The mature central nervous system contains a significant number of immature cells identified by the surface antigen ganglioside GD3. Most of these cells, when isolated and cultured, differentiate to become astrocytes, although some may become oligodendrocytes. The hypotheses to be tested are: that such precursor cells represent the small population of proliferating cells found in adult brain, and that they serve as a source of newly generated astrocytes and oligodendrocytes that appear following traumatic brain lesions and demyelinating events.
The specific aims designed to accomplish these goals are: (1) To determine the phenotype of proliferating cells in the normal adult rat CNS. (2) To determine the lineage of proliferating cells in the adult rat CNS following excision lesions and stab wounds. (3) To determine the lineage of proliferating cells in demyelinating and remyelinating lesions in the CNS of guinea pigs with chronic-relapsing experimental autoimmune encephalomyelitis (CREAE). The general experimental design is to identify proliferating cells in tissue sections by either 3H-thymidine incorporation or by the presence of proliferating cell nuclear antigen, a cyclin expressed only in Gl and S phase. In lesioned animals and in CREAE BrdU incorporation will also be used. The phenotype of such cells will then be determined immunohistochemically using stage-specific markers. Besides GD3, such markers will include vimentin, for precursor cells and immature astrocytes, antigen 04, for cells of the oligodendrocyte lineage, GFAP, for differentiated astrocytes, and galactosylceramide and myelin basic protein, for differentiated oligodendrocytes. In addition clonal analysis will be employed with lesioned animals. The BAG retrovirus, carrying the reporter gene, beta-galactosidase, will be injected into the brain at the site of lesion. Animals sacrificed at various times will be processed for immunohistochemistry and X-gal staining to identify the clonal type of cells proliferating in the lesion. Similar procedures will be used on sections of spinal cords of guinea pigs in different stages of CREAE, and in CREAE treated to prevent relapses, to identify the phenotypes of proliferating cells. The results of the proper application of these procedures will provide answers to the following questions. Are proliferating cells in normal and experimental animals solely immature precursors or can mature, differentiated cells also be induced to enter the cell cycle? Do the precursor cells generate astrocytes or oligodendrocytes or both? Are the increased numbers of oligodendrocytes in remyelinating lesions the result of cell proliferation of precursor cells or mature cells? Or do they appear through another mechanism, such as migration of non-proliferating differentiated cells? The health-related aspects of this work stem from the possibility that oligodendrocytes can be replenished in the adult for remyelination in demyelineated lesions such as occur in multiple sclerosis.
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