Decline in rate of colonization of oligodendrocyte progenitor cell (OPC)-depleted tissue by adult OPCs with age.

Rates of remyelination decline with age and this has been attributed to slower recruitment of oligodendrocyte progenitor cells (OPCs) into areas of demyelination and slower differentiation of OPCs into remyelinating oligodendrocytes. When considering causes for reduced recruitment rates, intrinsic causes (alterations in biological properties of OPCs) need to be separated from extrinsic causes (age-related differences in the lesion environment). Using 40 Gy of X-irradiation to deplete tissue of its endogenous OPC-population, we examined the effects of age on the rate at which adult rat OPCs colonize OPC-depleted tissue. We found a significant reduction in the rate of colonization between 2 and 10 months of age (0.6 mm/week versus 0.38 mm/week). To determine if this represented an intrinsic property of OPCs or was due to changes in the environment that the cells were recolonizing, OPCs from 10-month-old animals were transplanted into 2-month-old hosts and OPCs from 2-month-old animals were transplanted into 10-month-old hosts. These experiments showed that the transplanted OPCs retained their age-related rate of colonization, indicating that the decline in colonizing rates of OPCs with age reflects an intrinsic property of OPCs. This age-related decline in the ability of OPCs to repopulate OPC-depleted tissue has implications for understanding remyelination failure in multiple sclerosis (MS) and developing therapies for remyelination failure.

Effect of application of gamma amino butyric acid at the medial preoptic area on sleep-wakefulness.

Intracerebral microinjections of gamma amino butyric acid were given bilaterally at the medial preoptic area (mPOA) to determine the possible role of this neurotransmitter in the genesis and regulation of sleep-wakefulness. GABA (50 micrograms/0.2 microliters) when administered through chronically implanted cannulae in free moving rats, did not produce any significant alterations in sleep-wakefulness. This may be attributed either to the non-involvement of GABA at the level of mPOA in the regulation of sleep, or to other factors like the low dose and rapid breakdown of the injected drug.

A multicellular, neuro-mimetic model to study nanoparticle uptake in cells of the central nervous system.

Evaluating the uptake and handling of biomedically relevant nanoparticles by cells of the nervous system critically underpins the effective use of nanoparticle platforms for neuro-regenerative therapies. The lack of biologically relevant and 'neuromimetic' models for nanomaterials testing (that can simulate the cellular complexity of neural tissue) currently represents a bottleneck. Further, propagation of individual cell types, in different neural cell-specific media (as commonly occurs in the nanotechnology field), can result in non-standardised corona formation around particles, confounding analyses of intercellular differences between neural cells in nanoparticle uptake. To address these challenges, we have developed a facile multicellular model that broadly simulates the ratios of neurons, astrocytes and oligodendrocytes found in vivo. All cell types in the model are derived from a single neural stem cell source, and propagated in the same medium overcoming the issue of variant corona formation. Using a fluorescent transfection-grade magnetic particle (MP), we demonstrate dramatic differences in particle uptake and resultant gene transfer between neural cell subtypes, with astrocytes being the dominant population in terms of particle uptake and transfection. We demonstrate the compatibility of the model with a high resolution scanning electron microscopy technique, allowing for membrane features of MP stimulated cells to be examined. Using this approach, astrocytes displayed high membrane activity in line with extensive particle uptake/transfection, relative to neurons and oligodendrocytes. We consider that the stem cell based model described here can provide a simple and versatile tool to evaluate interactions of neural cells with nanoparticle systems developed for neurological applications. Models of greater complexity can be evolved from this basic system, to further enhance its neuromimetic capacity.

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.

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 oligodendroytes and OPCs. However, we suggest that for this approach to be effective, it will be critical that the environment is permissive for remyelination.

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.

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 Ralpha (PDGFRalpha) 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 (< or =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.

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.