Project 3 proposes to explore whether murine ES cells, induced down a neuroglial lineage by ex vivo pharmacological and/or genetic manipulation, can survive transplantation into the syrinx or a contused adult rat spinal cord; differentiate into neurons, oligodendrocytes. contused adult rat spinal cord; differentiate into neurons, oligodendrocytes, and astrocytes functional recovery. The particular focus of this grant is to understand the mechanisms of ES cell death following transplantation into the syrinx (9 days following contusion) and to reduce death by protective mechanisms, both pharmacologically and genetically based. Preliminary studies, while suggesting glial (predominantly) and neuronal (minor amounts) differentiation by the cells following engraftment into a contusion- induced syrinx, also demonstrated that substantial donor ES cell death occurred after transplantation. The investigators hypothesize that this cell death is likely limiting functional benefit from the engraftment procedure. Therefore, they propose how to investigate minimizing that death in situ, particularly death mediated by apoptotic (predominantly in the first post traumatic 48 hours) and excitotoxic (predominantly after the first 2 post-injury days) mechanisms, by specific pharmacological and genetic interventions speculating that such an outcome will optimize functional recovery. Genetic manipulations (e.g., over-expression of bcl-2 or deletion of Bax or caspase 9 genes) will be created in the ES Cell Genetic Engineering Core B; pharmacological approaches will be derived from the studies performed in Projects I & II (e.g., the pan-caspase inhibitor ZVAD or the glutamate receptor antagonist MK-801). The survival and differentiation fate of donor ES cells (identified by a number of marking techniques, including a lacZ transgene intrinsic to the cell or to cell type- specific structures, BrdU pre-incubation of donor cells, non-diffusible fluorescent cytoplasmic dyes, mouse-specific chromosomal sequences or cell surface markers that will not cross-react with rat host cells) will be assessed quantitatively at defined intervals after transplantation. The anatomical, physiological, and functional impact of ES transplantation will be determined in conjunction with Project IV (""""""""Integration of transplanted ESNLCs in spinal circuits) and the """"""""Injury and functional assessment core"""""""".
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