The research proposed is designed to address the unifying hypothesis that predominant prostanoids involved in control of the newborn cerebral microcirculation are synthesized by the endothelium. To test this hypothesis, three specific aims will be pursued using newborn pigs: 1) determine, in vivo, the affect of endothelial injury on prostanoid-dependent and prostanoid-independent cerebral microvascular responses, and 2) characterize, in vitro, prostanoid synthesis by cerebral cells stimulated by treatments that produce prostanoid dependent responses in vivo. These two specific aims lead logically to specific aim 3, which addresses the mechanisms involved in a dominant physiological regulator of cerebral vascular resistance: 3) investigate selected cellular mechanisms that may be involved in activation of cerebral prostanoid synthesis by hypercapnia. To accomplish these aims, techniques allowing investigation of intact cerebral microcirculation and primary culture of cells from newborn pig brain will be employed. Such research will be unique by studying the intact newborn cerebral circulation and the isolated components that contribute to control of that circulation. Methods will be used that have previously been employed to study the newborn cerebral circulation as well as ones that have not been used in newborn cerebral vascular research. Cranial windows allow observation of cerebral microcirculation, collection of cortical periarachnoid fluid, and selective endothelial damage, in vivo, using intravascular fluorescein activated by filtered light. Prostanoid synthesis by isolated cerebral microvascular endothelial cells, glia, microvascular smooth muscle, and neurons in primary culture will be investigated. Determination of whether hypercapnia-induced prostanoid synthesis relates to increasing intracellular or extracellular [H+] will be made by varying each independently. The [Ca2+]c signal in response to hypercapnia will be investigated by dual wavelength spectroscopy using fura-2 and Ca2+ fluxes using 45Ca2+; and the Ca2+ dependence of hypercapnia-induced prostanoid synthesis will be determined. The relationship between phosphoinositide turnover and prostanoid synthesis during exposure to hypercapnia will be explored using 3H-myoinositol prelabelling and HPLC separation of labelled inositol phosphates. Since disorders of cerebral circulation are major causes of morbidity and mortality in neonates and can result in lifelong disabilities in survivors, better understanding of factors controlling newborn cerebral hemodynamics is badly needed.
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