Neural stem cells (NSCs) represent a potential opportunity in the search for a cure of spinal cord injury (SCI), but mechanisms underlying NSC survival at injury sites remain unclear. Previous records showed that majority of NSCs implanted immediately after SCI die within one week before the onset of immunorejection. It is consequently crucial to understand the events that tiggers the early demise of NSCs. Secondary injury factors, such as excitotoxicity, cytokines, protein peroxidization and radical damage, have been speculated as the cause of acute death of NSC grafts. Unlike neurons, NSCs do not express the receptors (e.g., NMDA receptors, etc.) necessary for mediating lethal signals, suggesting an alternate mechanisms that do not involve membrane receptors. Among the possible candidates, nitric oxide (NO) is a highly conserved signal molecule. While NO is a major modulator of neurogenesis and NSC differentiation, excessive NO production has been shown to result in secondary injury processes after SCI including caspase activation and apoptosis. Therefore, we hypothesize that NO and its associated radical species, such as peroxynitrite, may trigger death of donor NSCs in SCI. Using our well established in vitro and in vivo systems, we aim to define the role of NO on the death, survival and differentiation of human NSC (hNSC) grafts in the injured spinal cord. In vitro studies such as apoptosis assays for hNSCs incubated with NO donors, and Western Blot analysis of NO mediated cytochrome c release and caspase activation will be performed to provide guidelines for constructing a retrievable biodegradable PLGA polymer scaffold to be used as hNSC implanting and host microenvironment reporting vehicles (Specific Aims I & II). Donors or scavengers of the reactive nitrogen species radicals embedded in these scaffolds will allow us to study the cellular response of hNSC grafts in a rat hemisection SCI model (Specific Aim III), as well as other neurological disorders in the future. If identified signaling pathways of NO-induced hNSC death may be used as targets for the development of new therapeutic strategies to enhance the efficacy of NSC-based SCI therapies. (For public statement: We will investigate the factors that hurt the survival of injected neural stem cells in the injured spinal cord. Since most stem cells die after injection, scientists and doctors must understand the mechanism of post-injection stem cell death before the stem cell technology can be used to treat spinal cord injury). [unreadable] [unreadable] [unreadable]