PROJECT SUMMARY The neonatal brain is very vulnerable to compromises in its blood supply. Cerebral vascular dysfunction is an important component in the development of neonatal brain injury. Neonatal cerebrovascular disease caused by oxidative stress during seizures, hypoxia/asphyxia, and ischemia frequently leads to lifelong neurological complications in babies. Maintaining astrocyte functionality is a key component in neuroprotection via cerebral blood flow regulation, blood-brain barrier (BBB) integrity, and glutamate clearance from the brain extracellular space. The project is focused on novel endogenous mechanisms that promote astrocyte survival during oxidative stress caused by seizures and glutamate excitotoxicity in the neonatal brain. Our preliminary data in newborn piglet brain suggest that hydrogen sulfide (H2S), a gasotransmitter produced by cortical astrocytes or released from the donor molecules, exhibits antioxidant and antiapopototic properties. We hypothesize that H2S preserves astrocyte functions during glutamate excitotoxicity in the neonatal brain. We will test three specific hypotheses using intact newborn pigs and freshly isolated and cultured astrocytes: 1) Astrocyte H2S production is activated during oxidative stress; 2) H2S protects astrocytes against excitotoxicity via mitochondrial targeting and mTORC1 signaling; 3) H2S preserves astrocyte functionality during neonatal seizures and glutamate excitotoxicity. We will use an exceptional combination of complementary techniques in a large animal model that allows in vivo and in vitro investigation of newborn circulation. Such research is unique, as it combines functional and mechanistic studies in intact cerebral circulation with investigation of the cellular and molecular defense mechanisms counteracting astrocyte injury. Gene silencing and pharmacological approaches will be used to pinpoint causative relationships among H2S, glutamate excitotoxicity, oxidative stress, and astrocyte functions. The project will lead to the development of novel translationally relevant treatments to provide neuroprotection to the neonatal brain during seizures. Importantly, we have collected sufficient preliminary data to demonstrate the feasibility of our proposal and to support our hypothesis on critical role of H2S in astrocyte survival during oxidative stress in the neonatal brain.