Hypoxia is a major cause of fetal brain injury, including cerebral palsy (CP). Recent reports suggest a role for Rho/Rho-kinase in neuronal degeneration, and our laboratory has shown that chronic hypoxia-induced fetal brain injury is associated with Rho family gene activation. The mechanisms underlying these fetal specific hypoxia-triggered processes are poorly understood. We hypothesize that chronic hypoxia, beginning preterm, causes fetal brain injury via activation of Rho/Rho-kinase through the nitric oxide (NO)-mitogen activated protein kinase (MAPKs) signaling pathway, and is an example of an adaptive response that, under common intrauterine/ intrapartum conditions, becomes maladaptive. We use a chronic hypoxia fetal mouse model and hypoxic primary cell cultures plus a ROCK Knock-out mice model to test the hypothesis in experiments performed under 3 specific aims: SPECIFIC AIM 1: Test the hypothesis that chronic hypoxia-induced brain injury is associated with Rho/Rho- kinase activation in a cell-specific manner. A fetal chronic hypoxia mouse will be generated, chronic hypoxia signaling cascade genes (Rho family genes, Rho kinases, NOSs, MAPKs, ICAM-1, NFAT) will be studied at two time points(10.5%O2-HPX-5d, and 10.5%O2-HPX-newborn). Co-localization of Rho genes and cell types (neuron or glial) will be studied by double-fluorescent immunostaining. In vivo studies will be confirmed by hypoxia primary cell models (neuron and glial) (1%O2 for 12 hours). SPECIFIC AIM 2: Test the hypothesis in primary cell cultures that chronic hypoxia activates Rho/Rho Kinase pathway through the NO-MAPK pathway, causing glial and neuronal injury in a cell-specific manner. Primary mouse neuron and glial cells will be cultured at 1%O2 for 12 hours. The mechanism of Rho/Rho kinase and its signaling cascade regulation mediated by hypoxia will be studied by inhibition/activation of these signaling elements (Rho family genes, Rho-kinase, NOS isoforms, MAPKs), respectively. Alternative mediators (ICAM-1, NFAT) mediated by Rho will be studied initially in hypoxia cell models. SPECIFIC AIM 3: Test the hypothesis that Rho is the key initiator in the chronic hypoxia signaling cascade by using a ROCK knock-out model. We will study chronic hypoxia-signaling mediators (Rho family genes, Rho kinase, total NO, NOSs, NO activity, MAPKs, cellular adhesion marker (ICAM-1), cytoskeletal regulator (NFAT) and fetal brain injury indices, using genome engineered mice to confirm the important role Rho/Rho-kinase plays in fetal brain injury. Chronic fetal hypoxia is a major risk for brain injury by mechanisms that are still poorly understood. These experiments will increase knowledge of mediators and interactions which contribute to fetal brain injury associated with chronic hypoxia, will expand applications of the core hypoxia animal model, and potentially lead to therapeutic interventions that ameliorate the sequelae of poor placental implantation. PUBLIC HEALTH RELEVANCE: In preliminary study, we find that chronic hypoxia triggers Rho/Rho-kinase in several mammals. We propose to test the hypothesis in the mouse that chronic hypoxia, beginning preterm, triggers fetal brain injury by up- regulating MAPKs-mediated release of NO via Rho/Rho-kinase activation. The study will increase knowledge of novel mediators and their interactions during fetal chronic hypoxia.