Strategies that may be effective for promoting regeneration of axons in the injured nervous system have been largely elusive. Recently, several lines of evidence suggest that a specific population of glia cells, ensheathing cells, may be uniquely effective in mediating axon regeneration. However, there is still relatively little known of these cells; the parameters that define their support of axon extension and the mechanisms that underlie that support remain speculative. To fill this gap in our knowledge and, specifically, to begin to explore these cells as an exciting and novel mechanisms for promoting recovery from spinal cord injury we propose to address the following questions: 1) Do neurites from DRG cells grow more effective on ensheathing cell substrates on other glial populations? We will compare neurite extension from primary cultures of rat DRG neurons plated on ensheathing cells compared with central astroglia and peripheral Schwann cells. To determine if diffusible substances are involved, we will also test neurite behavior in conditioned media that do not offer contact between the DRG neurons and glial substrate. We will compare axon extension from DRGs in vitro in the presence of blockers of ensheathing cell associated molecules that have been hypothesized to mediate axon growth. Although there are additional molecular differences, pivotal differences between astrocytes and ensheathing cells is the expression of the low affinity nerve growth factor receptor, PSA-NCAM and laminin. If these are blocked, are the favorable effects of ensheathing cells on neurite extension changed? Astrocytes are believed to influence neuronal function, in part, through the release of glutamate as well as by controlling extracellular concentrations of glutamate. Do ensheathing cells communicate with growing neurites and, are the dynamics of communication for different glial substrates equivalent? What are the effects of blocking glutamate-mediated mechanisms on neurite extension? 2) Do suspensions of ensheathing cells implanted into transected spinal cord promote axon extension through the lesion? We will compare axons extension through a transection site following infections of ensheathing cell versus Schwann cell suspensions. To address further the parameters that define the effects of ensheathing cells in vivo, we will vary the number of ensheathing cells implanted, the age of the ensheathing cells and the interval between injury and implantation. We will assess the functional status with electrically evoked responses proximal and distal, to transection site at different timepoints following the introduction of the cell suspensions. Together, these experiments will constitute a good test of the hypothesis that ensheathing cells are effective promoters of axon regeneration and, the molecular mechanisms that may underlie their effects.
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