W. F. Blakemore

Response of the oligodendrocyte progenitor cell population (defined by NG2 labelling) to demyelination of the adult spinal cord

Elucidation of the response of oligodendrocyte progenitor cell populations to demyelination in the adult central nervous system (CNS) is critical to understanding why remyelination fails in multiple sclerosis. Using the anti‐NG2 monoclonal antibody to identify oligodendrocyte progenitor cells, we have documented their response to antibody‐induced demyelination in the dorsal column of the adult rat spinal cord. The number of NG2+ cells in the vicinity of demyelinated lesions increased by 72% over the course of 3 days following the onset of demyelination. This increase in NG2+ cell numbers did not reflect a nonspecific staining of reactive cells, as GFAP, OX‐42, and Rip antibodies did not co‐localise with NG2+ cells in double immunostained tissue sections. NG2+ cells incorporated BrdU 48–72 h following the onset of demyelination. After the onset of remyelination (10–14 days), the number of NG2+ cells decreased to 46% of control levels and remained consistently low for 2 months. When spinal cords were exposed to 40 Grays of x‐irradiation prior to demyelination, the number of NG2+ cells decreased to 48% of control levels by 3 days following the onset of demyelination and remained unchanged at 3 weeks. Since 40 Grays of x‐irradiation kills dividing cells, these studies illustrate a responsive and nonresponsive NG2+ cell population following demyelination in the adult spinal cord and suggest that the responsive NG2+ cell population does not renew itself. GLIA 22:161–170, 1998.

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.

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 demyclination 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.

Lines of Murine Oligodendroglial Precursor Cells Immortalized by an Activated neu Tyrosine Kinase Show Distinct Degrees of Interaction with Axons In Vitro and In Vivo

Replication‐defective retroviruses expressing the t‐neu oncogene, or a hybrid protein with the neu tyrosine kinase linked to the external region of the human epidermal growth factor receptor (egfr‐neu), were used to establish lines of murine oligodendroglial precursor cells. Differentiation of the t‐neu lines into myelin‐associated glycoprotein (MAG)‐positive oligodendrocytes was induced by dibutyryl cAMP, and the egfr‐neu line showed limited differentiation in vitro upon withdrawal of epidermal growth factor. Cerebellar granule cell neurons expressed mitogens for the cell lines. Upon transplantation into demyelinated lesions, t‐neu line cells engaged with the demyelinated axons whereas the egfr‐neu line cells differentiated further and ensheathed the axons. These cell lines thus interact with neurons in vitro and in vivo and can be used as tools to define the molecules involved in different stages of neuron‐glia interaction.

Remyelination occurs as extensively but more slowly in old rats compared to young rats following gliotoxin-induced CNS demyelination

Age is one of the many factors that influence remyelination following CNS demyelination, although it is not clear whether it is the extent or rate of remyelination that is affected. To resolve this issue we have compared remyelination in young and old adult rat CNS following gliotoxin‐induced demyelination. Remyelination of areas of ethidium bromide‐induced demyelination in the caudal cerebellar peduncle reached completion by 4 weeks in young adult rats (2 months) but was not complete until 9 weeks in old adult rats (9–12 months). We have also shown that remyelination of lysolecithin‐induced demyelination in the spinal white matter of old adult rats (18 months) can be extensive, with longer survival times (8 weeks) than have previously been examined. Thus, it is the rate rather than the extent of remyelination that changes in the ageing CNS. These results have important implications for understanding the mechanisms of remyelination, indicating that remyelination need not occur rapidly for it to be extensive. The capacity for the process of remyelination to continue over many weeks must also be borne in mind when assessing remyelination‐enhancement strategies either by transplantation or promotion of endogenous mechanisms.  GLIA 28:77–83, 1999.

Local recruitment of remyelinating cells in the repair of demyelination in the central nervous system.

The distance over which remyelinating cells within surrounding intact tissue are stimulated to respond to a demyelinating lesion and migrate toward it is unknown. To address this issue we have conducted a series of experiments in which the generation of remyelinating cells in tissue surrounding a spontaneously repairing area of demyelination induced in the adult rat spinal cord is suppressed by exposure to X‐irradiation. By regulating the area of X‐irradiation relative to the length of the demyelinating lesion within dorsal white matter we have shown that remyelinating cells are not recruited over distances greater than 2 mm into areas of demyelination, implying that most of the remyelinating cells are locally generated. This result indicates that there is only a narrow rim of normal tissue surrounding an area of demyelination from which remyelinating cells can be recruited. The depletion of cells within this rim may account for the poor remyelination associated with large areas of demyelination and following repeated episodes of demyelination. We have also shown that, in contrast to Schwann cells, oligodendrocyte lineage cells recruited into lesions have a limited ability to rapidly repopulate large areas of demyelination. Attempts to enhance remyelination in situations where it fails should therefore focus on increasing the size of the surrounding area from which remyelinating cells can be recruited by augmenting the level of recruitment signal, and preventing premature differentiation of oligodendrocytes so as to maximize their migratory and proliferative potential. J. Neurosci. Res. 50:337–344, 1997.

Transplanted CG4 Cells (an Oligodendrocyte Progenitor Cell Line) Survive, Migrate, and Contribute to Repair of Areas of Demyelination in X-Irradiated and Damaged Spinal Cord but Not in Normal Spinal Cord

In this study, we have examined the behavior of a lac-Z-transfected O- 2A progenitor cell line, CG4, following transplantation into normal and X-irradiated adult rat spinal cord, and we have also addressed the issue of whether CG4 cells transplanted remotely from ethidium bromide-induced demyelinating lesions in both X-irradiated and nonirradiated spinal cord are able to contribute to their repair. Following transplantation into X-irradiated spinal cord, CG4 cells survive, divide, and migrate extensively. The migration occurs mainly within the parenchymal tissue of the cord without preference for white or gray matter. Moreover, CG4 cells migrating away from their point of introduction are able to enter areas of demyelination and remyelinate the demyelinated axons therein. In contrast, when CG4 cells are transplanted into nonirradiated spinal cord, their survival is limited to areas of damage created by the injection procedure. The CG4 cells do not survive in undamaged, nonirradiated spinal cord. When transplanted remotely from areas of demyelination they are unable to traverse intervening areas of normal white matter, although they may enter lesions if transplanted into their close vicinity. These results have important implications for the development of potential therapeutic strategies for the treatment of multifocal demyelinating disorders that are based on glial cell transplantation.

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.

Requirements for schwann cell migration within cns environments: A viewpoint

Schwann cells are able to migrate into the CNS and myelinate CNS axons in a number of developmental and pathological situations. Morphological studies based on normal, mutant and experimentally‐lesioned tissue have indicated that Schwann cells are only able to enter the CNS when the integrity of the astrocytic glia limitans is disrupted. The significance and subtlety of the interactions between Schwann cells and astrocytes have been further explored by glial cell transplantation studies. These studies support in vitro observations on Schwann cell behaviour in highlighting the importance of extracellular matrix for both migration and myelin sheath formation. The failure of Schwann cells to intermix with astrocytes is an important aspect of glial cell biology which will have a bearing on efforts to remyelinate demyelinated axons by Schwann cell‐transplantation.

Efficient recolonisation of progenitor-depleted areas of the CNS by adult oligodendrocyte progenitor cells

A widely quoted hypothesis for the failure of remyelination in multiple sclerosis (MS) is the exhaustion of the oligodendrocyte progenitor cell (OPC) pool that is strongly implicated as the source of remyelinating oligodendrocytes in demyelinating lesions. Despite this, little is known about the responses of adult OPCs to adjacent areas of the CNS from which their numbers are depleted. We have developed an experimental model to study the pattern and rate of repopulation of OPC‐depleted zones, by endogenous OPCs in the adult rat spinal cord. By X‐irradiating short lengths of the spinal cord with 40 Gy of X‐irradiation, we were able to produce a highly localised depletion of OPCs that allowed us to study the responses of cells located in adjacent normal areas, to this local depletion. Using both NG2 immunohistochemistry and PDGFαR in situ hybridisation to identify OPCs, we demonstrate that endogenous OPCs repopulated the depleted areas slowly, but completely. This repopulation occurred at the rate of approximately 0.5 mm/week in the first month. Most cells at the leading edge of repopulation had complex, branching morphologies. The repopulation process was capable of restoring the density of progenitors in repopulated areas to that of normal tissue and was not associated with a secondary progenitor loss in tissue from which progenitor cells were generated. These findings indicate that depletion of the OPC population around lesions is not likely to be the primary explanation for remyelination failure in MS. GLIA 37:307–313, 2002.