Remyelination in experimental models of toxin-induced demyelination.

Remyelination is the regenerative process by which demyelinated axons are reinvested with new myelin sheaths. It is associated with functional recovery and maintenance of axonal health. It occurs as a spontaneous regenerative response following demyelination in a range of pathologies including traumatic injury as well as primary demyelinating disease such as multiple sclerosis (MS). Experimental models of demyelination based on the use of toxins, while not attempting to accurately mimic a disease with complex etiology and pathogenesis such as MS, have nevertheless proven extremely useful for studying the biology of remyelination. In this chapter, we review the main toxin models of demyelination, drawing attention to their differences and how they can be used to study different aspects of remyelination. We also describe the optimal use of these models, highlighting potential pitfalls in interpretation, and how remyelination can be unequivocally recognized. Finally, we discuss the role of toxin models alongside viral and immune-mediated models of demyelination.

Remyelination protects axons from demyelination-associated axon degeneration.

In multiple sclerosis, demyelination of the CNS axons is associated with axonal injury and degeneration, which is now accepted as the major cause of neurological disability in the disease. Although the kinetics and the extent of axonal damage have been described in detail, the mechanisms by which it occurs are as yet unclear; one suggestion is failure of remyelination. The goal of this study was to test the hypothesis that failure of prompt remyelination contributes to axonal degeneration following demyelination. Remyelination was inhibited by exposing the brain to 40 Gy of X-irradiation prior to cuprizone intoxication and this resulted in a significant increase in the extent of axonal degeneration and loss compared to non-irradiated cuprizone-fed mice. To exclude the possibility that this increase was a consequence of the X-irradiation and to highlight the significance of remyelination, we restored remyelinating capacity to the X-irradiated mouse brain by transplanting of GFP-expressing embryo-derived neural progenitors. Restoring the remyelinating capacity in these mice resulted in a significant increase in axon survival compared to non-transplanted, X-irradiated cuprizone-intoxicated mice. Our results support the concept that prompt remyelination protects axons from demyelination-associated axonal loss and that remyelination failure contributes to the axon loss that occurs in multiple sclerosis.

Effects of direct transplantation of multipotent mesenchymal stromal/stem cells into the demyelinated spinal cord.

The adult bone marrow contains a population of multipotent mesenchymal stromal cells (MSCs), defined by plastic adherence, expression of stromal cell surface markers, and differentiation into mesenchymal lineages. There has been much interest in the possible therapeutic use of MSCs in the treatment of demyelinating diseases of the central nervous system. One therapeutic possibility is that these cells may be able to remyelinate when directly injected into the demyelinated spinal cord. Here we examine the effects of direct transplantation of green fluorescent protein (GFP)-labeled MSCs into a model of focal spinal cord demyelination induced by ethidium bromide. We demonstrate that direct intralesional injection of undifferentiated MSCs does not lead to remyelination. Furthermore, we report that transplanted MSCs migrate into areas of normal tissue, deposit collagen, and are associated with axonal damage. These findings support the need for further experimental evaluation of the safety and efficacy of direct parenchymal injection of MSCs into demyelinated lesions and highlight an important issue regarding potential clinical consequences of culture heterogeneity of MSCs between centers.

Regeneration and repair in multiple sclerosis: the view of experimental pathology.

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.

Endogenous or exogenous oligodendrocytes for remyelination.

The relative merits of endogenous and exogenous oligodendrocyte progenitor cells (OPCs) for remyelination are compared in terms of their ability to repopulate OPC-depleted tissue and generate remyelinating oligodendrocytes. Exogenous neonatal OPCs can repopulate OPC-depleted tissue 5-10 times faster than endogenous cells and as a result are capable of more extensive remyelination. Both endogenous and exogenous cells will only repopulate normal tissue if there is extensive depletion of the local OPC population and both show reduced ability to generate remyelinating cells in the absence of acute inflammation. When endogenous OPCs are depleted by X-irradiation during cuprizone intoxication, where there is a combination of astrocytosis and acute demyelination, endogenous but not exogenous embryo-derived OPCs fail to repopulate the OPC-depleted cortex.

Schwann cell precursors: a favourable cell for myelin repair in the Central 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.

Transplantation of glial cells into the CNS.

Glial cell transplantation into the CNS offers an experimental approach to help us unravel the complex interactions that occur between CNS glia, Schwann cells and axons during repair and development. This article reviews recent advances that have been made in our understanding of the nature and potential of CNS repair using this approach, and introduces the idea of using transplantation to address broader issues in glial biology.

Glial-cell transplantation and plasticity in the O-2A lineage--implications for CNS repair.

The close association that exists between development and regeneration stems from the many shared functions and the similar mechanisms by which these functions are achieved. For this reason, advances made in developmental neurobiology have provided important insights for neurobiologists interested in repair of the damaged CNS. Through the technique of transplantation, it has been possible to demonstrate that the differentiation of the 'O-2A' glial progenitor cell in vitro into oligodendrocytes and astrocytes described initially by developmental biologists can also occur within pathological states of the adult CNS.

Age increases axon loss associated with primary demyelination in cuprizone-induced demyelination in C57BL/6 mice.

Axon loss is recognised as a significant contributor to the progression of the disability associated with multiple sclerosis. Although evidence of axon damage is found in areas of chronic demyelination it is more frequently seen in association with acute demyelination. This study compares the incidence of axon degeneration associated with the areas undergoing demyelination in young adult (8-10 weeks) and aged (6-7 months) C57BL/6 mice in cuprizone intoxication; a widely used model of demyelination. The incidence of axon transection, as indicated by the presence of SMI 32 positive axonal spheroids, and evidence of axon loss in the medial corpus callosum, were significantly greater in aged mice, as was the magnitude of the macrophage and astrocyte response to demyelination. Aged C57BL/6 mice are thus more prone to axon degeneration in association with demyelination than young adult mice. A retrospective study indicated that the incidence of axon degeneration was much higher in C57BL/6 mice than in the Swiss albino mice used in the early cuprizone intoxication studies which were fed much higher doses of cuprizone. These results indicate both a genetic and age susceptibility to demyelination-associated axon transection.

Inflammation stimulates remyelination in areas of chronic demyelination.

A major challenge in multiple sclerosis research is to understand the cause or causes of remyelination failure and to devise ways of ameliorating its consequences. This requires appropriate experimental models. Although there are many models of acute demyelination, at present there are few suitable models of chronic demyelination. The taiep rat is a myelin mutant that shows progressive myelin loss and, by 1 year of age, its CNS tissue has many features of chronic areas of demyelination in multiple sclerosis: chronically demyelinated axons present in an astrocytic environment in the absence of acute inflammation. Using the taiep rat and a combination of X-irradiation and cell transplantation, it has been possible to address a number of questions concerning remyelination failure in chronic multiple sclerosis lesions, such as whether chronically demyelinated axons have undergone changes that render them refractory to remyelination and why remyelination is absent when oligodendrocyte progenitor cells (OPCs) are present. Our experiments show that (i) transplanted OPCs will not populate OPC-containing areas of chronic demyelination; (ii) myelination competent OPCs can repopulate OPC-depleted chronically demyelinated astrocytosed tissue, but this repopulation does not result in remyelination--closely resembling the situation found in some multiple sclerosis plaques; and (iii) the induction of acute inflammation in this non-remyelinating situation results in remyelination. Thus, we can conclude that axonal changes induced by chronic demyelination are unlikely to contribute to remyelination failure in multiple sclerosis. Rather, remyelination fails either because OPCs fail to repopulate areas of demyelination or because if OPCs are present they are unable to generate remyelinating oligodendrocytes owing to the presence of inhibitory factors and/or a lack of the stimuli required to activate these cells to generate remyelinating oligodendrocytes. This non-remyelinating situation can be transformed to a remyelinating one by the induction of acute inflammation.

Polysialylated neural cell adhesion molecule-positive CNS precursors generate both oligodendrocytes and Schwann cells to remyelinate the CNS after transplantation.

Transplantation offers a means of identifying the differentiation and myelination potential of early neural precursors, features relevant to myelin regeneration in demyelinating diseases. In the postnatal rat brain, precursor cells expressing the polysialylated (PSA) form of the neural cell adhesion molecule NCAM have been shown to generate mostly oligodendrocytes and astrocytes in vitro (Ben-Hur et al., 1998). Immunoselected PSA-NCAM+ newborn rat CNS precursors were expanded as clusters with FGF2 and grafted into a focal demyelinating lesion in adult rat spinal cord. We show that these neural precursors can completely remyelinate such CNS lesions. While PSA-NCAM+ precursor clusters contain rare P75+ putative neural crest precursors, they do not generate Schwann cells in vitro even in the presence of glial growth factor. Yet they generate oligodendrocytes, astrocytes, and Schwann cells in vivo when confronted with demyelinated axons in a glia-free area. We confirmed the transplant origin of these Schwann cells using Y chromosome in situ hybridization and immunostaining for the peripheral myelin protein P0 of tissue from female rats that had been grafted with male cell clusters. The number and distribution of Schwann cells within remyelinated tissue, and the absence of P0 mRNAs in donor cells, indicated that Schwann cells were generated by expansion and differentiation of transplanted PSA-NCAM+ neural precursors and were not derived from contaminating Schwann cells. Thus, transplantation into demyelinated CNS tissue reveals an unexpected differentiation potential of a neural precursor, resulting in remyelination of CNS axons by PNS and CNS myelin-forming cells.

Neuropathological assessment of artemether-treated severe malaria.

In animals, high doses of intramuscular artemether and artemotil have been shown to cause an unusual pattern of selective damage to certain brainstem nuclei, especially those implicated in hearing and balance. We aimed to investigate whether a similar pattern arises in human adults. We examined the brainstems of adults who died after treatment with high dose artemether or quinine for severe falciparum malaria for evidence of a pattern of selective neuronal damage. Neuropathological findings were similar in recipients of quinine (n=15) and artemether (n=6; total artemether doses received 4-44 mg/kg). No evidence was recorded for artemether-induced neurotoxic effects.

Identification of post-mitotic oligodendrocytes incapable of remyelination within the demyelinated adult spinal cord.

In order to investigate the remyelinating potential of mature oligodendrocytes in vivo, we have developed a model of demyelination in the adult rat spinal cord in which some oligodendrocytes survive demyelination. A single intraspinal injection of complement proteins plus antibodies to galactocerebroside (the major myelin sphingolipid) resulted in demyelination followed by oligodendrocyte remyelination. Remyelination was absent when the spinal cord was exposed to 40 Grays of x-irradiation prior to demyelination, a procedure that kills dividing cells. Quantitative Rip immunohistochemical analysis revealed a similar density of surviving oligodendrocytes in x-irradiated and nonirradiated lesions 3 days after demyelination. Rip and bromodeoxyuridine double immunohistochemical analysis of demyelinated lesions indicated that Rip+ oligodendrocytes did not divide as an acute response to demyelination. Oligodendrocytes were also identified by Rip immunostaining and electron microscopy at late time points (3 weeks) within x-irradiated areas of demyelination. These oligodendrocytes extended processes that engaged axons, and on occasion formed myelin membranes, but did not lay down new myelin sheaths. These studies demonstrate that (a) oligodendrocytes that survive within a region of demyelination are not induced to divide in the presence of demyelinated axons, and (b) fully-differentiated oligodendrocytes are therefore postmitotic and do not contribute to remyelination in the adult CNS.

Decline in rate of colonization of oligodendrocyte progenitor cell (OPC)-depleted tissue by adult OPCs with age.

Rates of remyelination decline with age and this has been attributed to slower recruitment of oligodendrocyte progenitor cells (OPCs) into areas of demyelination and slower differentiation of OPCs into remyelinating oligodendrocytes. When considering causes for reduced recruitment rates, intrinsic causes (alterations in biological properties of OPCs) need to be separated from extrinsic causes (age-related differences in the lesion environment). Using 40 Gy of X-irradiation to deplete tissue of its endogenous OPC-population, we examined the effects of age on the rate at which adult rat OPCs colonize OPC-depleted tissue. We found a significant reduction in the rate of colonization between 2 and 10 months of age (0.6 mm/week versus 0.38 mm/week). To determine if this represented an intrinsic property of OPCs or was due to changes in the environment that the cells were recolonizing, OPCs from 10-month-old animals were transplanted into 2-month-old hosts and OPCs from 2-month-old animals were transplanted into 10-month-old hosts. These experiments showed that the transplanted OPCs retained their age-related rate of colonization, indicating that the decline in colonizing rates of OPCs with age reflects an intrinsic property of OPCs. This age-related decline in the ability of OPCs to repopulate OPC-depleted tissue has implications for understanding remyelination failure in multiple sclerosis (MS) and developing therapies for remyelination failure.

The use of transplanted glial cells to reconstruct glial environments in the CNS.

Transplantation studies have demonstrated that glia-depleted areas of the CNS can be reconstituted by the introduction of cultured cells. Thus, the influx of Schwann cells into glia-free areas of demyelination in the spinal cord can be prevented by the combined introduction of astrocytes and cells of the O-2A lineage. Although Schwann cell invasion of areas of demyelination is associated with destruction of astrocytes, the transplantation of rat tissue culture astrocytes ("type-1") alone cannot suppress this invasion, indicating a role for cells of the O-2A lineage in reconstruction of glial environments. By transplanting different glial cell preparations and manipulating lesions so as to prevent meningeal cell and Schwann cell proliferation it is possible to demonstrate that the behaviour of tissue culture astrocytes ("type-1") and astrocytes derived from O-2A progenitor cells ("type-2") is different. In the presence of meningeal cells, tissue culture astrocytes clump together to form cords of cells. In contrast, type-2 astrocytes spread throughout glia-free areas in a manner unaffected by the presence of meningeal cells or Schwann cells. Thus, progenitor-derived astrocytes show a greater ability to fill glia-free areas than tissue culture astrocytes. Similarly, when introduced into infarcted white matter in the spinal cord, progenitor-derived astrocytes fill the malacic area more effectively than tissue culture astrocytes, although axons do not regenerate into the reconstituted area.

The response of oligodendrocytes to chemical injury.

Oligodendrocytes establish relationships with axons at myelination which commit the cell to make and then maintain certain volumes of myelin. As a result of this oligodendrocytes are a heterogenous population of cells. At one extreme, large cells support a single internode on large diameter axons while at the other, small cells support many internodes on small diameter axons. Although it is common practice to separate chemicals which cause vacuolation of myelin sheaths from those which bring about cell death and thus demyelination, many compounds produce vacuolation and/or cell degeneration depending on concentration; an observation which suggests that myelin sheath-associated vacuolation reflects oligodendrocyte toxicity rather than a specific myelinopathy. The restoration of myelin sheath-axon relationships following chemically induced demyelination requires a complex sequence of cell-cell interactions to occur in an orderly manner if new myelin sheaths are to be formed. Recruitment of new oligodendrocytes can be separated from the interaction of oligodendrocytes with axons which results in the laying down of a myelin sheath. The latter event can only take place in the absence of demyelinating agents and in the presence of astrocytes.

The origin of remyelinating cells in the central nervous system.

A clear understanding of the cellular events underlying successful remyelination of demyelinating lesions is a necessary prerequisite for an understanding of the failure of remyelination in multiple sclerosis (MS). The potential for remyelination of the adult central nervous system (CNS) has been well-established. However, there is still some dispute whether remyelinating oligodendrocytes arise from dedifferentiation and/or proliferation of mature oligodendrocytes, or are generated solely from proliferation and differentiation of glial progenitor cells. This review focuses on studies carried out on remyelinating lesions in the adult rat spinal cord produced by injection of antibodies to galactocerebroside and serum complement that show: (1) oligodendrocytes which survive within an area of demyelination do not contribute to remyelination, (2) remyelination is carried out by oligodendrocyte progenitor cells, (3) recruitment of oligodendrocyte progenitors to an area of demyelination is a local response, and (4) division of oligodendrocyte progenitors is symmetrical, resulting in chronic depletion of the oligodendrocyte progenitor population in the normal white matter around an area of remyelination. Such results suggest that repeated episodes of demyelination could lead to a failure of remyelination due to a depletion of oligodendrocyte progenitors.

Lymphocyte-myelin sheath interactions in acute experimental allergic encephalomyelitis.

Using a passively transferred acute model of experimental allergic encephalomyelitis (EAE) in the rat, inflammatory central nervous system (CNS) lesions were shown to develop rapidly, peak and then resolve. An unusual feature of the lesions in the CNS was the presence of pyknotic cells within myelin sheaths. A sequence of observations indicated that such cells were lymphocytes which had insinuated themselves into the myelin sheath by passage along the interperiod line. The presence of lymphocytes within myelin sheaths, a process which did not lead to demyelination, was considered to represent a change which reflects the specificity of the immune response in this disease. The detection of this change in other CNS autoimmune diseases, notably those associated with virus infections, may be important as an indicator of pathogenetically relevant lymphocyte-myelin interactions.

The differentiation of glial cell progenitor populations following transplantation into non-repairing central nervous system glial lesions in adult animals.

The non-repairing nature of the locally x-irradiated ethidium bromide (EB)-induced demyelinating white matter lesion has been further validated by showing that injections of two cultures which promote host remyelination of EB lesions in normal tissue do not do so in x-irradiated lesions. The behaviour of an oncogene-immortalized glial cell line and a growth-factor-expanded glial progenitor population have been examined following transplantation into the non-repairing EB lesion. Our studies indicate that the selected glial cell populations were each capable of establishing glial environments around demyelinated axons. Extensive oligodendrocyte remyelination with little astrocytic presence was observed in lesions transplanted with growth-factor-expanded optic nerve progenitors, while less extensive oligodendrocyte remyelination with the establishment of astrocyte-like cells was found in lesions transplanted with ts A58-SV40T immortalized glial cells. Prolonged expansion of both populations resulted in a loss of differentiation to normal glial phenotypes.

Rejection of wild-type and genetically engineered major histocompatibility complex-deficient glial cell xenografts in the central nervous system results in bystander demyelination and Wallerian degeneration.

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.