Divya M. Chari

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

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

Modelling large areas of demyelination in the rat reveals the potential and possible limitations of transplanted glial cells for remyelination in the CNS

Transplantation of myelin‐forming glial cells may provide a means of achieving remyelination in situations in which endogenous remyelination fails. For this type of cell therapy to be successful, cells will have to migrate long distances in normal tissue and within areas of demyelination. In this study, 40 Gy of X‐irradiation was used to deplete tissue of endogenous oligodendrocyte progenitors (OPCs). By transplanting neonatal OPCs into OPC‐depleted tissue, we were able to examine the speed with which neonatal OPCs repopulate OPC‐depleted tissue. Using antibodies to NG‐2 proteoglycan and in situ hybridisation to detect platelet‐derived growth factor alpha‐receptor Rα (PDGFRα) mRNA to visualise OPCs, we were able to show that neonatal OPCs repopulate OPC‐depleted normal tissue 3–5 times more rapidly than endogenous OPCs. Transplanted neonatal OPCs restore OPC densities to near‐normal values and when demyelinating lesions were made in tissue into which transplanted OPCs had been incorporated 1 month previously, we were able to show that the transplanted cells retain a robust ability to remyelinate axons after their integration into host tissue. In order to model the situation that would exist in a large OPC‐depleted area of demyelination such as may occur in humans; we depleted tissue of its endogenous OPC population and placed focal demyelinating lesions at a distance (≤1 cm) from a source of neonatal OPCs. In this situation, cells would have to repopulate depleted tissue in order to reach the area of demyelination. As the repopulation process would take time, this model allowed us to examine the consequences of delaying the interaction between OPCs and demyelinated axons on remyelination. Using this approach, we have obtained data that suggest that delaying the time of the interaction between OPCs and demyelinated axons restricts the expression of the remyelinating potential of transplanted OPCs. GLIA 38:155–168, 2002.

Remyelination in multiple sclerosis.

Remyelination is the phenomenon by which new myelin sheaths are generated around axons in the adult central nervous system (CNS). This follows the pathological loss of myelin in diseases like multiple sclerosis (MS). Remyelination can restore conduction properties to axons (thereby restoring neurological function) and is increasingly believed to exert a neuroprotective role on axons. Remyelination occurs in many MS lesions but becomes increasingly incomplete/inadequate and eventually fails in the majority of lesions and patients. Efforts to understand the causes for this failure of regeneration have fueled research into the biology of remyelination and the complex, interdependent cellular and molecular factors that regulate this process. Examination of the mechanisms of repair of experimental lesions has demonstrated that remyelination occurs in two major phases. The first consists of colonization of lesions by oligodendrocyte progenitor cells (OPCs), the second the differentiation of OPCs into myelinating oligodendrocytes that contact demyelinated axons to generate functional myelin sheaths. Several intracellular and extracellular molecules have been identified that mediate these two phases of repair. Theoretically, the repair of demyelinating lesions can be promoted by enhancing the intrinsic repair process (by providing one or more remyelination‐enhancing factors or via immunoglobulin therapy). Alternatively, endogenous repair can be bypassed by introducing myelinogenic cells into demyelinated areas; several cellular candidates have been identified that can mediate repair of experimental demyelinating lesions. Future challenges confronting therapeutic strategies to enhance remyelination will involve the translation of findings from basic science to clinical demyelinating disease.

Corticosteroids delay remyelination of experimental demyelination in the rodent central nervous system

High dose corticosteroid (CS) administration is a common mode of therapy in treatment of acute relapses in multiple sclerosis (MS) but the effects of CS on remyelination and the cellular mechanisms mediating this repair process are controversial. We have examined CS effects on repair of toxin‐induced demyelinating lesions in the adult rat spinal cord. Corticosteroids reduced the extent of oligodendrocyte remyelination at 1 month post lesion (whereas Schwann‐cell mediated repair was unaffected). However, CS did not cause permanent impairment of remyelination as lesions were fully remyelinated at 2 months after cessation of treatment. The delay in oligodendrocyte mediated repair could be attributed to inhibition of differentiation of oligodendrocyte progenitor cells (OPCs) into oligodendrocytes, with no effect of CS treatment observed on OPC colonisation of the lesions. No differences were observed in animals treated with methylprednisolone succinate alone or with a subsequent prednisone taper indicating that CS effects occur at an early stage of repair. The potential consequences of delayed remyelination in inflammatory lesions are discussed.

New insights into remyelination failure in multiple sclerosis: implications for glial cell transplantation.

This review considers aspects of remyelination that require further clarification if successful strategies are to be devised to enhance remyelination in multiple sclerosis (MS). We speculate, based on our understanding of the rate with which oligodendrocyte progenitor cells (OPCs) repopulate OPC-depleted tissue in adult rats, that OPC depletion during the demyelination process could explain why remyelination fails in MS. We show that loss of OPCs in the context of large areas of demyelination would have serious consequences for remyelination as the rates of colonization of tissue by adult OPCs would lead to a situation where the cellular events associated with demyelination become uncoupled from the interaction of OPCs with demyelinated axons. Experimental studies indicate that transplanted neonatal OPCs would be able to repopulate large areas of demyelination with much greater efficiency than endogenous OPCs. This suggests that cell transplantation will have considerable potential to achieve remyelination in situations where the endogenous repair process is failing due to concurrent death of oligodendrocytes and OPCs. However, we suggest that for this approach to be effective, it will be critical that the environment is permissive for remyelination.

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.

Robust Uptake of Magnetic Nanoparticles (MNPs) by Central Nervous System (CNS) Microglia: Implications for Particle Uptake in Mixed Neural Cell Populations

Magnetic nanoparticles (MNPs) are important contrast agents used to monitor a range of neuropathological processes; microglial cells significantly contribute to MNP uptake in sites of pathology. Microglial activation occurs following most CNS pathologies but it is not known if such activation alters MNP uptake, intracellular processing and toxicity. We assessed these parameters in microglial cultures with and without experimental ‘activation’. Microglia showed rapid and extensive MNP uptake under basal conditions with no changes found following activation; significant microglial toxicity was observed at higher particle concentrations. Based on our findings, we suggest that avid MNP uptake by endogenous CNS microglia could significantly limit uptake by other cellular subtypes in mixed neural cell populations.

Differences in magnetic particle uptake by CNS neuroglial subclasses: implications for neural tissue engineering

AIM To analyze magnetic particle uptake and intracellular processing by the four main non-neuronal subclasses of the CNS: oligodendrocyte precursor cells; oligodendrocytes; astrocytes; and microglia. MATERIALS & METHODS Magnetic particle uptake and processing were studied in rat oligodendrocyte precursor cells and oligodendrocytes using fluorescence and transmission electron microscopy, and the results collated with previous data from rat microglia and astrocyte studies. All cells were derived from primary mixed glial cultures. RESULTS Significant intercellular differences were observed between glial subtypes: microglia demonstrate the most rapid/extensive particle uptake, followed by astrocytes, with oligodendrocyte precursor cells and oligodendrocytes showing significantly lower uptake. Ultrastructural analyses suggest that magnetic particles are extensively degraded in microglia, but relatively stable in other cells. CONCLUSION Intercellular differences in particle uptake and handling exist between the major neuroglial subtypes. This has important implications for the utility of the magnetic particle platform for neurobiological applications including genetic modification, transplant cell labeling and biomolecule delivery to mixed CNS cell populations.

Dysfunctional oligodendrocyte progenitor cell (OPC) populations may inhibit repopulation of OPC depleted tissue

We have attempted to extend a previously described rat model of focal oligodendrocyte progenitor cell (OPC) depletion, using 40 Gy X‐irradiation (Chari and Blakemore [2002] Glia 37:307–313), to the adult mouse spinal cord, to examine the ability of OPCs present in adjacent normal areas to colonise areas of progenitor depletion. In contrast to rat, OPCs in the mouse spinal cord appeared to be a comparatively radiation‐resistant population, as 30–35% of OPCs survived in X‐irradiated tissue (whereas <1% of OPCs survive in X‐irradiated rat spinal cord). The numbers of surviving OPCs remained constant with time indicating that this population was incapable of regenerating itself in response to OPC loss. Additionally, these OPCs did not contribute to remyelination of axons when demyelinating lesions were placed in X‐irradiated tissue, suggesting that the surviving cells are functionally impaired. Importantly, the length of the OPC‐depleted area did not diminish with time, as would be expected if progressive repopulation of OPC‐depleted areas by OPCs from normal areas was occurring. Our findings therefore raise the possibility that the presence of a residual dysfunctional OPC population may inhibit colonisation of such areas by normal OPCs.

Uptake of systemically administered magnetic nanoparticles (MNPs) in areas of experimental spinal cord injury (SCI)

The regenerative potential of the adult central nervous system (CNS) is limited, contributing to poor recovery from neurological insult. Many genes have been identified that promote neural regeneration, but the current viral methods used to mediate neural gene transfer have a range of drawbacks, notably safety. Non‐viral magnetic nanoparticle (MNP)‐based vector systems offer significant advantages over viral systems, including: (a) safety; (b) flexibility of functionalization with genetic material; (c) potential for non‐invasive magnetic targeting; and (d) imaging potential. The applications of such a system to promote intrinsic neural regeneration have not been assessed. We examined uptake of intravenously administered MNPs (diameter, 320 nm) into areas of experimental rodent spinal cord injury (SCI), using a transection model. We found focal uptake of MNPs in areas of SCI associated with breakdown of the blood–brain barrier (BBB) within 6 h of injury; a spatial association was observed between MNPs and nuclei in lesions, suggesting that particle uptake was occurring in cells within injury sites. Our data suggest that there may be a ‘therapeutic window of opportunity’ during post‐injury BBB compromise within which MNPs can be used to mediate gene transfer to sites of spinal cord trauma. Taking into account the relatively superficial anatomical location of the spinal cord, these findings also raise the possibility that the spinal cord could be an attractive target for MNP‐based delivery of biomolecules, particularly when combined with magnetic targeting strategies. We discuss factors that will need to be addressed in order to optimize such an approach. Copyright