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 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.

Redistribution of bisbenzimide Hoechst 33342 from transplanted cells to host cells.

Identification of transplanted cells within host tissue is an important component of many transplantation experiments. In this study, Schwann cells labelled with the fluorochrome bisbenzimide (Hoechst 33342, H33342) and transduced with the lac-Z gene were introduced into normal white matter and their distribution was examined 2 h, 24 h and 4 weeks after transplantation. At 2 and 24 h following transplantation, H33342-labelled cells were more widely distributed than lac-Z-labelled cells in both longitudinal and transverse directions. By 4 weeks following transplantation, no lac-Z-labelled cells could be found. However, H33342-labelled cells were observed in and around the glial scar. Therefore, labelling of host cells by transfer of H33342 dye from transplanted cells has to be considered whenever this dye is used as a transplant marker.

Remyelination of demyelinated CNS axons by transplanted human schwann cells: the deleterious effect of contaminating fibroblasts.

Areas of demyelination can be remyelinated by transplanting myelin-forming cells. Schwann cells are the naturally remyelinating cells of the peripheral nervous system and have a number of features that may make them attractive for cell implantation therapies in multiple sclerosis, in which spontaneous but limited Schwann cell remyelination has been well documented. Schwann cells can be expanded in vitro, potentially affording the opportunity of autologous transplantation; and they might also be spared the demyelinating process in multiple sclerosis. Although rat, cat, and monkey Schwann cells have been transplanted into rodent demyelinating lesions, the behavior of transplanted human Schwann cells has not been evaluated. In this study we examined the consequences of injecting human Schwann cells into areas of acute demyelination in the spinal cords of adult rats. We found that transplants containing significant fibroblast contamination resulted in deposition of large amounts of collagen and extensive axonal degeneration. However, Schwann cell preparations that had been purified by positive immunoselection using antibodies to human low-affinity nerve growth factor receptor containing less than 10% fibroblasts were associated with remyelination. This result indicates that fibroblast contamination of human Schwann cells represents a greater problem than would have been appreciated from previous studies.

Repair of demyelinated lesions by glial cell transplantation.

Transplantation of oligodendrocyte lineage cells results in myelination of naked axons. The most extensive remyelination is achieved using A2B5 positive progenitor cells and these observations encourage the view that glial transplantation may be a useful treatment in human demyelinating diseases. However, several issues need to be resolved before glial cell transplantation can be applied clinically; this review focuses on the choice of cells, their source, whether chronically demyelinated axons can be repaired by transplantation, and whether the principles of glial transplantation established in small rodents are applicable to other mammalian species and to man.

The effect of the number of oligodendrocytes transplanted into X-irradiated, glial-free lesions on the extent of oligodendrocyte remyelination.

Central nervous system mixed glia cell cultures were prepared which contained varying proportions of galactocerebroside-positive oligodendrocytes and high proportions of GFAP-positive flat astrocytes. When these cell preparations were transplanted into areas of persistent demyelination produced in the dorsal columns of adult rat spinal cord, it was found that the number of oligodendrocyte internodes formed and the extent of CNS reconstruction achieved was directly related to the number of oligodendrocytes introduced to the lesioned area. With transplants containing low numbers of oligodendrocytes, astrocytes appeared unable to establish relationships within the lesioned areas and only Schwann cells, which comprise less than 5% of the total cell preparation, effected significant remyelination. These results indicate that a minimum number of oligodendrocytes are required to support significant reconstruction of CNS territories in the face of competition-for-axons from contaminating Schwann cells.

Schwann cell remyelination of CNS axons following injection of cultures of CNS cells into areas of persistent demyelination.

The injection of suspensions of central nervous system (CNS) cells, prepared by standard methods from 4-day-old rat brain and maintained in vitro for 10 days, into areas of persistent demyelination in rat spinal cord resulted in extensive remyelination of axons by Schwann cells. As control lesions injected with medium showed no remyelination, the most likely explanation of this finding is that 'CNS cultures' contain a small population of Schwann cells which are stimulated to proliferate by the demyelinated axons.

Rat Schwann cell remyelination of demyelinated cat CNS axons: evidence that injection of cell suspensions of CNS tissue results in Schwann cell remyelination.

Injection of a suspension of embryonic rat central nervous system (CNS) cells into an area of persistent demyelination, produced in the cat spinal cord by injecting ethidium bromide into an area previously exposed to 40 Grays of x-irradiation, results, initially, in Schwann cell remyelination of the demyelinated axons. However, the Schwann cells are subsequently rejected; a response which confirms that the remyelinating cells are of rat origin.

The use of cultured autologous Schwann cells to remyelinate areas of persistent demyelination in the central nervous system.

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.

Molecular organisation in central nerve myelin.

Pertinent data from the literature and in press is summarised and used to construct a model for the molecular arrangement of lipid and protein in the lamellae of compact central nerve myelin. For the lipid phase of myelin the available data is best interpreted in terms of a bilayer arrangement while physical studies suggest that the lipids are in an intermediate fluid state maintained by the presence of cholesterol and water in the system. Lipids will interact to maintain this condition. The proteins of myelin differ in their membrane locations. The high molecular weight proteins are considered to be intrinsic components with at least part of their polypeptide chains in the lipid phase. The proteolipid protein is also intrinsic and may be completely buried in the lipid phase. The basic protein of myelin is an extrinsic component and must be localised at the surface of the lipid phase at either the external or cytoplasmic face of the lamellae. Present results suggest an elusive location at the cytoplasmic apposition region. The lipid-interacting properties of the basic protein are segregated on the polypeptide chain of the molecule and this may be important for the possible role of the basic protein in bridging adjacent lamellae at the cytoplasmic apposition. It is speculated that association of the proteolipid protein with the basic protein in a 1:1 molar ratio would form an effective lipid-complexing nucleus in the lipid rich myelin lamellae but experimental data to support this idea is lacking at present.

Covalent probe investigations with isolated central nerve myelin preparations.

The interaction of the covalently reacting probes dansyl chloride, fluorodinitrobenzene and trinitrobenzene sulphonic acid with isolated central nerve myelin sheath preparations has been studied. The three probes interact preferentially with accessible amino groups on lipid and protein in the membrane. With isolated myelin some 13% of the total phosphatidyl ethanolamine is labelled with dansyl chloride while the figure is 66% with fluorodinitro benzene and 47% with trinitrobenzene sulphonic acid. Lower levels of phosphatidyl serine are labelled. Phosphatidyl ethanolamine seems to be more accessible to probes in the myelin sheath than is phosphatidyl serine perhaps because the ethanolamine-containing lipid class is localised partially at the external apposition surfaces of the membrane which are most accessible to the probes. The serine phospholipids may not react so well because they are preferentially distributed at the cytoplasmic surface of the system. Analysis of protein labelling patterns after reaction of intact myelin with dansyl chloride indicates that the high molecular weight proteins and the proteolipid protein is accessible to the probe while the basic protein is not, even though this latter component is readily labelled with dansyl choride in purified form. It is suggested that the inability of the basic protein to react with myelin is perhaps due to the fact that it is occluded from interaction with the probe at the cytoplasmic apposition surfaces of the lamellae.

Observations on the migratory behaviour of Schwann cells from adult peripheral nerve explant cultures.

The migration of Schwann cells from adult sciatic nerve explant cultures has been examined by time-lapse photomicrography. Analysis of Schwann cell migratory behaviour indicates that the initial outwandering by individual Schwann cells was random. Although chance cell-cell contacts resulted in temporary immobilization of pairs of cells, stable multicellular structures did not form during this initial phase. As local cell densities increased, Schwann cells assembled networks within which Schwann cell movement continued to be observed. A second form of Schwann cell outgrowth was observed from degenerating fibres in which arrays of highly oriented Schwann cells migrated away from their basal lamina tubes onto the culture dish. These observations of Schwann cell random migration, network self-assembly and coordinated extratubal migration are considered to highlight aspects of Schwann cell behaviour, independent of axonal influences, which may have relevance to their role in peripheral nerve repair following nerve section.

Extensive oligodendrocyte remyelination following injection of cultured central nervous system cells into demyelinating lesions in adult central nervous system.

Following the injection of central nervous system (CNS) cell cultures, prepared from 1-day-old rats and maintained in vitro for 7 days, into irradiated, demyelinating lesions in the spinal cord of adult isologous animals, extensive remyelination of axons by oligodendrocytes was observed. In addition, astrocytes, within the transplanted cell suspension, established normal relationships with oligodendrocytes, axons and other tissue elements, which led to the establishment of large CNS territories throughout the lesions. Outside these CNS domains, Schwann cells, which are present in the transplanted cell suspension, myelinated groups of axons. These observations indicate that the irradiated, ethidium bromide lesion provides an in vivo environment, devoid of the influences of host glia, in which to examine the interactions of transplanted glial cells with demyelinating axons.

Observations on Wallerian degeneration in explant cultures of cat sciatic nerve.

Wallerian degeneration was studied in vitro using explant cultures of cat sciatic nerve. As these cultures contain no macrophages they highlight the responses of Schwann cells to myelin sheath breakdown. Although there were regional variations in the changes observed in these cultures with respect to time, the sequence of events which lead to Schwann cell proliferation and to fragmentation and liberation of myelin debris into the endoneurial space was established. The initial event was rejection of myelin sheaths by Schwann cells. Liberated Schwann cells then proliferated within the basal lamina tube. In nerve fibres containing proliferating Schwann cells, myelin debris passed through breaks in the basal lamina tube into the endoneurial space. Schwann cells also escaped from the basal lamina tube with the myelin debris. Following the loss of the luminal contents the basal lamina tube collapsed and the intratubular Schwann cells formed bands of Büngner. The Schwann cells which migrated into the endoneurial space and subsequently onto the culture dish retained contact with each other. These studies indicate that rejection of myelin internodes by their supporting Schwann cells set in train a series of events in which Schwann cells and degenerating myelin behaved as separate components. Schwann cells were not involved in phagocytosis or degeneration of myelin. We conclude that Schwann cell proliferation in Wallerian degeneration is directed towards re-establishing cellular continuity within the basal lamina tube which is lost when Schwann cells reject their myelin sheaths.

Remyelination of demyelinated rat axons by transplanted mouse oligodendrocytes.

The injection of the gliotoxic agent ethidium bromide (EB) into spinal white matter produces a CNS lesion in which it is possible to investigate the ability of transplanted glial cells to reconstruct a glial environment around demyelinated axons. This study demonstrates that transplanted mouse glial cells can repopulate EB lesions in rats provided tissue rejection is controlled. In X-irradiated EB lesions in cyclosporin-A-treated rats, mouse oligodendrocytes remyelinated rat axons and, together with mouse astrocytes, re-established a CNS environment. When transplanted into nonirradiated EB lesions in nude rats, mouse glial cells modulated the normal host repair by Schwann cells to remyelination by oligodendrocytes. In both X-irradiated and non-irradiated EB lesions, transplanted mouse glial cells behaved similarly to isogenic rat glial cell transplants (Blakemore and Crang Dev Neurosci, 1988;10:1-10; J Neurocytol, 1989;18:519-528). These findings indicate that the cell-cell interactions involved in reconstruction of a glial environment are common to both mouse and rat.

Attempts to produce astrocyte cultures devoid of oligodendrocyte generating potential by the use of antimitotic treatment reveal the presence of quiescent oligodendrocyte precursors.

The presence of oligodendrocyte precursor cells which cannot be removed from primary cultures by antibody-dependent techniques complicates the interpretation of transplantation experiments designed to examine the potential of astrocytes to influence remyelination (Blakemore et al.; Glia 13:79-91, 1995). In the present series of experiments we have investigated the use of the antimitotic cytosine arabinoside to eliminate oligodendrocyte precursors from mixed glial cell cultures following immunolytic removal of both oligodendrocytes and oligodendrocyte progenitors using A2B5 and O4 monoclonal antibodies. Our results indicate that not all oligodendrocyte precursors are involved during the subsequent regeneration of oligodendrocytes since a population of precursors survive 3-day and 12-day exposure to cytosine arabinoside. Maintaining immunolysed cultures in serum-free medium containing cytosine arabinoside for 23 days, removed the potential to generate large clones of oligodendrocytes both in vitro and following transplantation. However, a small number of oligodendrocyte precursors survived this treatment and generated single oligodendrocytes in vitro and isolated clusters of oligodendrocyte remyelination following transplantation. Overall, these results indicate that oligodendrocyte precursors have considerable potential to generate oligodendrocytes, but, since they can also survive for long periods in a quiescent state, their complete elimination from immunolysed astrocyte cultures by the use of an antimitotic is unreliable, if not impossible.

Solubilization of isolated central-nervous-system myelin preparations by the amniotic detergent sodium dodecyl sulphate.

The mechanism for the solubilization of isolated central-nervous-system myelin by sodium dodecyl sulphate was studied in detail. The release of protein and phospholipid to the 100000 g x 1 h supernatant fraction is dependent on the total amount of detergent relative to the amount of membrane present and on the ionic strength of the solubilization system. Gel-filtration analysis of supernatant fractions indicate that at suboptimal concentrations of detergent these contain lipid-protein complexes. The complete dissociation of the individual protein components from lipid is dependent on the total amount of sodium dodecyl sulphate present in the system. The results indicate that for the analysis of membrane components in sodium dodecyl sulphate it is essential that sufficient detergent is present.