Since sufficient remyelination results in prolonged neurological impairment in demyelinating disease states, such as multiple sclerosis (MS), it is important to discern the cellular and molecular mechanisms that bring about remyelination. The proposed experiments will identify molecules that can regulate repopulation of demyelinated lesions by surviving and/or newly generated oligodendrocyte lineage cells (OLCs). The long term objective of this work is to provide the information necessary to design treatments to optimize functional recovery in patients with demyelinating diseases after the disease process is abated. To examine successful remyelination, the proposed studies will use two established methods of demyelination of murine spinal cord, lysolecithin (LPC) injection and coronavirus (MHV-A59) infection. Both demyelination models result in extensive remyelination but each model is specifically suited for different proposed experiments. Initially, sections of LPC- demyelinated areas will be examined by immunohistochemistry and in situ hybridization to identify putative OLC mitogens or chemoattractants that are increased in response to demyelination. The expression of the corresponding receptors will be examined in proximate tissue sections. In addition, immunohistochemical detection of proliferating cell nuclear antigen will assess the proliferative response to demyelination. In each case, the analysis will be combined with cell type-specific immunochemical markers to identify the cell type(s) synthesizing the ligand and the OLC type(s) expressing the corresponding receptor and/or proliferating. The potential function of the relevant growth factors will be determined using 3 in vitro bioassays to distinguish mitogenic and chemoattractant responses of OLCs from remyelinating CNS. OLCs will be cultured, from spinal cords of MHV-infected mice during early remyelination, and mitogenic effects of exogenous growth factors will be tested using a bromodeoxyuridine incorporation assay. Soluble factors will be screened in a microchemotaxis chamber for the ability to induce directed migration of similarly prepared OLCs. Membrane-bound factors and cell adhesion molecules will also be examined for their potential to modulate OLC migration using explants of LPC-demyelinated spinal cord cultured on tissue sections of demyelinated or normal spinal cord. The proposed experiments will identify potential mitogens and chemoattractants for remyelinating OLCs. These studies will also reveal the cells that have active roles in remyelination, i.e. cells in a lesion that produce relevant growth factors and those that respond by repopulating the lesion.
