were no longer present in the distal stump, and only a few unmyelinated fibres remained. After 10-13 days, the distal nerve consisted mainly of a core of reactive astrocytes with enlarged processes and scattered oligodendrocytes which persisted throughout the degenerative period. Regenerating axons traversed the site of the lesion and extended into the distal stump within 13-15 days. As regeneration progressed, astrocytic processes extended radially from the optic nerve's central cellular core and formed longitudinal compartments for regenerating axons. Between 15-19 days, a few regenerating fibres were remyelinated and by 35 days, more axons were surrounded either by thin collars of oligodendrocyte cytoplasm or by 1-3 spiral turns of myelin membrane. By 95 days, the number of myelinated fibres had increased to about 50% of those present in control nerves. Their myelin sheaths were normal in appearance and thickness relative to their respective axon diameters. The largest axons were surrounded by compact sheaths with 4-9 lamellae.     

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.     

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 and remyelination in the regenerating visual system of the goldfish

Summary The remyelination of regenerated optic axons was investigated in goldfish following either optic nerve crush or ouabain retinal intoxication. Axons grown after nerve crushing acquire thinner myelin sheaths than axons originating from reconstituted ganglion cells. If axons of reconstituted ganglion cells are crushed and allowed to regenerate, the subsequent myelination is weaker than that of control axons not interrupted by crushing, but stronger than that of axons of preexisting retinal ganglion cells.The present results suggest that a neuron is capable of inducing a normally developed myelin sheath when its axon contacts an oligodendrocyte the first time, whereas a neuron whose axon contacts an oligodendrocyte the second time is not capable of forming a normal myelin sheath in the adult animal. The present results also support the notion that the oligodendrocyte requires a neuronal signal for myelin sheath formation.Supported by the Deutsche Forschungsgemeinschaft (Wo 215/5)     

Biphasic cellular response to transection in the newt optic nerve: Glial reactivity precedes axonal degeneration

Summary Morphological interactions between axons and glia within the lesioned newt optic nerve were studied at time periods prior to the onset of Wallerian degeneration. Optic nerves were transected 0.5 mm from the eye, animals were killed at 5,10,20 and 30 min post-lesion, and the intracranial half of the tract was examined with light and electron microscopy. A sequence of structural changes was observed within the time interval 5–30 min post-lesion. Over the first 20 minutes these changes primarily involved the endogenous neuroglia; there was a displacement of glial nuclei from the center to the periphery of the nerve and an increase of 50–100% in glial cytoplasmic and nuclear area. Nuclei of reactive glia were euchromatic and surrounded by a high density of Golgi, vesicles, mitochondria and filaments, the last of which extended throughout the expanded glial processes. Optic axons appearintact at 20 min post-lesion except for some separation between the axolemma and myelin sheath in some of the myelinated fibres. By 30 min post-lesion both myelinated and non-myelinated fibres were found in various stages of lysis. Many of the expanded glial processes contained a population of vesicles aggregated adjacent to the glial plasmalemma. Profiles of infolded glial membranes suggested the opening of such vesicles into the extracellular space around degenerating axons. We conclude that, after optic nerve injury, there are very rapid reactive changes in glia and axons, with the changes in glia preceding the degenerative events in axons.     

视神经损伤后视神经及其内毛细血管的早期超微结构的变化

目的观察视神经损伤后视神经和神经内毛细血管的超微结构变化,探讨视神经损伤的机制。方法建立家兔视神经损伤的动物模型,利用透射电子显微镜观察视神经及神经内毛细血管超微结构的变化。结果视神经损伤0.5h后,轴突部分肿胀,髓鞘疏松,微管、微丝排列出现紊乱,线粒体肿胀,毛细血管内皮细胞中的吞饮小泡和微绒毛明显减少;损伤6h后,线粒体出现髓样变和空泡样变性,血管内皮细胞肿胀,管腔变窄;损伤12h后,轴突空泡样变性,髓鞘脱失,毛细血管周围间隙增宽;损伤48h后,轴质密度增加,部分髓鞘板层完全分离,微管、微丝及线粒体发生颗粒性溶解,内皮细胞中的线粒体出现广泛变性;损伤96h时,轴索崩解呈空泡状变性,髓鞘更广泛崩解,毛细血管扩张破裂,红细胞外溢。结论视神经损伤早期轴突肿胀、空泡样变性,线粒体水肿变性,微管、微丝数量减少;视神经内毛细血管扩张,通透性增加。     

Wallerian degeneration in the optic nerve of the rabbit

Progressive anterograde axonal degeneration is known to follow after transection of the axon from the soma, which to some extent correlates with the passage of time after the lesion. However, the minimum time required for such changes to begin remains unresolved. In this study, 20 young adult rabbits of either sex underwent experimental monocular enucleation (left eye) under general anaesthesia. Left optic nerves from such animals were treated as experimental and those from either side of non-operated animals served as controls. Animals were sacrificed postoperatively at periods ranging from 12 h to 3 months. Brains were fixed with 10% formalin and Karnovsky fixatives by an intracardiac perfusion method. Light microscopy of 8-microm paraffin sections and 0.5-microm araldite sections from the optic nerves did not reveal any changes at 12 h. At 24 h, focal minute cavities appeared across the optic nerves. Those nerves from late postoperative stages revealed such cavities with increasing dimensions, disarray of fascicular organization, fragmentation, ovoid formation and finally dissolution of the myelin sheaths. There was an appreciable increase in the number, size and aggregation of glia cells. The debris of degeneration remained prominent even 3 months after enucleation. Electron microscopy revealed splitting of myelin, intramyelinic and periaxonal oedema and occurrence of amorphous and electron-dense materials in the degenerating nerve fibres. It was concluded that while the optic nerve showed degenerative changes as early as 24 h after enucleation, debris of degeneration was only partly removed even after 3 months.     

Studies on the control of myelinogenesis. I. Myelination of regenerating axons after entry into a foreign unmyelinated nerve.

The proximal stump of a predominantly myelinated nerve (sternohyoid) was anastomosed in a tube to the distal stump of a largely unmyelinated nerve (cervical sympathetic) in order to explore the role of the axon in activating Schwann cells to produce myelin. The two nerves were examined after being united for periods between 3 and 15 weeks. At these times, regenerating myelinated and nonmyelinated fibres, originating from the proximal stump, were seen within the original fascicle in the distal stump of the sympathetic trunk. There was a significant (P less than 0.01) increase in the mean number of myelinated fibres in the anastomosed sympathetic nerves from the mean number of myelinated fibres in the normal sympathetic nerve. The histological and ultrastructural features of the anastomosed nerves are described and the differences between the morphology of the intratubal and extratubal regions are highlighted. The results of this study may indicate that the axon instructs the Schwann cell to produce myelin, but before this conclusion is reached, the origin of the myelinating cells has to be determined.     

纤溶酶原激活剂及其相关分子在小鼠视神经损伤再生中的变化

目的:检测细胞外基质(ECM)中各蛋白酶在视神经损伤后的变化,分析ECM蛋白酶活性的变化与小鼠视神经损伤和损伤后再生之间的关系。方法:本实验采用建立小鼠视神经钳夹伤的动物模型,用WesternBlot方法检测小鼠视神经损伤后不同时间点神经丝(NF)、金属基质蛋白酶-9(MMP-9)、IgG的表达变化。同时采用原位酶谱分析法检测纤溶酶原激活剂(PA)活性在视神经损伤后各阶段的变化,并分析这种变化与纤维蛋白(原)沉积、髓鞘碎片清除等影响神经再生的因素之间的关系。结果:小鼠视神经损伤后发生进行性Wallerian变性,血-神经屏障(BNB)修复迟缓,沉积的纤维蛋白(原)于损伤后第2d清除。MMP-9在损伤后2d达到高峰,以后仍呈现高水平的表达,且均以前体形式出现。PA活性在损伤后第7d达到高峰,并持续至第28d。结论:视神经损伤后,损伤部位BNB重建、PA激活、纤维蛋白(原)的清除以及MMP-9的表达与周围神经截然不同,正是由于微环境的迥然差异,导致了中枢神经系统(CNS)髓鞘碎片清除不利、轴突再生障碍。     

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.     

Peripheral nerve fibres regenerate through myenteric plexus

The ability of myenteric glia and neurons to support peripheral nerve regeneration was tested by grafting pieces of muscularis externa 5 mm long from the distal colon of inbred CBA mice adjacent to the proximal stump of cut common peroneal nerves. By two weeks after operation many axons had invaded the plexus and after 3 weeks regeneration common peroneal nerve fibres could be identified in all parts of the plexus throughout the grafts. Some axonal profiles within the plexus appeared to be in the early stages of myelination by enteric glia. Axons surrounded by compact myelin were found at the periphery of ganglia, but the cells involved resembled Schwann cells and could not be positively identified as enteric glia. Profiles similar to those of regenerating axons were only very rarely seen in control experiments in which grafts were placed adjacent to intact common peroneal nerves. It is suggested that the cellular elements of the myenteric plexus can support peripheral nerve regeneration.     

Quantification of N-CAM and N-cadherin expression in axotomized and crushed rat sciatic nerve

Adhesion molecules are important in supporting axonal regeneration. Qualitative studies have described increased expression of neural cell adhesion molecule (NCAM) and N-cadherin in models of nerve injury allowing active regeneration. In this study we have used quantitative immunohistochemistry to compare expression of NCAM and N-cadherin after nerve injury either with active regeneration (crush) into the distal stump or without (axotomy and capping). Quantification was performed 15 days after axotomy in proximal and distal stumps. Quantification after crush either proximal, distal or within the crushed area was performed at 2, 7, 15 and 30 days after injury. Axotomy induced increases in expression in proximal stumps between two and three times those in uninjured nerves for both molecules. In distal stumps, N-cadherin levels increased seven-fold, yet NCAM levels did not exceed control values. After crush, NCAM immunoreactivity increased in the crushed area and distal stump in contrast to axotomy and NCAM-positive axons co-localized with PGP9.5. N-cadherin levels in the distal stump increased above control levels, but the magnitude of the increase seen after crush was different to those seen after axotomy. In conclusion, increased expression of adhesion molecules, particularly NCAM, in the distal stump of injured nerves is dependent upon the presence of regenerating axons.     

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.