This is a competitive renewal application that has been revised in response to all the critiques of previous reviewers and includes new preliminary data. Perinatal hypoxic-ischemic brain injuries are an important cause of cerebral palsy. To understand the mechanisms for these brain injuries and develop neuroprotective strategies, we use rodent models in which one carotid artery is ligated at around 7 days of age followed by exposure to a period of hypoxia. Hypoxia-ischemia leads to NMDA receptor-channel opening and activation of neuronal nitric oxide synthase, which synthesizes nitric oxide leading to injury from peroxynitrite. During the last period of funding, we hypothesized that these events lead to brain injury in the neonate by activating poly (ADP-ribose) polymerase-1 (PARP 1). We established a model of unilateral carotid artery ligation plus hypoxia in mice, and used this model to determine if knockout (KO) of the (Parp 1) gene reduced damage. We discovered that Parp 1 KO produced a gene dose-dependent reduction in damage in male, but not female mice. The gender dependent difference we found in neonatal mice has also been confirmed by others in adult rodents. For the next period of support, we propose to focus on the signaling pathways involved in cell death from hypoxia-ischemia and the mechanisms for the gender difference in effect of Parp 1 KO. Recent evidence from neonatal rodent and in vitro models indicates that cell death pathways are sexually dimorphic. We will examine gender differences in the intracellular translocation of apoptosis inducing factor (AIF) and cytochrome C, as well as activation of caspases in male versus female mice that are wild type (wt) vs. KO for Parp 1 and exposed to hypoxia-ischemia. We also plan to examine gender- specific differences in cell death pathways in vitro in cultured cerebellar granule and hippocampal neurons taken from these groups of mice in the neonatal period. This will provide confirmation of the data from mice, and will also allow measurement of mitochondrial membrane potential and levels of ATP, NAD+, and glutathione. Gender dependent differences in vulnerability to NMDA, AMPA and Fas receptor stimulation will also be assessed in vitro. In a third set of experiments, we will determine if female neurons that are not protected by Parp 1 KO can be rescued by treatment with EPO or fibroblast growth factor-1 (FGF-1). We hypothesize that these substances will activate protective pathways in both males and females. These studies are directly relevant to previously reported gender differences in the outcome of perinatal brain injuries in human infants, as well as to the development of clinical therapies to salvage brain tissue in these infants. Detailed information about the molecular pathways that mediate cell death from hypoxia-ischemia is critically important for developing and testing neuroprotective interventions.
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