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Biomedical subjects

R J Franklin

Publications and source records attributed to R J Franklin.

At least 19 recordsLinked to original sources
PubMed2001

The effect of immunosuppressive protocols on spontaneous CNS remyelination following toxin-induced demyelination.

Glial cell transplantation is a potential therapy for human demyelinating disease, though obtaining large numbers of human myelinating cells for transplantation remains a major stumbling block. Autologous transplantation is currently not possible, since the adult human CNS is not a good source of oligodendrocyte precursors, and long-term immunosuppression of engrafted allogeneic or xenogeneic cells is therefore likely to be necessary. Immunosuppressive drugs may need to be used in situations where more recent, active areas of demyelination are undergoing endogenous remyelination. It is therefore pertinent to establish the extent to which immunosuppressive protocols will suppress spontaneous remyelination. In order to investigate this issue, we created demyelinating lesions in the spinal cord of adult rats and compared the extent of remyelination in animals receiving different immunosuppressive treatments. In animals given only cyclosporin A, there was no difference in the extent of either Schwann cell or oligodendrocyte remyelination of ethidium bromide-induced demyelinating lesions. However, in animals given cyclophosphamide, either alone or in combination with cyclosporin, there was a significant reduction in the extent of oligodendrocyte-mediated remyelination. These results demonstrate that cyclophosphamide is deleterious to oligodendrocyte remyelination and for this reason should be used with caution in patients with demyelinating disease.

Animals↗
PubMed2001

Progressive encephalomyelopathy and cerebellar degeneration in 10 captive-bred cheetahs.

Progressive ataxia, with head tremor, developed in 10 captive-born cheetah cubs under six months of age. The condition was usually preceded by coryza and an ocular discharge. Initially the ataxia and weakness affected the hindquarters, then the forelegs, and head tremor developed later. Significant pathological changes were confined to the central nervous system. There was widespread Wallerian degeneration in the funiculi of the spinal cord (except those in the dorsal columns), in the medulla and in the cerebellum. In the cerebellum there was degeneration of Purkinje cells and of the molecular and granular cell layers. There was chromatolysis in the Purkinje cells, the ventral horn cells of the spinal cord and in the neurons of the lateral vestibular nucleus. The olivary nucleus was necrotic. There were foci of inflammatory cells in the molecular layer of the cerebellum and in the medulla. The cause of the disease remains unknown.

Acinonyx↗
PubMed2001

Expression of dominant-negative and chimeric subunits reveals an essential role for beta1 integrin during myelination.

Myelination represents a remarkable example of cell specialization and cell-cell interaction in development. During this process, axons are wrapped by concentric layers of cell membrane derived either from central nervous system (CNS) oligodendrocytes or peripheral nervous system Schwann cells. In the CNS, oligodendrocytes elaborate a membranous extension with an area of more than 1000 times that of the cell body. The mechanisms regulating this change in cell shape remain poorly understood. Signaling mechanisms regulated by cell surface adhesion receptors of the integrin family represent likely candidates. Integrins link the extracellular environment of the cell with both intracellular signaling molecules and the cytoskeleton and have been shown to regulate the activity of GTPases implicated in the control of cell shape. Our previous work has established that oligodendrocytes and their precursors express a limited repertoire of integrins. One of these, the alpha6beta1 laminin receptor, can interact with laminin-2 substrates to enhance oligodendrocyte myelin membrane formation in cell culture. However, these experiments do not address the important question of integrin function during myelination in vivo, nor do they define the respective roles of the alpha and beta subunits in the signaling pathways involved. Here, we use a dominant-negative approach to provide, for the first time, evidence that beta1 integrin function is required for myelination in vivo and use chimeric integrins to dissect apart the roles of the extracellular and cytoplasmic domains of the alpha6 subunit in the signaling pathways of myelination.

Animals↗
PubMed2001

Histological diagnosis of mucopolysaccharidosis IIIA in a wire-haired dachshund.

A four-year-old wire-haired dachshund developed progressive neurological signs of ataxia, intention tremor and finally dysuria. Two years later, histopathology showed that neurons throughout the brain and spinal cord were distended with lipopigment which was also present in macrophages. Ultrastructurally, the pigment in the neurons occurred predominantly as electron-dense membranous whorls and stacks. There were a few vacuolated macrophages in the meninges. Hepatocytes were highly vacuolated and electron microscopy suggested that they were empty membrane-bound vesicles. The disease was diagnosed as mucopolysaccharidosis IIIA because of its similarity to other biochemically confirmed cases in the same breed and in a New Zealand huntaway dog. Additional lesions included calcium oxalate uroliths, severe secondary calcification of tissues including the brain and storage deposits in some neurons, and lesions which may have been associated with high levels of the substrate, heparan sulphate.

Animals↗
PubMed2001

Macrophage depletion impairs oligodendrocyte remyelination following lysolecithin-induced demyelination.

An association between macrophages and remyelination efficiency has been observed in a variety of different models of CNS demyelination. In order to test whether this association is causal or coincidental, we have examined the effects of macrophage depletion on the rate of remyelination of lysolecithin-induced demyelination in the spinal cord of young adult female rats. Macrophage depletion was achieved by reducing the monocyte contribution to the macrophages within the lesion using the clodronate-liposome technique. This technique not only resulted in a decrease in Ox-42-positive cells in the spleen of treated animals but also in the levels of macrophage scavenger receptor type B mRNA expression within the demyelinating lesion. In animals treated with clodronate-liposomes throughout the remyelination process, there was a significant decrease in the extent of oligodendrocyte remyelination at 3 weeks after lesion induction, but no effect on Schwann cell remyelination. If macrophage depletion was delayed until the second half of the remyelination phase, then there was no effect on the repair outcome, implying that macrophages are required for the early stages of CNS remyelination. The results of this study indicate that the macrophage response is an important component of successful CNS remyelination and that approaches to the treatment of demyelinating disease based on inhibition of the inflammatory response may also impair regenerative events that follow demyelination.

Analgesics, Non-Narcotic↗
PubMed2001

SpL201: a conditionally immortalized Schwann cell precursor line that generates myelin.

Dramatic progress has been made over recent years toward the elucidation of the mechanisms regulating lineage determination and cell survival in the developing peripheral nervous system. However, our understanding of Schwann cell development is limited. This is partly due to the difficulties in culturing primary Schwann cell precursor cells, the earliest developmental stage of the Schwann cell lineage defined to date. Both the inability to maintain cultured Schwann cell precursor cells in an undifferentiated state and the technical difficulties involved in their isolation have hampered progress. We have conditionally immortalized rat Schwann cell precursor cells using a retrovirally encoded EGFR/neu fusion protein to circumvent these problems and to generate a source of homogeneous cells. The resulting SpL201 cell line expresses p75 and nestin, two proteins expressed by neural crest-derived cells, as well as peripheral myelin protein 22, protein zero, and Oct-6 as markers of the Schwann cell lineage. When cultured in EGF-containing medium, the SpL201 cells proliferate and maintain an undifferentiated, Schwann cell precursor cell-like state. The cell line is dependent on EGF for survival but can differentiate into early Schwann cell-like cells in response to exogenous factors. Like primary rat Schwann cells, SpL201 cells upregulate Oct-6 and myelin gene expression in response to forskolin treatment. Furthermore, the SpL201 cell line can form myelin in the presence of axons in vitro and is capable of extensively remyelinating a CNS white matter lesion in vivo. Thus, this cell line provides a valuable and unique tool to study the Schwann cell lineage, including differentiation from the Schwann cell precursor cell stage through to myelination.

Animals↗
PubMed2001

SCIP/Oct-6, Krox-20, and desert hedgehog mRNA expression during CNS remyelination by transplanted olfactory ensheathing cells.

Olfactory ensheathing cells (OECs), although having a separate developmental origin to Schwann cells, are able to generate myelin sheaths following transplantation into areas of CNS demyelination that are remarkably similar to those made by Schwann cells. The transcriptional control of Schwann cell myelination has been well documented, in particular the role of SCIP/Oct-6 and Krox-20. It is not known, however, whether these transcription factors are also expressed when OECs assume a myelinating phenotype. In this study, we addressed this question by using a transplantation approach to generate myelinating OECs and then examined the expression of SCIP/Oct-6 and Krox-20 mRNA by in situ hybridization using oligonucleotide probes. We also examined the expression of desert hedgehog (Dhh), a Schwann cell-derived signaling molecule that is responsible for regulating the development of the connective tissue elements in peripheral nerve, which bear similarities to the morphologies adopted by nonmyelinating transplanted cells. Our results indicate that both Krox-20 and Dhh mRNA are strongly expressed by transplanted OECs, with SCIP mRNA present at much lower levels. The expression of Krox-20 relative to the expression of P0 mRNA by the transplanted OECs is consistent with its playing a similar role in OEC myelination to that in Schwann cell myelination, while the expression of Dhh suggests a possible mechanism for the diverse morphologies that cells adopt following OEC transplantation into the damaged CNS. Taken together, our results provide further evidence for the close similarity of OECs and Schwann cells and suggest that, despite their separate origins, the manner in which they generate a peripheral-type myelin sheath involves similar regulatory mechanisms.

Animals↗
PubMed2001

Depletion of endogenous oligodendrocyte progenitors rather than increased availability of survival factors is a likely explanation for enhanced survival of transplanted oligodendrocyte progenitors in X-irradiated compared to normal CNS.

Oligodendrocyte progenitors (OPs) survive and migrate following transplantation into adult rat central nervous system (CNS) exposed to high levels of X-irradiation but fail to do so if they are transplanted into normal adult rat CNS. In the context of developing OP transplantation as a potential therapy for repairing demyelinating diseases it is clearly of some importance to understand what changes have occurred in X-irradiated CNS that permit OP survival. This study addressed two alternative hypotheses. Firstly, X-irradiation causes an increase in the availability of OP survival factors, allowing the CNS to support a greater number of progenitors. Secondly, X-irradiation depletes the endogenous OP population thereby providing vacant niches that can be occupied by transplanted OPs. In situ hybridization was used to examine whether X-irradiation causes an increase in mRNA expression of five known OP survival factors, CNTF, IGF-I, PDGF-A, NT-3 and GGF-2. The levels of expression of these factors at 4 and 10 days following exposure of the adult rat spinal cord to X-irradiation remain the same as the expression levels in normal tissue. Using intravenous injection of horseradish peroxidase, no evidence was found of X-irradiation-induced change in blood-brain barrier permeability that might have exposed X-irradiated tissue to serum-derived survival factors. However, in support of the second hypothesis, a profound X-irradiation-induced decrease in the number of OPs was noted. These data suggest that the increased survival of transplanted OPs in X-irradiated CNS is not a result of the increases in the availability of the OP survival factors examined in this study but rather the depletion of endogenous OPs creating 'space' for transplanted OPs to integrate into the host tissue.

Animals↗
PubMed2001

Dyskinesia in an adult bichon frise.

Dyskinesias are disorders of the central nervous system that result in involuntary movements in a fully conscious individual. This report describes a disorder in a five-year-old male neutered bichon frise characterised by episodic involuntary skeletal muscle activity with normal levels of consciousness that bears some similarity to the previously described movement disorder in boxer puppies and to the human condition descriptively referred to as paroxysmal dystonic choreoathetosis. The disorder was differentiated from partial motor seizure activity by the character of the episodes, absence of identifiable preceding aura, absence of autonomic signs and the fact that multiple limbs were affected in a varying pattern without generalisation and loss of consciousness. Movement disorders are a well documented group of disorders in human neurology, but only rarely described in the veterinary literature. The purpose of this report is to contribute to an increased awareness of movement disorders within veterinary practice.

Animals↗
PubMed2001

Bridging the divide: spinal cord repair by cellular transplantation--from research laboratory to therapeutic application.

Spinal cord injury remains a clinical problem for which new therapeutic options are required. Recent developments in spinal cord injury research have suggested that promotion of axonal regeneration by cellular transplantation may be a feasible future treatment modality and it is possible that small animal clinical patients may be the first to benefit from these new approaches. This article describes the experiments which have generated this optimism and considers the steps that will be required to make the transition from laboratory experiments to clinical application.

Animals↗
PubMed2000

Schwann cell remyelination is restricted to astrocyte-deficient areas after transplantation into demyelinated adult rat brain.

The ability to generate large numbers of Schwann cells from a peripheral nerve biopsy makes them potential candidates for the clinical application of cell transplantation to enhance remyelination in human demyelinating disease. Transplant-derived Schwann cell remyelination has previously been demonstrated in the spinal cord but not for demyelinated axons in the brain, a more likely site for initial clinical intervention. We have transplanted Schwann cells from male neonatal rat sciatic nerves into ethidium bromide-induced areas of demyelination in the deep cerebellar white matter of adult female rats. The extent of Schwann cell remyelination 28 days after transplantation was significantly increased in lesions that received direct injections of Schwann cells compared with non-transplanted lesions. Using in situ hybridisation to identify the rat Y chromosome, transplanted male cells were found to co-localise with the P0 immunoreactive area of Schwann cell remyelination. Combined immunohistochemistry and in situ hybridisation confirmed that many remyelinating Schwann cells were transplant-derived. P0 immunoreactivity and transplanted male cells were found in GFAP-negative, astrocyte-free areas. Transplanted Schwann cells were not identified outside of transplanted lesions, nor did they did not contribute to remyelination of a lesion at a distance from the site of transplantation. Our findings indicate that demyelinated axons in the adult brain can be remyelinated by transplanted Schwann cells but that migration and remyelination are restricted to areas from which astrocytes are absent.

Animals↗
PubMed2000

Remyelinating the demyelinated CNS.

The CNS has an inherent capacity to generate remyelinating cells following episodes of myelin loss. However, persistent demyelination is the major pathology of multiple sclerosis and the leucodystrophies, and is also a feature of spinal cord trauma. There are potentially two approaches for achieving remyelination in situations where it fails; enhancement of the inherent remyelinating capacity of the CNS, or transplantation of an exogenous source of myelin forming cells. In experimental animals it is possible to remyelinate demyelinated CNS axons by transplanting cultures containing central or peripheral myelinogenic cells. Glial cell transplantation may thus provide a therapeutic strategy for remyelinating areas of chronic demyelination as well as for stimulating axon regeneration. This presentation will review four issues that have to be addressed before glial transplantation can be undertaken in humans: is the procedure safe, what cells would be used, where would the cells come from and can we predict how much remyelination will be achieved? It concludes that the most promising approach will be to use multipotent neural precursor cells that have been committed to oligodendrocyte lineage differentiation prior to implantation. However, even with such preparations, which have considerable myelinating potential, the extent of remyelination that would be achieved can not yet be predicted with any degree of certainty.

Animals↗
PubMed2000

Olfactory ensheathing cells and Schwann cells differ in their in vitro interactions with astrocytes.

Transplanted olfactory ensheathing cells (OECs) are able to remyelinate demyelinated axons and support regrowth of transected axons after transplantation into the adult CNS. Transplanted Schwann cells (SCs) share these repair properties but have limitations imposed on their behavior by the presence of astrocytes (ACs). Because OECs exist alongside astrocytes in the olfactory bulb, we have hypothesized that they have advantages over SCs in transplant-mediated CNS repair due to an increased ability to integrate and migrate within an astrocytic environment. In this study, we have tested this hypothesis by comparing the interactions between astrocytes and either SCs or OECs, using a range of in vitro assays. We have shown that (1) astrocytes and SCs segregate into defined non-overlapping domains in co-culture, whereas astrocytes and OECs freely intermingle; (2) both SCs and OECs will migrate across astrocyte monolayers, but only OECs will migrate into an area containing astrocytes; (3) SCs spend less time in contact with astrocytes than do OECs; and (4) astrocytes undergo hypertrophy when in contact with SCs, but not with OECs. Expression of N-cadherin has been implicated as a key mediator of the failure of SCs to integrate with astrocytes. However, we found no differences in the intensity of N-cadherin immunoreactivity between SCs and OECs, suggesting that it is not the adhesion molecule that accounts for the observed differences. In addition, the number of astrocytes expressing chondroitin sulfate proteoglycans (CSPG) is increased when astrocytes are co-cultured with Schwann cells compared with the number when astrocytes are grown alone or with OECs. Taken together, these data support the hypothesis that OECs will integrate more extensively than Schwann cells in astrocytic environments and are therefore better candidates for transplant-mediated repair of the damaged CNS.

Animals↗
PubMed2000

Robust regeneration of CNS axons through a track depleted of CNS glia.

Transected CNS axons do not regenerate spontaneously but may do so if given an appropriate environment through which to grow. Since molecules associated with CNS macroglia are thought to be inhibitory to axon regeneration, we have tested the hypothesis that removing these cell types from an area of brain will leave an environment more permissive for axon regeneration. Adult rats received unilateral knife cuts of the nigrostriatal tract and ethidium bromide (EB) was used to create a lesion devoid of astrocytes, oligodendrocytes, intact myelin sheaths, and NG2 immunoreactive cells from the site of the knife cut to the ipsilateral striatum (a distance of 6 mm). The regenerative response and the EB lesion environment was examined with immunostaining and electron microscopy at different timepoints following surgery. We report that large numbers of dopaminergic nigral axons regenerated for over 4 mm through EB lesions. At 4 days postlesion dopaminergic sprouting was maximal and the axon growth front had reached the striatum, but there was no additional growth into the striatum after 7 days. Regenerating axons did not leave the EB lesion to form terminals in the striatum, there was no recovery of function, and the end of axon growth correlated with increasing glial immunoreactivity around the EB lesion. We conclude that the removal of CNS glia promotes robust axon regeneration but that this becomes limited by the reappearance of nonpermissive CNS glia. These results suggest, first, that control of the glial reaction is likely to be an important feature in brain repair and, second, that reports of axon regeneration must be interpreted with caution since extensive regeneration can occur simply as a result of a major glia-depleting lesion, rather than as the result of some other specific intervention.

Animals↗
PubMed2000

Schwann cells transplanted into normal and X-irradiated adult white matter do not migrate extensively and show poor long-term survival.

Although Schwann cells are able to enter the central nervous system (CNS) when the integrity of the glia limitans is disrupted, their ability to migrate through intact CNS remains unclear. We have addressed this issue by transplanting lacZ-labeled Schwann cells into normal adult spinal cord white matter, and into X-irradiated spinal cord (an environment that, unlike normal spinal cord, permits the migration of transplanted oligodendrocyte progenitors). Schwann cell cultures, obtained from neonatal rat sciatic nerve and expanded using bovine pituitary extract and forskolin, were transfected by repeated exposure to retroviral vectors encoding the Escherichia coli lacZ gene. The normal behavior of the transduced cells was confirmed by transplantation into a nonrepairing area of demyelination in the spinal cord, where they formed myelin sheaths around demyelinated axons. A single microliter containing 4 x 10(4) cells was then transplanted into unlesioned normal and X-irradiated white matter of the spinal cord of adult syngeneic rats. One hour after injection, blue cells were observed as a discrete mass within the dorsal funiculus with a longitudinal distribution of 2-3 mm, indicating the extent of passive spread of the injected cells. At subsequent survival times (1, 2, and 4 weeks posttransplantation) blue cells had a distribution that was no more extensive than that seen 1 h after transplantation. However, the number of Schwann cells declined with time following transplantation such that at 4 weeks there were few surviving Schwann cells in both X-irradiated and nonirradiated spinal cord. These results indicate that transplanted Schwann cells do not migrate extensively and show poor long-term survival when introduced into a normal CNS environment.

Animals↗
PubMed2000

Delayed changes in growth factor gene expression during slow remyelination in the CNS of aged rats.

In this study we have examined whether the slower rate of CNS remyelination that occurs with age is associated with a change in growth factor expression patterns, an association that would provide further support for a causal relationship between growth factors and remyelination. Using quantitative in situ hybridization we have shown that there are differences in IGF-I, TGF-beta 1, and PDGF-A mRNA expression during remyelination of lysolecithin-induced demyelination in the spinal cord of young adult and old adult rats. IGF-I and TGF-beta1 mRNA expression in old rats had a delayed and lower peak expression compared to young rats. The initial increase in PDGF-A mRNA expression was delayed in old rats compared to young rats, but after 5 days both age groups had similar patterns of expression, as was the expression pattern of FGF-2 mRNA at all survival times. In neither age group were increases in CNTF, NT-3, or GGF-2 mRNA expression detected. An analysis of the macrophage response using oligonucleotide probes for scavenger receptor-B mRNA indicated that differences in the macrophage response in young and old animals was the likely cause of the age related change in IGF-I and TGF-beta 1 mRNA expression patterns. On the basis of these data we suggest a model of remyelination in which PDGF is involved in the initial phase of oligodendrocyte progenitor recruitment, while IGF-I and TGF-beta 1 trigger the differentiation of the recruited cells into myelinating oligodendrocytes.

Aging↗
PubMed2000

GM1-gangliosidosis in a cross-bred dog confirmed by detection of GM1-ganglioside using electrospray ionisation-tandem mass spectrometry.

The post-mortem diagnosis of lysosomal storage diseases can be confounded by the unavailability of suitable material. Here we report the diagnosis of GM1-gangliosidosis in a cross-bred dog, from which only formalin-fixed brain was available, by a combination of electron microscopy and the detection of elevated levels of GM1-ganglioside within the tissue using the novel technique of electrospray ionisation tandem mass spectrometry. Electron microscopic examination of ultrathin sections of resin-embedded tissue revealed cytoplasmic inclusions (membranous cytoplasmic and zebra bodies) in brain stem and cerebellar neurons that were characteristic of a gangliosidosis. Glycolipids were extracted from the fixed tissue and analysed by tandem mass spectrometry. Two major ions were detected, which corresponded to GM1 (d18:1-C18:0) and Gm1 (d20:1-C18:0). Their identity was confirmed by comparison of their fragmentation patterns with those of authentic standards. The concentration of GM1 was approximately sixfold higher on a wet weight basis than in the brain of a normal control dog, confirming the diagnosis of GM1-gangliosidosis.

Animals↗