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.     

Defective myelination in the optic nerve of the Browman-Wyse (BW) mutant rat

Summary The Browman-Wyse (BW) rat displays a spectrum of ocular abnormalities which include myelination by Schwann cells of retinal ganglion cell (RGC) axons within the retina. Immunohistochemical and ultrastructural studies of the optic nerves of adult BW rats (30–60 days of age) with myelinated intraretinal axons were performed. Although individual nerves displayed considerable morphological variability, all were characterized by an initial dysmyelinated proximal segment which was separated from a normally myelinated distal segment by a transitional junctional zone. The proximal segment contained axons which were predominantly unmyelinated: where myelination occurred, almost all sheaths were P_o-positive, proteolipid protein-negative, and the myelinating cell was a Schwann cell. In the distal segment the distribution of myelinated axons appeared to be normal, sheaths were PLP⁺, and the myelinating cell was an oligodendrocyte. Within the proximal segment, axons that were myelinated by Schwann cells were isolated by a basal lamina and expanded extracellular spaces from the bulk of other RGC axons within the optic nerve. Few carbonic anhydrase (CAII)⁺ or GalC⁺ oligodendrocytes were seen in proximal segments that contained Schwann cells: anti-CAII antibody stained atypical cells within the proximal segments which did not resemble CAII⁺ oligodendrocytes in the distal segment, and which were probably GalC^–. Astrocytes appeared normal throughout the length of the nerve, and there was no morphological specialization at the junctional zone similar to that at the lamina cribrosa. The possible source (s) of the intraneural Schwann cells, and the pathogenetic mechanisms underlying the aberrant myelination of RGC axons within the BW optic nerve are discussed.     

Schwann cell myelin ensheathing C.N.S. axons in the nerve fibre layer of the cat retina

Summary In the retina of the cat the axons of the nerve fibre layer are unmyelinated and are provided with a C.N.S. myelin sheath only in the extraocular part of the optic nerve. The present study demonstrates that in the apparently normal cat retina close to the optic disc, some axons of the nerve fibre layer run for a short distance in the perivascular space of the retinal arteries. While coursing in the perivascular space, these C.N.S. axons become transiently myelinated by Schwann cells, which form a typical P.N.S. myelin sheath. These P.N.S. myelin sheaths terminate at a heminode in the transitional zone in which the C.N.S. axons penetrate the perivascular glial sheath in order to leave or to re-enter the nerve fibre layer. It is suggested that the Schwann cells, which elaborate the P.N.S. myelin around C.N.S. axons, are descendants of the Schwann cells of the perivascular autonomie nerves. The present study shows that Schwann cells are able to provide previously unmyelinated C.N.S. axons with a P.N.S. myelin sheath.     

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.     

Development of astrocytes in the lamina cribrosa sclerae of the mouse optic nerve,with special reference to myelin formation

In the mouse optic nerve, the optic nerve fiber layer in the retina, the optic papilla and the lamina cribrosa sclerae (LCS) just after penetrating the eyeball failed to generate myelin, whereas the optic nerve proper in the orbit was occupied by myelinated nerve fibers. The present study investigated development of the architecture of LCS, where the axons develop from unmyelinated to myelinated type, to elucidate how the initial part of axons was unmyelinated. At the LCS of the adult optic nerve, well developed astrocytes densely formed a cytoplasmic mesh-like frame through which unmyelinated fibers passed. The astrocytes here contained numerous and densely packed intermediate glial filaments and cell organelles. This framework formed by astrocytes appeared to be completed between 7 and 14 postnatal days before oligodendrocyte progenitors, migrated from the chiasm side, reached the proximal end of LCS, and began myelin formation. Thus the failure in myelin formation at the intraocular part and LCS possibly depended upon unsuccessful migration of oligodendrocytes beyond LCS constructed by specialized astrocytes, although other inhibitory factors for myelin formation, such as adhesion molecules distributed around LCS, may be unsolved.     

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.     

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.     

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 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.     

Response of axons and glia at the site of anastomosis between the optic nerve and cellular or acellular sciatic nerve grafts

Summary Axonal and glial reactions at the site of optic nerve section and at the junctional zone between optic nerve and normal or acellular peripheral nerve grafts have been studied. Following optic nerve section, no axons grew into the distal optic nerve stump. Similarly, no axons invaded the acellular peripheral nerve grafts, although in both instances fibres did regenerate into the junctional zone and a few remained there at least until 30 days post lesion (dpl, the duration of the experiments). Axons regenerated into normal peripheral nerve grafts by 3–5 dpl and by 10 dpl large numbers had penetrated deeply into the grafts. The glial response to injury appeared similar in both groups of grafted animals. Astrocytes and oligodendrocytes grew out into the junctional zone over the 5–7 day period and invaded the margins of the cellular grafts by 10 dpl. They did not penetrate the acellular nerves or distal optic nerve stumps. We were unable to determine whether Schwann cells invaded the junctional zone from the normal peripheral nerve grafts. Schwann cells are both GFAP⁺ and Vim⁺, especially when reacting after injury, and Lam^– when not associated with axons: it is therefore possible that Schwann cells from the cellular grafts contributed to the population of GFAP⁺, Vim⁺ cells in the junctional zone of the cellular grafts. Anti-laminin immuno-reactivity persisted in the basal lamina tubes of both the normal and acellular peripheral nerve grafts. Thus, the failure of axon regeneration into acellular peripheral nerve grafts can be correlated with the absence of Schwann cells and does not appear to be related to the presence of laminin.     

Immunocytochemical analysis of glial cells in the hypomyelinated optic nerve of the BW mutant rat

Summary The Browman-Wyse (BW) rat is a mutant with structural defects of the visual system, including a failure of the proximal (retinal) end of the optic nerve to myelinate. This latter abnormality is correlated with an absence of CAII+ oligodendrocytes, but we have previously shown that astrocytes are normally distributed, as judged by morphological characteristics of GFAP+ cellsin vivo. We have further examinedin vitro the immunohistochemical characteristics of macroglia isolated from the BW optic nerve, either as cell suspensions or after 4 days in culture.Cell cultures derived from the hypomyelinated proximal segment of BW optic nerves contained very few 0–2A progenitor cells (from which oligodendrocytes and cells with the GFAP+/A2B5+ phenotype develop), whereas over 90% of the glia were Schwann cells. A proportion of these few 0–2A progenitor cells differentiated normally after 4 daysin vitro into both progeny phenotypes in appropriate media. Accordingly, we conclude that the myelination deficiency in the BW optic nerve could be explained as a failure of 0–2A progenitor cells to populate fully the proximal extremity of the nerve during development.Since most glia isolated from adult optic nerves did not adhere to the culture substrate, we analysed the phenotypes of freshly isolated cells in suspension. Comparing optic nerves of normal adult rats with those of BW mutants, a significantly higher fraction of the GFAP+ cells reacted with A2B5 in cell suspensions of the latter. The double-labelled cells which are present in abnormally high numbers may be the differentiated progeny of 0–2A progenitors in the hypomyelinated segment of nerve. One explanation for these findings is that Schwann cells within the BW nerve induce the differentiation of 0–2A progenitor cells to the GFAP+/A2B5+ phenotype. We investigated this possibility using conditioned medium from cultured Schwann cells which increased tenfold the frequency of GFAP+/A2B5+ cells in normal neonatal rat optic nerve cultures. Oligodendrocyte numbers showed a concomitant decline with increasing concentration of Schwann cell conditioned medium.Hypomyelination in the BW rat optic nerve may therefore arise because Schwann cells, present in the proximal segment of the nerve, not only impede the migration of 0–2A progenitor cells but also release a factor which induces those 0–2A progenitor cells which arrive in the proximal segment of the nerve to differentiate into GFAP+ cells at a critical stage in oligodendrocyte development.     

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.     

Lesion-induced myelin formation in the retina

Summary In the normal rat retina ganglion cell axons are not myelinated until they enter the optic nerve. After a lesion to the retina made via the sclera and choroid, Schwann cells invade the retina and myelinate ganglion cell axons. The lesion-induced myelin formation is most conspicuous in animals operated between the day of birth and 20 days of age. A lesion to the retina made from the vitread surface does not produce Schwann cell invasion. We suggest that the Schwann cells migrate into the retina from extraocular structures via the sclera. These observations provide a valuable system for the study of interactions between CNS axons and Schwann cells.     

Laminin in traumatized peripheral nerve: Basement membrane changes during degeneration and regeneration

Summary The changes in Schwann cell basement membrane associated with degeneration and regeneration during 50 weeks after transection of rat sciatic nerve were studied immunohistochemically with antibodies to laminin. In half of the animals, regeneration was prevented by suturing the nerve stumps aside, whereas in the rest spontaneous regeneration was allowed. Axonal regeneration was monitored with anti-neurofilament protein antibodies.In control nerves, basement membranes surrounding Schwann cells were visualized as circular, laminin-positive structures within the endoneurium. By 8 weeks after transection, Schwann cells had formed columns which were laminin-positive throughout their cross-sectional area and indistinguishable from basement membrane zones in both non-regenerating and regenerating nerves. As axons repopulated the distal stump, the normal shape of Schwann cell basement membrane tubes was slowly restored in freely regenerating nerves. In non-regenerating nerves, however, a striking atrophy of Schwann cell columns was observed. Regenerating axons were only seen inside laminin-positive tubular structures in all phases after 8 weeks in regenerating nerves. On the other hand, restoration of normal shape in laminin-positive basement membrane zones was coincident with appearance of axons in the distal stump, but it did not take place in chronically degenerating nerves.The results show that chronic degeneration leads to an atrophy of Schwann cell columns and results in a decrease in laminin immunoreactivity associated with them.     

Highly sialylated N-CAM is expressed in adult mouse optic nerve and retina

Summary The localization of the neural cell adhesion molecule (N-CAM) and its highly sialylated form, which is prevalent in young tissues and has therefore been called embryonic neural cell adhesion molecule, was studied in the developing and adult mouse optic nerve and retina immunohistologically and immunochemically. At embryonic and early postnatal ages, neuroblasts and young postmitotic neurons, Müller cells and astrocytes in the retina, and retinal ganglion cell axons and all glial cells in the optic nerve express highly sialylated neural cell adhesion molecule. Beginning with the third postnatal week, highly sialylated neural cell adhesion molecule disappears from retinal ganglion cell axons in the optic nerve and from neuronal cell bodies and processes in the retina. In addition, it is not detectable on oligodendrocytes in 3-week-old animals. However, highly sialylated neural cell adhesion molecule continues to be expressed in the adult optic nerve and retina by astrocytes and Müller cells. On these cells it is only absent from cell membranes contacting basal lamina. Weakly sialylated neural cell adhesion molecule, in contrast, is expressed by all cell types of retina and optic nerve during development and in the adult. The loss of highly sialylated neural cell adhesion molecule from neurons and oligodendrocytes must therefore be considered as a cell type-specific conversion of the so-called embryonic to the adult form of neural cell adhesion molecule and does not simply reflect the disappearance of neural cell adhesion molecule from these cells. Weakly sialylated neural cell adhesion molecule, however, is absent from outer segments of photoreceptor cells and, as is the case for the highly sialylated form, from glial cell surfaces contacting basal lamina. Thus, the expression of highly sialylated neural cell adhesion molecule by pre- and postmitotic neurons and by oligodendrocytes is restricted mainly to the period of histogenetic events in retina and optic nerve, i.e. cell division, cell migration, dendritic and axonal growth and synaptogenesis. In addition to the observation that this form of neural cell adhesion molecule is less adhesive than the weakly sialylated, adult form, it is likely that highly sialylated neural cell adhesion molecule plays an important role during dynamic morphogenetic events. Furthermore, the expression of highly sialylated neural cell adhesion molecule by astrocytes and Müller cells in adult optic nerves and retinae suggests some histogenetically plastic functions for these cells in the adult mouse visual system.     

Interaction of regrowing PNS axons with transplanted aggregates of cultured CNS gliain vivo

Summary Aggregates of cultured neonatal mouse cerebellar astrocytes were implanted into adult mouse sciatic nerves. Two different experimental models were used: aggregates were either placed between proximal and distal stumps of totally transected nerves, or were placed in gaps in partially transected nerves in direct apposition with the cut surface of the proximal stumps. In the model where aggregates were not placed in contact with the proximal stump, regrowing axons rarely entered the aggregates. Where aggregates were placed in contact with the proximal stumps, axons entered the astrocyte-rich environment. Experimental depression of the supply of Schwann cells available to comigrate with regenerating axons proved to be unnecessary: astrocytes provided an alternative substrate for axons. Some axons became myelinated by oligodendrocytes which differentiated within the aggregates; however, few axons remained, unmyelinated, in long-term association with the transplanted astrocytes.     

Effects of neonatal transection on glial cell development in the rat optic nerve: evidence that the oligodendrocyte-type 2 astrocyte cell lineage depends on axons for its survival

Summary We have previously provided evidence that the rat optic nerve contains three types of macroglial cells that develop as two distinct lineages: one lineage comprises type 1 astrocytes, which develop before birth, while the other comprises oligodendrocytes and type 2 astrocytes, which develop after birth from a common, bipotential glial progenitor cell. In the present study we have examined the influence of axons on the development of these two glial cell lineages by cutting the optic nerve at birth so that the retinal ganglion cell axons in the nerve degenerate. Using antibodies to distinguish the different types of glial cells in suspensions and semithin frozen sections of cut and uncut optic nerves, we show that neonatal transection results in a striking decrease in the total number of oligodendrocytes, type 2 astrocytes and their progenitor cells but has much less effect on the number of type 1 astrocytes. Since the [³H]thymidine labelling indices of oligodendrocytes and their progenitor cells were not significantly decreased in cut nerves, our results suggest that the progenitor cells and/or their progeny die in large numbers following neonatal nerve transection. We conclude that axons are required for the survival of cells of the oligodendrocyte-type 2 astrocyte lineage, at least during postnatal development.     

Schwann cell myelination in the nerve fibre layer of the BW rat retina

Summary Schwann cells are frequently present in retinae of a partially inbred strain of rats, the BW rats. In affected animals, most if not all axons in the nerve fibre layer, immediately adjacent to the optic disc, are ensheathed by Schwann cells. This can occur in the form of a distinct myelin sheath or a simple envelopment of the axons by Schwann cells. Some axons lose their Schwann cell sheath within a few hundred micrometers of the optic discs while others retain the sheath out to the mid-retina. Although the pathophysiological basis of the defect is unknown, it appears to be related to a genetic defect that has widespread C.N.S. expression.     

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.     

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.