The research proposed is designed to address the unifying hypothesis that mechanisms involved in cerebrovascular regulation change with age from perinatal to juvenile period. Specifically, the principle endothelial dependent dilator influence is transformed from prostanoid dominance in the newborn to NO dominance in later life. To test this hypothesis three specific aims will be addressed using neonatal and juvenile pigs: 1) Compare cerebral microvascular responses of newborns and juveniles to specific dilator and constrictor stimuli, 2) Investigate endothelial involvement, in general, and prostanoids and NO, in particular, in these responses in newborns and juveniles, and 3) Determine the cellular mechanisms involved in the age-dependent variance of endothelial-derived mediators. To accomplish these aims techniques allowing investigation of intact cerebral microcirculation, isolated cerebral microvessels, and primary culture of cells from newborn and juvenile pig brain will be employed. Such research will be unique by studying intact cerebral circulation in newborns and older individuals in a consistant model and investigating, at the cellular and molecular levels, the mechanisms responsible for ontogenic divergence of paracrine mediators. Cranial windows allow observation of cerebral circulation, collection of cortical periarachnoid fluid, selective endothelial damage in vivo, and topical applications of agonists and inhibitors. Levels and cellular distribution of mRNA transcripts and protein expression for COX and NOS, as well as the relative activities of the enzymes in cerebral microvessels of newborn and juvenile pigs will be examined. Whether difference in mRNA, protein, and enzymatic activity are maintained by cells in primary culture will be determined, as will the potential mechanisms responsible for changes in expression and/or activity of COX and NOS. Responses of newborn and juvenile vascular smooth muscle to the products of these enzymes will be compared. Using single age and cross cocultures of microvascular smooth muscle and endothelial cells from newborns and juveniles, age dependence of intercellular communication involving mediator production and cellular second messenger responses to stimuli that produce vascular actions via endothelium dependent mechanisms will be examined. Since endothelial control of cerebral circulation is easily impaired by pathological conditions in both newborn and older individuals and cerebrovascular disease is a major health care problem from pediatrics to geriatrics, better understanding of the nature of endothelial humoral function and how it is altered during postnatal development is badly needed.
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