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W F Blakemore

Publications and source records attributed to W F Blakemore.

At least 73 records · Page 4Linked to original sources
PubMed1996

Failure to achieve remyelination of demyelinated rat axons following transplantation of glial cells obtained from the adult human brain.

The ability of transplanted glial cells to myelinate axons in experimental animals offers the prospect that it may be possible to achieve remyelination in human demyelinating disease by the implantation of oligodendrocyte lineage cells. Autologous normal white matter could represent a potential source of cells whose use would avoid tissue rejection and overcome ethical and practical constraints associated with the use of fetal tissue. To determine the remyelinating potential of cells isolated from adult human CNS, a cell preparation prepared from adult human white matter which contained 56% oligodendrocytes, 3% preoligodendrocytes and 1% precursor cells was transplanted into non-repairing demyelinating lesions in immunosuppressed rats created by the injection of ethidium bromide into x-irradiated spinal cord white matter. The extent of remyelination was examined 3 and 5 weeks after transplantation. Although the transplanted oligodendrocytes survived in the area of demyelination, associated with demyelinated axons and produced myelin membranes, no myelin sheaths were produced and there was no evidence of cell migration or division. The failure of human oligodendrocytes to form myelin sheaths may reflect the poor remyelinating potential of post mitotic oligodendrocytes, and the failure of the small number of co-transplanted bipotential oligodendrocyte progenitor cells to differentiate and myelinate axons may be a consequence of lack of appropriate environmental factors within the rat lesion required for expansion and differentiation of these cells.

Adult↗
PubMed1995

Reconstruction of the glia limitans by sub-arachnoid transplantation of astrocyte-enriched cultures.

Lesions in CNS white matter involving loss of glial cells with concurrent destruction of the glia limitans lead to widespread remyelination of CNS axons by Schwann cells. Previous studies have demonstrated that this situation can be changed by transplanting cultured CNS glial cells into lesions early on in the repair process. In this study we have transplanted cultured astrocytes into the sub-arachnoid space above such a lesion in order to (1) influence the normal repair process by transplant-assisted reconstruction of the glia limitans, and (2) explore the potential of a minimally invasive route for introducing cells to white matter lesions. In some cases, it proved possible to influence normal repair by transplanting cells via the sub-arachnoid route, although the results were inconsistent. However, the experiment permitted observations to be made on the migration of transplanted astrocytes across the surface of and within the spinal cord.

Animals↗
PubMed1995

Differentiation of the O-2A progenitor cell line CG-4 into oligodendrocytes and astrocytes following transplantation into glia-deficient areas of CNS white matter.

The in vitro properties of the CG4 cell line have led to its increasing use as a cell line with which to study the behaviour of the O-2A progenitor cell. In this study we have examined the in vivo behaviour of the CG4 cell line following transplantation into areas of adult rat spinal cord white matter which have been permanently depleted of glial cells by the combination of local X-irradiation and direct injection of 0.1% ethidium bromide. Twenty-one days after transplantation, both myelin-forming oligodendrocytes and glial fibrillary acidic protein-positive astrocytes were identified within the lesion, indicating that the CG4 cell line has bipotential differentiation properties when introduced into a pathological environment consisting of demyelinated axons but devoid of oligodendrocytes or astrocytes. In this respect, the CG4 cell line resembles other glial progenitor cell lines that have been transplanted into similar lesions. In some areas of the lesion, remyelination was observed that was similar in extent to that achieved by growth factor-expanded populations of O-2A progenitor cells. The transplant origin of the cell types within the lesion was confirmed by retroviral incorporation of the lacZ marker gene, the expression of which allowed their identification by histochemistry. In conclusion, the in vivo properties of the CG4 cell line make it a highly suitable line with which to study the behaviour of O-2A progenitors following transplantation into normal and damaged CNS.

Animals↗
PubMed1995

Glial cell transplants that are subsequently rejected can be used to influence regeneration of glial cell environments in the CNS.

Transplantation of glial cells into demyelinating lesions in CNS offers an experimental approach which allows investigation of the complex interactions that occur between CNS glia, Schwann cells, and axons during remyelination and repair. Earlier studies have shown that 1) transplanted astrocytes are able to prevent Schwann cells from participating in CNS remyelination, but that they are only able to do so with the cooperation of cells of the oligodendrocyte lineage, and 2) transplanted mouse oligodendrocytes can remyelinate rat axons provided their rejection is controlled by immunosuppression. On the basis of these observations, we have been able to prevent the Schwann cell remyelination that normally follows ethidium bromide demyelination in the rat spinal cord by co-transplanting isogeneic astrocytes with a potentially rejectable population of mouse oligodendrocyte lineage cells. Since male mouse cells were used it was possible to demonstrate their presence in immunosuppressed recipients using a mouse Y-chromosome probe by in situ hydridisation. When myelinating mouse cells were rejected by removal of immunosuppression, the demyelinated axons were remyelinated by host oligodendrocytes rather than Schwann cells, whose entry was prevented by the persistence of the transplanted isogeneic astrocytes. The oligodendrocyte remyelination was extensive and rapid, indicating that the inflammation associated with cell rejection did not impede repair. If this host oligodendrocyte remyelination was prevented by local X-irradiation, the lesion consisted of demyelinated axons surrounded by processes from the transplanted astrocytes. By this approach, it was possible to create an environment which resembled the chronic plaques of multiple sclerosis. Thus, these experiments demonstrate that in appropriate circumstances the temporary presence of a population of glial cells can alter the outcome of damage to the CNS.

Animals↗
PubMed1995

Monocyte recruitment into the scrapie-affected brain.

The recruitment of monocytes into the scrapie-affected brain was investigated in female mice reconstituted with male bone marrow, using a Y-chromosome-specific probe and F4/80 immunocytochemistry. Recruitment of monocytes could be demonstrated in six out of eight animals and the number of recruited cells correlated with the severity of vacuolation in most, but not all, animals. The proportion of microglia derived from recruited monocytes varied between individual animals, did not correlate with the increase in cellularity (glia) in affected areas of brain and did not affect the length of incubation period. Thus, it is unlikely that the recruitment of monocytes is a pivotal event in the development of early pathological changes in scrapie. The morphology of recruited cells in scrapie lesions, as revealed by F4/80 immunoreactivity, was indistinguishable from that of activated resident microglia.

Animals↗
PubMed1995

Remyelination of mouse spinal cord axons demyelinated by local injection of lysolecithin.

Local injections of lysolecithin are commonly used to produce areas of demyelination in the CNS. For the mouse, the demyelinating initial phase of lesion development has been described previously but the spontaneous repair which follows has not. In this study we describe the morphological sequelae which result from the injection of 2 microliters of 1% lysolecithin into the spinal cord of adult mice. Lesion length was variable, extending 8 mm or more in 33% of lesioned animals. By 7 days after injection very little extracellular myelin debris was detected and remyelination had commenced. Remyelination progressed rapidly so that almost all axons were invested by myelin sheaths by 23 days. Remyelination was accompanied by a prominent astrocytosis. The long lesion length and the rapidity of repair has important implications for studies designed to assess the ability of transplanted myelinogenic cells to migrate towards demyelinating lesions.

Animals↗
PubMed1995

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.

Animals↗
PubMed1995

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.

Animals↗
PubMed1994

In vitro and in vivo characterisation of glial cells immortalised with a temperature sensitive SV40 T antigen-containing retrovirus.

An oncogene-carrying replication-defective retrovirus was used to establish immortalised lines of murine glial cells. Primary cultures of early postnatal cerebellar cells were infected with a retrovirus based on the Murine Moloney Leukemia Virus containing a temperature-sensitive mutant of the Simian Virus 40 large T antigen (SV40 T) oncogene and a gene coding for resistance to the antibiotic G418. Infected cells were selected in G418 and after several in vitro passages cells expressing the O4 antigen were established as a cell line. At a later time point O4-positive single-cell clones were established. Two different types of clones were obtained: 1) "plastic" clones consisting of cells which initially had a morphological and antigenic phenotype of young glial precursor cells but which gradually lost these features, and 2) "stable" cell clones including a clone with the immunological and electrophysiological characteristics of Schwann cells. Culture of the latter cells in the presence of 1 mM dibutyryl cyclic adenosine monophosphate for a period of at least 10 days induced a change in shape and a shift in antigen expression towards a more "differentiated" maturation stage. When the SV40 T O4-positive immortalised cell line isolated on the cell sorter was transplanted into demyelinated lesions in adult rats, cells were observed ensheathing axons and forming limited amounts of PNS-type myelin. Glial cells immortalised with a temperature-sensitive mutant of the SV40 T oncogene thus retain many physiological properties of their primary culture counterparts and can be induced to undergo limited differentiation in vitro and in vivo. These cell lines, which represent immature CNS glia or Schwann cells, are providing useful tools for investigating the role of cell surface antigens involved in neuron-glial interactions.

Animals↗
PubMed1994

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.

Animals↗
PubMed1994

The use of xenografting to evaluate the remyelinating potential of glial cell cultures.

Experiments in rodents have shown a potential role for glial cell transplantation as a means of influencing repair in the central nervous system of man. A crucial step in developing human therapy is to establish whether knowledge gained from studies in rodents is applicable to larger mammalian species. In order to explore this issue we examined the ability of cat glial cell cultures to remyelinate areas of ethidium-bromide-induced demyelination in the spinal cord of immunosuppressed rats and cats. Transplantation of density-gradient-isolated glial cells obtained from the forebrain of 7-day-old kittens resulted in enhanced oligodendrocyte remyelination in the rat but failed to enhance oligodendrocyte remyelination in the cat. The feasibility of enhancing oligodendrocyte remyelination in the cat lesion was demonstrated by transplanting a rat culture containing a high proportion of cells of the oligodendrocyte lineage. Tissue culture of the density-gradient-isolated cell preparations suggested that the failure of the kitten glial preparation to enhance oligodendrocyte remyelination in the cat was most probably due to its poor oligodendrocyte-generating capacity. However, our lack of understanding of the biology of feline glial cells precludes a full understanding of these experiments.

Animals↗
PubMed1994

Generalised ceroid-lipofuscinosis and brown bowel syndrome in Cocker spaniel dogs.

The ceroid-lipofuscinoses comprise a group of inherited storage diseases of human beings and animals that are characterised by progressive neurodegenerative disease and the accumulation within cells of a fluorescent lipopigment. A distinct syndrome occurs in Cocker spaniel dogs in which there is a generalised accumulation of a lipofuscin-like pigment, with such a heavy accumulation in smooth muscle that the intestine and other organs have a brown discolouration. Such discolouration is not observed in other forms of ceroid-lipofuscinosis. Dogs are clinically affected in adulthood and show progressive hind limb paresis, incoordination, and deficient postural reactions and proprioception. Spinal reflexes may be exaggerated. Behavioural or temperament changes, seizures or blindness may occur.

Journal Article↗
PubMed1993

In vitro and in vivo analysis of a rat bipotential O-2A progenitor cell line containing the temperature-sensitive mutant gene of the SV40 large T antigen.

Primary cultures from neonatal optic nerve contain pluripotential O-2A progenitor cells that are capable of differentiating into oligodendrocytes, type-2 astrocytes or adult O-2A progenitors (O-2Aadult). Since primary optic nerve cultures contain a mixture of glial cell types of which only a small number are O-2A progenitors, experiments on cell lineage and differentiation carried out using these cultures are both intrinsically limited and difficult to interpret. Ideally, cells from a clonal cell population would provide the optimal starting material for biological studies. In this paper we describe the creation of an O-2A progenitor cell line using a retrovirus carrying a temperature-sensitive mutant SV40 large T antigen gene. This cell line has provided sufficient numbers of cells to allow analysis of their in vitro properties and their behaviour following transplantation into an in vivo environment. At the non-permissive temperature (39 degrees C), these cells differentiate into oligodendrocytes and type-2 astrocytes in a similar fashion to O-2A progenitor cells from primary cultures (O-2Aprim). When grown in media containing platelet-derived growth factor and basic fibroblast growth factor, the cell numbers can be expanded in culture without differentiating, consistent with the behaviour of O-2Aprim progenitor cells. By exploiting this property, it has been possible to culture large numbers of O-2A progenitors for in vivo analysis. In this study we have shown that transplantation of this O-2A cell line into glia-free areas in adult rat spinal cord results in differentiation of a proportion of cells into oligodendrocytes which are capable of myelinating axons. Furthermore, differentiation of O-2A cells into astrocytes was also observed, indicating that the bipotentiality of these cells in vitro can also be demonstrated in vivo.

Animals↗
PubMed1993

Repair of demyelinated lesions by transplantation of purified O-2A progenitor cells.

The transplantation of well defined populations of precursor cells offers a means of repairing damaged tissue and of delivering therapeutic compounds to sites of injury or degeneration. For example, a functional immune system can be reconstituted by transplantation of purified haematopoietic stem cells, and transplanted skeletal myoblasts and keratinocytes can participate in the formation of normal tissue in host animals. Cell transplantation in the central nervous system (CNS) has been proposed as a means of correcting neuronal dysfunction in diseases associated with neuronal loss; it might also rectify glial cell dysfunction, with transplanted oligodendrocyte precursor cells eventually allowing repair of demyelinating damage in the CNS. Here we use co-operating growth factors to expand purified populations of oligodendrocyte type-2 astrocyte (O-2A) progenitor cells for several weeks in vitro. When injected into demyelinating lesions in spinal cords of adult rats, created in such a way as to preclude host-mediated remyelination, these expanded populations are capable of producing extensive remyelination. In addition, transplantation of O-2A progenitor cells genetically modified to express the bacterial beta-galactosidase gene gives rise to beta-galactosidase-positive oligodendrocytes which remyelinate demyelinated axons within the lesion. These results offer a viable strategy for the manipulation of neural precursor cells which is compatible with attempts to repair damaged CNS tissue by precursor transplantation.

Animals↗
PubMed1993

Lines of glial precursor cells immortalised with a temperature-sensitive oncogene give rise to astrocytes and oligodendrocytes following transplantation into demyelinated lesions in the central nervous system.

Immortalised lines of murine glial precursor cells expressing the neomycin resistance gene and a temperature-sensitive mutation of the SV 40 T oncogene were established from cultures of oligodendrocytes and precursor cells infected with a replication-incompetent, helper-free retrovirus. At the permissive temperature (33 degrees C), they could be continually propagated in vitro and cells were present expressing the 04 antigen specific for glial precursor cells and oligodendrocytes. At 38 degrees C, where the expression of the T antigen is down regulated, cell division largely ceased. During early passage in vitro, limited differentiation to a more mature phenotype, as evidenced by expression of GFAP and the oligodendrocyte marker 01 was observed at both 33 degrees C and 38 degrees C. When transplanted into demyelinating lesions in the spinal cords of adult rats early passages of the lines yielded myelin-forming oligodendrocytes and astrocytes. Cells from later passages of the lines although failing to synthesise myelin still associated specifically with the demyelinated axons. These experiments demonstrate the retention of physiological properties of these oncogene-carrying glial cells when transplanted in vivo and suggest that such immortalised populations can be used for the isolation of molecules regulating glial cell function.

Animals↗
PubMed1993

The reconstruction of an astrocytic environment in glia-deficient areas of white matter.

Injection of ethidium bromide into X-irradiated spinal cord white matter produces a lesion in which demyelinated axons reside in an environment that is permanently depleted of glial cells. By transplanting defined populations of glial cells into this lesion it is possible to recreate normal or novel glial environments. In this study we have transplanted cultures of astrocytes into the X-irradiated ethidium bromide lesion in order to (1) assess the ability of these cells to relate to components within the lesion environment and thereby contribute to tissue reconstruction and (2) establish an astrocytic environment around demyelinated axons that resembles pathological states such as the chronic demyelinated plaques of multiple sclerosis. In order to focus attention on the interactions between astrocytes and demyelinated axons we developed a protocol for depleting astrocyte cultures of oligodendrocyte lineage cells and Schwann cells based on complement-mediated immunocytolysis and in vitro X-irradiation. In addition to establishing the ability of transplanted astrocytes to form an astrocytic matrix around demyelinated axons, this study has also revealed the diversity of cell types present within neonatal forebrain cultures.

Animals↗
PubMed1993

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.

Animals↗