Biomedical subjects

W F Blakemore

Publications and source records attributed to W F Blakemore.

At least 55 records · Page 3Linked to original sources

PubMed1998

The demonstration by transplantation of the very restricted remyelinating potential of post-mitotic oligodendrocytes.

To examine the remyelinating ability of post-mitotic oligodendrocytes, we subjected cell preparations derived from neonatal and adult rats to 40 Grays of X-irradiation to remove mitotically active cells and injected them into areas of demyelination in which the inherent ability to generate remyelinating cells had been inhibited. The extensive remyelination seen following implantation of non-irradiated neonatal and adult cells was almost completely abolished when the transplanted cell suspension was exposed to 40 Grays of X-irradiation, demonstrating that effective remyelination requires the generation of cells by mitosis. Radiation-resistant and therefore non-dividing oligodendrocytes were detected in areas of demyelination following transplantation of neonatal cultures and oligodendrocyte preparations derived from the adult nervous system. However, the pattern of myelin formation associated with the radiation-resistant oligodendrocytes from the two sources was different. Following implantation of X-irradiated neonatal cultures, a small number of oligodendrocytes could be found within the area of demyelination, and although these cells formed sheets of myelin membrane, they did not form myelin sheaths. After implantation of X-irradiated adult cells, in addition to the aberrant myelin formation seen with the neonatal cells, some myelin sheaths were observed. Our findings confirm that effective remyelination requires cell division and suggest that there may be diverse populations of radiation-resistant oligodendrocytes in the adult nervous system, some of which can form myelin sheaths and others of which can only make myelin sheets. Important for the interpretation of our previous studies is the demonstration here that 40 Grays of X-irradiation per se does not inhibit oligodendrocytes from remyelinating axons.

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PubMed1998

Investigation of the role of delayed-type-hypersensitivity responses to myelin in the pathogenesis of Theiler's virus-induced demyelinating disease.

The contribution of autoimmune responses to the pathogenesis of Theiler's virus-induced demyelinating disease was investigated. Delayed-type hypersensitivity responses to myelin were examined in both symptomatic and asymptomatic mice at different times post-infection, in order to determine whether autoreactivity correlates with the development of demyelination. The results indicate that although autoimmune responses probably do not play a major role in the initiation of demyelination at early times post-infection, autoreactivity to myelin antigens dose eventually develop in symptomatic animals, perhaps through the mechanism of epitope spreading. Autoimmunity to myelin components is therefore an additional factor that may contribute to lesion progression in chronically diseased animals.

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PubMed1998

Trapidil-mediated inhibition of CNS remyelination results from reduced numbers and impaired differentiation of oligodendrocytes.

In a previous study, we described the inhibitory effects of the growth factor-antagonist, trapidil, on spontaneously occurring oligodendrocyte remyelination in the rat spinal cord following lysolecithin-induced demyelination [30]. The objective of the present study was to further investigate the mechanisms of trapidil-mediated impairment of remyelination and thus obtain greater insight into the steps at which growth factors may be involved in remyelination. To this end, an ultrastructural analysis of the cellular composition of lesions from control and trapidil-treated animals was undertaken. Demyelination was created in the dorsal funiculus of 6-week-old female rats by the injection of 1.0 microliter of 1% lysolecithin. The animals received daily intraperitoneal injections of trapidil (80 mg/kg) or saline for 21 days, beginning on the day of lesion induction. Quantitative electron microscopic examination of lesions from both groups of animals showed that trapidil-treated lesions had reduced numbers of oligodendrocytes (P = 0.02) with a higher relative proportion of immature phenotypes, but increased numbers of microglia (P = 0.0009) and dystrophic axons (P0.02). In addition, the numbers of myelin lamellae around remyelinated axons were fewer in trapidil-treated animals. These results suggest that trapidil-mediated impairment of CNS remyelination is due to a blockage of growth factor-mediated proliferation and/or recruitment of remyelinating cells. Furthermore, the presence of oligodendrocytes with a more immature phenotype and the decreased thickness of the myelin sheaths of remyelination in the trapidil-treated animals indicate an impairment of growth factor-mediated differentiation.

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PubMed1998

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.

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PubMed1997

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.

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PubMed1997

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.

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PubMed1997

Oligodendrocyte precursors survive poorly and do not migrate following transplantation into the normal adult central nervous system.

Cell cultures enriched for oligodendrocyte precursors were prepared for male neonatal rat pups and transplanted into the spinal cord white matter of normal and x-irradiated syngeneic adult female rats. Transplanted cells were detected using a probe specific for the rat Y chromosome immediately after transplantation and 14 days later. In non-x-irradiated tissue, significantly fewer cells were seen at 14 days compared with time zero, and no cell migration was observed. In x-irradiated tissue, cells both survived and migrated into the surrounding grey and white matter. The observed behaviour of oligodendrocyte precursors in normal adult tissue is in contrast to their behaviour in myelin mutants and neonates, where migration and survival have been well documented (Warrington et al., 1993; Lachapelle et al., 1994), but mimics the behaviour of the O-2A progenitor-like cell line, CG4, following transplantation into similar environments (Franklin et al., 1996). The findings in this study have profound implications for the use of grafts of oligodendrocyte precursors as a therapy in human demyelinating diseases, because they indicate that grafts will need to be introduced directly into each clinically relevant area of demyelination.

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PubMed1997

Use of a rat Y chromosome probe to determine the long-term survival of glial cells transplanted into areas of CNS demyelination.

A lack of suitable markers for cells which undergo division following transplantation has hindered studies assessing the long-term survival of glial cell grafts in the CNS. A probe specific to the rat Y chromosome was used to identify male glial cells grafted into an area of ethidium bromide-induced demyelination in syngeneic adult female rat spinal cord 4 weeks, 6 months and 12 months post-transplantation. At all time points there was extensive oligodendrocyte remyelination of transplanted lesions, and graft-derived cells were present within the lesion up to 12 months post-transplantation. In order to demonstrate graft-derived oligodendrocytes in the remyelinated region at 6 and 12 months, double-labelling studies were performed using the oligodendrocyte-specific antibodies carbonic anhydrase II or phosphatidyl ethanolamine-binding protein in combination with the Y chromosome probe. It was found that the majority of oligodendrocytes in the transplanted region were graft-derived. Graft-mediated remyelination was associated with a reduction in myelin sheath thickness and increase in nodal frequency similar to that observed in spontaneous remyelination, suggesting that, like axons remyelinated spontaneously, axons remyelinated by grafted cells will be capable of secure conduction. An alteration in the immunoreactivity of oligodendrocytes from carbonic anhydrase II-negative in the unlesioned dorsal funiculus to carbonic anhydrase II-positive in the remyelinated dorsal funiculus was considered to reflect a reduction in the amount of myelin supported by each oligodendrocyte, leading to the proposal that carbonic anhydrase II immunoreactivity may provide a means of identifying areas of remyelination in normally carbonic anhydrase II-negative white matter tracts.

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PubMed1997

Transplanting oligodendrocyte progenitors into the adult CNS.

This review covers a number of aspects of the behaviour of oligodendrocyte progenitors following transplantation into the adult CNS. First, an account is given of the ability of transplanted oligodendrocyte progenitors, grown in tissue culture in the presence of PDGF and bFGF, to extensively remyelinate focal areas of persistent demyelination. Secondly, we describe how transplanted clonal cell lines of oligodendrocyte progenitors will differentiate into astrocytes as well oligodendrocytes following transplantation into pathological environments in which both oligodendrocytes and astrocytes are absent, thereby manifesting the bipotentially demonstrable in vitro but not during development. Finally, a series of studies examining the migratory behaviour of transplanted oligodendrocyte progenitors (modelled using the oliodendrocyte progenitor cell line CG4) are described. These show that CG4 cells do not survive (or migrate) when transplanted into the normal adult CNS. However, if they are transplanted into CNS tissue that has previously been exposed to 40 Gy of x-irradiation then transplanted CG4 cells survive, divide and migrate over large distances. Moreover, within an x-irradiated environment, migrating transplanted CG4 cells are able to enter remotely located foci of demyelination and contribute to the remyelination of the demyelinated axons within. These studies demonstrate that although the normal adult CNS does not appear to support survival and migration of the CG4 cell line, it is possible to manipulate the environment in such a way that these nonpermissive properties of the environment can be overcome.

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PubMed1997

Locomotor deficits induced by experimental spinal cord demyelination are abolished by spontaneous remyelination.

Demyelinating lesions induced by intraspinal injection of gliotoxin have been studied for many years in order to gain insights into reasons for failure of remyelination and to improve understanding of the axonal conduction disorders in multiple sclerosis. Although the electrophysiological correlates of experimental demyelination and remyelination are well established, the behavioural effects have not been investigated. In this study we aimed to determine whether behavioural deficits could be detected during spinal cord demyelination, and furthermore, whether remyelination was associated with return of lost function. We used injections of the gliotoxin ethidium bromide into the dorsal funiculus of the cervical spinal cord of the rat to induce zones of demyelination and compared the effects on locomotion with those resulting from saline injections. The resulting locomotor deficits were quantified by analysis of foot placement during traverse of a horizontal 18 mm diameter wooden beam. Following ethidium bromide injection there was a decrease in security of foot placement, that recovered by approximately 5 weeks post-injection. In a second experiment, remyelination was prevented by exposure of the spinal cord to 40 Gy of X-irradiation. Behavioural deficits were induced as before, but the animals failed to recover throughout the duration of the experiment. Saline-injected animals in both experiments exhibited minimal deficits and quickly recovered. We conclude that demyelination produces detectable behavioural deficits which disappear following spontaneous remyelination.

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PubMed1997

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.

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PubMed1997

To what extent is oligodendrocyte progenitor migration a limiting factor in the remyelination of multiple sclerosis lesions?

In this article we describe a series of experimental approaches, involving the use of gliotoxin-induced demyelination, X-irradiation and glial cell transplantation, which examine the size of the area around demyelinating lesions from which new remyelinating cells are generated, and the distance over which they are able to migrate. Taken together, these studies suggest that the recruitment of remyelinating cells takes place over a very limited area and that long distance migration of remyelinating cells is not a feature of remyelination. The implications of these findings for spontaneous remyelination of multiple sclerosis plaques, and the development of strategies for enhancing remyelination are discussed.

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PubMed1997

The effects of the growth factor-antagonist, trapidil, on remyelination in the CNS.

In this study we describe the effects of trapidil, a putative platelet-derived growth factor-antagonist, on spontaneously occurring remyelination in rat spinal cord. Demyelination was created in the dorsal funiculus of 6- and 11-week-old female rats by the direct injection of 1.0 microliter of 1% lysolecithin. The animals received daily intra-peritoneal injections of either trapidil or saline for 21 days, commencing on the day of lesion induction. The 11-week-old rats receiving trapidil (60 mg/kg) showed a significant decrease in the extent of oligodendrocyte remyelination. Moreover, those axons that were remyelinated by oligodendrocytes tended to have thinner myelin sheaths than axons remyelinated by oligodendrocytes in the control group. In the 6-week-old group, the dose of trapidil which inhibited oligodendrocyte remyelination in the 11-week-old animals had a minimal effect on the extent of oligodendrocyte remyelination and no effect on the quality of myelin sheath formation. A higher dose of trapidil (80 mg/kg) was required before significant impairment of oligodendrocyte remyelination was achieved in the younger age group, implying an age-dependent effect of growth factor-inhibition of CNS remyelination. These results indicate an important role for growth factors, and in particular PDGF, in the orchestration of oligodendrocyte remyelination in the rodent CNS.

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PubMed1996

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.

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PubMed1996

A new method of quantifying the extent of tissue loss following spinal cord injury in the rat.

Many agents are being tested for their ability to limit the development of secondary tissue damage following trauma to the spinal cord by comparing the extent of trauma-induced tissue destruction between groups of animals receiving different treatments. However, the changes that occur following spinal concussion result in initial swelling of the cord and then progressive shrinkage. It is therefore impossible to determine the preinjury cross-sectional area of cord once it has been damaged, making accurate calculations of the extent of tissue destruction difficult. To overcome this problem, a method for predicting the preinjury cross-sectional area of damaged spinal cord was developed in the rat by establishing correction factors that related the cross-sectional area of a reference transverse section to the area of sections at other points along the cord. This method was used to calculate the volume of tissue destruction in groups of rats 48 h, 4 days, and 4 weeks after photochemically induced cord injury. The results were compared with the volume of tissue destruction obtained when calculated from measured rather than predicted cord areas. One month following photochemically induced injury, the spinal cord had shrunk significantly. This resulted in significant underestimation of the lesion volume when calculated from the measured area of injured cord. This demonstrates that the preinjury area of the cord must be established in order to calculate the full extent of tissue destruction and that it can be estimated accurately using the method described in this study.

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PubMed1996

Primary demyelination and regeneration of ascending axons in the dorsal funiculus of the rat spinal cord following photochemically induced injury.

The extent of primary demyelination and regeneration of ascending axons in the dorsal funiculus of the rat spinal cord was investigated following photochemically-induced ischaemic injury. Groups of rats were killed at intervals from 48h to 1 month after injury and a combination of light and electron microscopy and counting of axons in specific sites was used to study the axonal changes. Unmyelinated axons were noted in the dorsal rim of the lesion at its centre and at the centre of the gracile fasciculus at the caudal end of the lesion 7 days after injury. By 1 month, axons in these sites were thinly myelinated by Schwann cells or oligodendrocytes. In order to differentiate between remyelination of demyelinated axons and myelination of regenerated axons, axon counts were performed. The number of sub-pial axons present at the lesion centre did not change significantly from 48h to 1 month after injury, whereas the number of axons at the caudal end of the lesion increased significantly from 4 to 10 days after injury. We therefore conclude that sub-pial axons at the lesion centre are demyelinated between 4 and 7 days after injury and subsequently remyelinated by Schwann cells. At the caudal end of the lesion, a specific population of small diameter axons located at the centre of the gracile fasciculus regenerates for a distance of approximately 1 mm between 4 and 10 days after injury; these axons are then myelinated by oligodendrocytes or Schwann cells. In contrast, larger diameter axons of the gracile fasciculus do not show a regenerative response, demonstrating the variability of axonal responses to injury.

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PubMed1996

Reconstruction of the glial environment of a photochemically induced lesion in the rat spinal cord by transplantation of mixed glial cells.

It is becoming increasingly apparent that the astrocytic environment is critical to the normal development and functioning of the CNS, and that acute injury to the spinal cord causes destruction of glial cells in addition to neurones and axons. The aims of this study were to assess the viability of reconstructing the astrocytic environment of a cystic spinal cord lesion by transplantation of glial cells and to examine the effect of the transplanted cells on meningeal cell invasion and revascularisation of the lesion and on axonal regeneration. Neonatal rat and kitten mixed glial cells and the CG-4 rat O-2A progenitor cell line were transplanted into a lesion produced in the dorsal funiculus of the rat spinal cord by photochemical infarction. The animals were killed 4 weeks after injury, their cords examined with light and electron microscopy and compared with control animals that were injected with medium alone. Transplantation of all three preparations resulted in increased numbers of astrocytes in the area of Wallerian degeneration cranial to the lesion and within the cyst. Mixed glial cell cultures prepared from neonatal rat forebrain contained cells with in vitro characteristics of type-1 astrocytes, and produced dense clusters of astrocytes that were surrounded by meningeal cells, resulting in a fragmented environment in the cyst. In contrast, glial cell cultures prepared from kitten forebrain and the CG-4 cell line produced cells that filled the cyst with a loose network of fine processes and reduced meningeal cell infiltration of the lesion. The CG-4 cell line significantly increased the density of blood vessels in the centre of the lesion and the number of spared axons present dorsal to the lesion, but none of the preparations significantly increased the number of axons regenerating at the caudal end of the lesion. We conclude that O-2A progenitor-derived astrocytes are more suitable for reconstruction of the glial environment of a cystic lesion in the rat spinal cord than 'type-1 like' astrocytes and would therefore be the cell of choice to engineer to produce factors that promote axonal regeneration.

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PubMed1996

A study of pathology of a bovine primary peripheral myelinopathy with features of tomaculous neuropathy.

Cases of a bovine neuropathy are reported in which peripheral nerves show "sausage-shaped" thickenings of the myelin sheaths at different sites of the internode. Clinical signs of dysphagia and chronic rumenal bloat developed after weaning which were attributable to bilateral vagus nerve degeneration. Trunks of the sciatic nerves and brachial plexuses were similarly affected with the animal adopting a weak shuffling gait. Affected animals were the progeny of sire-daughter matings. The lesions are similar to those seen in the tomaculous neuropathies of man. The present study is believed to be the first report of this lesion occurring in domestic animals.

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