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In order to devise a strategy to enhance remyelination in multiple sclerosis (MS) it is necessary to understand the cause of remyelination failure in MS. A case is made that areas of chronic demyelination arise because of concurrent loss of oligodendrocyte progenitor cells (OPCs) and oligodendrocytes and that because of the slow rate of repopulation that occurs in old individuals the recruited OPCs are not exposed to the acute inflammatory environment required to generate remyelinating oligodendrocytes. Based on this analysis the case is made that only areas of acute demyelination will be amenable to transplant-mediated remyelination. An analysis of the many cells that could be used to provide a source of remyelinating cells would indicate that structural repair of the CNS in MS would likely only be possible if neural precursors were used and the most promising route for their introduction would appear to be by intraventricular injection. Both neural precursors and mesenchymal stromal cells can be immunomodulatory and neuroprotective following intravenous injection; however, only neural precursors are likely to be able to contribute to structural repair of the damaged nervous system.

Cell transplant therapies are currently under active consideration for a number of degenerative diseases. In the immune-mediated demyelinating-neurodegenerative disease multiple sclerosis (MS), only the myelin sheaths of the CNS are lost, while Schwann cell myelin of the PNS remains normal. This, and the finding that Schwann cells can myelinate CNS axons, has focussed interest on Schwann cell transplants to repair myelin in MS. However, the experimental use of these cells for myelin repair in animal models has revealed a number of problems relating to the incompatibility between peripheral glial cells and the CNS glial environment. Here, we have tested whether these difficulties can be avoided by using an earlier stage of the Schwann cell lineage, the Schwann cell precursor (SCP). For direct comparison of these two cell types, we implanted Schwann cells from post-natal rat nerves and SCPs from embryo day 14 (E14) rat nerves into the CNS under various experimental conditions. Examination 1 and 2 months later showed that in the presence of naked CNS axons, both types of cell form myelin that antigenically and ultrastructurally resembles that formed by Schwann cells in peripheral nerves. In terms of every other parameter we studied, however, the cells in these two implants behaved remarkably differently. As expected from previous work, Schwann cell implants survive poorly unless the cells find axons to myelinate, the cells do not migrate significantly from the implantation site, fail to integrate with host oligodendrocytes and astrocytes, and form little myelin when challenged with astrocyte-rich environment in the retina. Following SCP implantation, on the other hand, the cells survive well, migrate through normal CNS tissue, interface smoothly and intimately with host glial cells and myelinate extensively among the astrocytes of the retina. Furthermore, when implanted at a distance from a demyelinated lesion, SCPs but not Schwann cells migrate through normal CNS tissue to reach the lesion and generate new myelin. These features of SCP implants are all likely to be helpful attributes for a myelin repair cell. Since these cells also form Schwann cell myelin that is arguably likely to be resistant to MS pathology, they share some of the main advantages of Schwann cells without suffering from the disadvantages that render Schwann cells less than ideal candidates for transplantation into MS lesions.

Following spinal cord trauma there is controversy as to whether myelin-supporting oligodendrocytes at a distance from areas of spinal cord damage undergo apoptosis. To examine the response of oligodendrocytes to axon degeneration, we counted the number of oligodendrocytes and oligodendrocyte precursors in the dorsal funiculi during the course of Wallerian degeneration. Axons were disrupted at T13 and the number of labelled cells in the dorsal funiculi counted at T12, 4 days and 2, 4, and 8 weeks after injury using riboprobes to exon-3b of the PLP gene whose expression is considered to restricted to myelin-supporting oligodendrocytes, PDGFRalpha which is regarded as a marker of oligodendrocyte precursors, and MOG a marker previously used to identify myelin-supporting oligodendrocytes. We found that the number of PLP-exon-3b labelled cells remained constant during the course of Wallerian degeneration while the number of cells labelled with the riboprobes to PDGFRalpha and MOG increased. Significantly the number of MOG-positive cells was increased at times when the number of PDGFRalpha labelled cells was highest. The number of PDGFRalpha labelled cells decreased with time while the number PLP-exon-3b labelled cells remained constant. It is therefore possible that the apoptotic oligodendrocytes identified in previous studies could represent degenerating oligodendrocyte precursors or their progeny rather than degenerating myelin-supporting oligodendrocytes.

Publisher Summary This chapter examines and evaluates the therapeutic potential of achieving remyelination by transplantation of myelin-forming cells. It also illustrates the power of glial-cell transplantation for examining aspects of glial-cell biology during development and in pathological situations. The normal glial populations of the CNS and PNS have an intrinsic replacement capacity. Two major issues must be addressed when undertaking and interpreting glial-cell transplantation studies. First, there is the question of identifying the transplanted cells and, second, there is the question of having a clear knowledge of the nature of the cells being transplanted. The first issue has led to the development of various experimental models and the use of cellular markers to track the fate of transplanted cells. The second has led to the development of different cell isolation and expansion procedures that result in enrichment for cells of a particular phenotype. It must also be appreciated that one may document results of glial-cell transplantation in terms of presence of myelin-forming oligodendrocytes or Schwann cells. Furthermore, the chapter also discusses target diseases and clinical situations where transplant-mediated remyelination would be beneficial.

Mixed glial cell cultures prepared from neonatal wild type and mutant male mice lacking either major histocompatibility complex class I, class II or both class I and II molecules (major histocompatibility complex class I o/o II o/o ), and from syngeneic male rats were transplanted into female rat spinal cord white matter. Graft survival was monitored using DNA probes specific to the Y chromosome. Survival of major histocompatibility complex class-deficient grafts was not prolonged compared to wild-type grafts and in most cases grafts could not be detected at 28 days post-transplantation, at which time syngeneic grafts were still present. However, rejection of xenografts resulted in significant bystander damage to host tissue. In recipients of wild-type and major histocompatibility complex class I o/o xenografts the predominant pathology was demyelination. Demyelination was also observed in recipients of major histocompatibility complex class II o/o and major histocompatibility complex class I o/o II o/o xenografts, however in addition there was marked collagen deposition and meningeal cell invasion. Significantly more axons had undergone Wallerian degeneration in recipients of major histocompatibility complex class II o/o and major histocompatibility complex class I o/o II o/o xenografts than recipients of wild-type and major histocompatibility complex class I o/o xenografts. These findings were interpreted as evidence of a more destructive immune response associated with rejection of grafts lacking major histocompatibility complex class II molecules. It was proposed that the difference in the severity of bystander damage may be related to the previously demonstrated ability of xenogeneic major histocompatibility complex class II molecules to activate host T cells directly, whereas xenografts lacking major histocompatibility complex class II molecules were capable of activating host T cells only by the indirect pathway.

Areas of persistent demyelination were created in the dorsal columns of the cat spinal cord by injecting ethidium bromide into white matter which had previously been exposed to 40 Grays of X-irradiation. In the centre of such lesions demyelinated axons occurred in a glial-free area while axons next to normal tissue were separated by astrocyte processes. No remyelination occurs in such lesions (Blakemore 1984). Autologous Schwann cells and fibroblasts cultured from a peripheral nerve biopsy were injected into such lesions and the extent of Schwann cell remyelination examined. Only lesions injected with viable cells showed remyelination by Schwann cells; in no lesion were all the demyelinated axons remyelinated. Three forms of association of Schwann cell with axons were detected. In the centre of the lesions Schwann cells either remyelinated axons around or near to blood vessels, or lay next to demyelinated axons and did not form myelin. Schwann cell remyelination was also detected in the astrocyte-containing areas around the edges of some lesions. It was concluded that the extent of Schwann cell remyelination was influenced by the mode of entry of the cells into the lesion and by the architecture of the lesion. The presence or absence of stable extracellular matrix is believed to be the prime factor which influenced Schwann cell remyelination. The relevance of these observations to artificial repair of the lesions of multiple sclerosis is discussed.

Chronically demyelinated lesions of cat dorsal columns were created by focal injection of the glial toxin ethidium bromide. Freeze-fracture studies show that the center of the lesion, which is devoid of glial cells and processes, contains axons having neither node-like nor paranodal-type membrane specializations. Near the margin of the lesion,, however, where axons are in contact with glial cells, the axolemma sometimes displays focal accumulations of E- and P-face particles resembling those at nodes of Ranvier. In cases where the adjacent cell could be identified, it had the characteristics of an astrocyte. Linear indentations of the axolemma displaying a paracrystalline pattern like that of the paranodal axolemma also occur in the marginal region. Here, the adjacent cell had the characteristics of an oligodendrocyte. These specializations may be closely associated with each other or spatially separate. Normal nodal and paranodal specializations were absent throughout the lesion at all time periods examined. These findings support the view that both the formation and the maintenance of nodal and paranodal axon membrane specialization require contact with glial cells.

The ultrastructural appearance of the spinal cord of 3 piglets affected with congenital tremor type AIV was examined and compared with normal litter mates. In affected pigs some axons, of all diameters, were unmyelinated and the myelin sheaths of the remainder were much thinner than normal. Signs of myelin breakdown were present and oligodendrocytes contained autophagic vacuoles; some degenerating oligodendrocytes were also found. These observations indicated that demyelination, myelination and/or remyelination were occurring concurrently and support an hypothesis suggesting that the abnormal fatty acid metabolism present in this condition renders myelin unstable.

Summary The relationship between internodal length and external fibre diameter in the spinal cord was examined on glutaraldehyde perfused material from a number of cats. The relationship in all samples was highly significant and there was no significant difference either between animals or between the lateral and dorsal columns. However, there was considerable variability, some internodes being twice and others less than half as long as the length predicted by the regression equation. Our results showed that the relationship between internodal length and fibre diameter was essentially a linear function. No internodes shorter than 100 μ m were seen.

The ultrastructural appearance of the spinal cord of a Landrace piglet with a sex-linked abnormality of myelination (congenital tremor type AIII) was examined and compared with a clinically normal litter mate. The white matter of the former contained some myelinated axons and a large number of unmyelinated axons. All axons below 1 μm in diameter were unmyelinated, as were 93% of axons between 1–2 μm, 72% of axons between 2–3 μm and 19% of axons between 3–4 μm; all axons above 4 μm in diameter were myelinated. Some myelin sheaths were of normal thickness but most were thinner than normal. There was a reduction in the number of oligodendrocytes but no evidence of demyelination. This condition in the pig has similarities to Pelizaeus-Merzbacher disease in man, and the ‘jimpy’ mutant in mice.

When young or adult mice were infected experimentally with isolates of Streptococcus suis type 2 of known pathogenicity for pigs, the organisms produced disease, young mice being more susceptible than adults. An isolate of S suis type 2 less pathogenic for pigs also appeared less pathogenic in mice. The organism could be reisolated from infected mice for up to 42 days. Inoculum size was one factor influencing the likelihood of an individual mouse developing meningitis following intravenous inoculation. Transmission of the organism from inoculated to in-contact mice occurred in young mice after intravenous or oral administration. These studies indicate that the behaviour of S suis type 2 in mice resembles its behaviour in pigs.

Internodes from normal, remyelinated and regenerated nerve fibres have been isolated from rat spinal roots and sciatic nerve. The internodes have been examined quantitatively by light and electron microscopy to determine their internodal length, myelin thickness, and the circumference and cross-sectional area of both the axons and fibre. Comparison of these measurements of the axon and myelin sheath has revealed a close relationship between the volume of myelin comprising the internode and the area over which the Schwann cell and axon are in close proximity, i.e. the surface area of the axolemma beneath the internodal myelin sheath. The same relationship described not only the internodes on normal nerve fibres, where internodal length is proportional to axon diameter, but also the short and thinly myelinated internodes formed in the adult animal on remyelinated and on regenerated axons. Examination of data presented by Berthold (1978) revealed that a closely similar relationship is also present in feline nerve fibres. In view of the constancy of the relationship between such different types of internode it is suggested that the regulation of myelin volume, and thereby of myelin thickness, may be mediated via the area of the axolemma or of the Schwann cell membrane beneath the myelin sheath.

The superior cerebellar peduncles of adult mice were demyelinated by feeding a diet containing cuprizone for 10 weeks. When mice were taken off this diet the tract underwent complete remyelination similar to that found in younger animals. It was concluded that age was not a factor which limits extensive remyelination in mice, rather that the limiting factor must be the supply of cells capable of repopulating demyelinated areas. Such cells appeared to be available throughout life in the mouse brain.

The internodal length of remyelinated internodes was examined by observations on teased CNS nerve fibres following primary demyelination induced by intraspinal injections of lysolecithin into the white matter of cats. A remyelinated internode was identified as a thinly-myelinated internode, where a node of Ranvier was bounded by a thickly-myelinated internode on one side and a less-thickly-myelinated internode on the other; as an internode of less than 100 microns in length or below 2 standard deviations from the normal regression line of internodal length against fibre diameter; as an internode joined to one of similar myelin sheath thickness which fulfilled either or both of the previous two criteria. Using the above criteria, remyelinated internodes were found to be shorter than normal; some were very short with no relationship to axon diameter while others were longer, falling within the predicted range for a given fibre diameter. This study illustrates that examination of teased CNS fibres in pathological situations can yield valuable information. However, sampling and technical difficulties are far greater than in comparable studies on peripheral nerve fibres.

The superior cerebellar peduncles of mice fed a diet containing 0.5% cuprizone since weaning were examined. It was found that this tract was demyelinating after 4 weeks on the diet, a process which was almost complete after 5 weeks. Demyelination followed degeneration of oligodendrocytes. Extensive remyelination did not occur if the animals were kept on the diet. The reproducibility of demyelination was such that quantitative studies on remyelination can be undertaken. The genotype of the mice used in the present experiments was considered to be of importance in the aetiology of the lesion.

A case of GM 1 gangliosidosis in a 3-month-old cat is described. The clinical, chemical and histopathological features closely resemble those seen in this condition in man. There is an increase in GM 1 ganglioside and ceramide tetrahexoside in the brain and liver. There is also storage of an extremely labile substance in the liver and to a lesser extent in the perivascular pericytes of the cerebral white matter. Membranous cytoplasmic bodies are seen in neurones and glia, but large cytoplasmic bodies are the more common type of glial inclusion. There is a paucity of myelin and oligodendrocytes in the cortical white matter.

The retinas from normal sheep, sheep in the early stages of retinal degeneration induced experimentally by feeding dried bracken, and “bright blind” sheep (clinical field cases of advanced retinal degeneration), have been examined. Differences in the LDH isoenzyme pattern and specific activity were seen in the advanced field cases but these did not occur consistently in the experimental animals. It is suggested that these changes are secondary in nature. Increases in the specific activity of G6PDH occurred in the experimental animals prior to gross histological damage. Although the overall respiratory and glycolytic ability of the retinas of these sheep was impaired when the histological changes were still minimal, the decrease was not observed to precede cellular damage.

The superior cerebellar peduncle (SCP) in mice was demyelinated by feeding them from weaning on a diet containing 0.5% cuprizone. When the mice were taken off this diet, after 6–7 weeks, remyelination occurred. After 4 weeks on a normal diet nearly all the axons in the scp were remyelinated (mean of 6 animals = 88%). Remyelinated sheaths were thinner than normal and the normal relationship between axon diameter and myelin sheath thickness had not been established by 6 weeks.

After portocaval anastomosis in rats ultrastructural changes have been observed in the inner and outer cytoplasmic tongues of the myelin sheaths and also in the paranodal cytoplasmic loops. Principally, these consist of aggregates of fibrils (4–5 nm wide) in various stages of orderly packing. Additionally, closely-packed hexagonal arrays of tubular structures (20 nm wide) were occasionally found, as well as banded and coiled arrangements. Certain minor abnormalities may be found in the large neurones of the dentate nucleus. These findings are in general conformity with the hypothesis that a disturbance to microtubular metabolism in the brain occurs in association with high plasma ammonium levels, probably due to reduction in size of the glutamate and aspartate pools in glial cells.

After portocaval anastomosis, rats develop focal vacuolation, with apparent homogenization and loss of Purkinje cells and accompanying gliosis, in the depths of the cerebellar folia. The vacuolation represents focal swelling of astrocyte processes. This appears from the 5th week and continues to occur up to 38 weeks after operation. The severity of the cerebellar changes appear to be directly related to the plasma NH 4 -N level. It is argued that these changes are closely similar to those found in human patients with acquired hepatic encephalopathy, and that they are part of a general metabolic stress induced by the persistently high plasma ammonia level on the astrocyte cellular compartment.

Conduction has been examined serially through the experimental demyelinating lesion produced by the direct micro-injection of lysophatidyl choline into the cat spinal cord. Conduction was blocked during the phase of demyelination. Remyelination commenced during the latter part of the second week, and conduction through the lesion was restored in some fibres at this time. The refractory period of transmission was initially prolonged. As remyelination proceeded, conduction was restored in increasing numbers of fibres. Within three months the responses were stable and the refractory periods of transmission had returned to normal even though the new internodes were still abnormally thin and were generally shorter than normal. Restoration of conduction by remyelination is likely to contribute to recovery from compressive and traumatic lesions of the central nervous system but probably plays little part in the remissions of multiple sclerosis.

Areas of primary demyelination which did not subsequently remyelinate spontaneously were prepared in the cat spinal cord by injecting small volumes of ethidium bromide into tissue which had previously been exposed to 40 Grays of X-irradiation. Autologous peripheral nerve tissue was placed in the sub-arachnoid space over such lesions, either at the time of injecting ethidium bromide, or at 14 days or 28 days after injecting ethidium bromide. The extent of Schwann cell remyelination was assessed 28 days after transplantation. In no case were all the demyelinated axons remyelinated; rather, remyelination was limited to axons near to blood vessels. It was concluded that Schwann cells migrated from the transplanted tissue into the lesion via the perivascular space and that they failed to remyelinate the bulk of demyelinated axons because of an absence within the CNS of suitable extracellular matrix.

Focal regions of node-like axonal undercoating occur occasionally in the myelin-deficient rat spinal cord. In the optic nerve of heterozygous myelin-deficient rats, areas of amyelination are present, within which many axons show regions of node-like undercoating. These structures were most easily seen when fixation resulted in round axonal profiles. In one-year-old rats, these structures were common. The ubiquitous association of patches of axonal undercoating with regions of chronic amyelination and demyelination indicates that chronically demyelinated axons differ structurally from recently demyelinated axons. It is possible that such axons may have lost their ability to recruit myelin-forming cells.

ONE of the outstanding characteristics of multiple sclerosis and other clinical and experimental diseases associated with demyelination, is the tendency to remit. Although there is good evidence that an important cause of functional loss in such conditions is conduction block produced by the selective destruction of the myelin sheath1, the mechanisms underlying remission are unknown2. It has long been recognised that in certain experimental lesions demyelinated central axons can be remyelinated by the normal myelin-forming cell, the oligodendrocyte3. The new sheaths are, however, abnormally thin and short and it is not known whether they are capable of restoring conduction. A previous investigation was inconclusive4. Because of the physiological and clinical importance of this problem, we have now examined conduction serially through the lesion induced by the direct microinjection of lysophosphatidyl choline (LPC) into the cat spinal cord5. This lesion has the advantage that the phases of active demyelination and remyelination are well separated, thus permitting a reasonable correlation of the morphological with the physiological changes. We have found that secure conduction is restored with remyelination.

The ultrastructural changes in astrocytes in the area of oedema which results from a small cortical cold lesion were studied from 1–18 days later. Their reaction in response to plasma in the extracellular space depended on their distance from the area of thermal necrosis. Those cells close to it became grossly swollen and lost their processes (clasmatodendrosis), and later became phagocytosed (dendrophagocytosis). Those cells further away became swollen without loss of their processes, and they then showed an increase in glycogen granules, mitochondria, endoplasmic reticulum and ribosomes; such cells commonly underwent division. Later this swelling subsided. The time course of the astrocytic reaction was brief. The appearance of the cells returned towards normality between 4 and 12 days after making the cold lesion. The extracellular space appeared normal at 4 days. It was concluded that these reactions of the astrocytes contributed first towards the removal of escaped plasma from the extracellular space and secondly towards restoring the internal milieu of the astrocyte.

Lesions were made in the spinal cord of cats by means of local injections of 6-aminonicotinamide. The concentration needed to induce primary demyelination also caused extensive axonal death. In the early stages following injection there was evidence of astrocyte and oligodendrocyte destruction and with longer survival times most of the surviving demyelinated axons were remyelinated by Schwann cells. This experimental system indicates that Schwann cell remyelination of central axons follows astrocyte destruction, but it was not considered a suitable model system for the study of cellular relationships in remyelination because of the extensive concomitant axonal damage.