The overall objective of this proposal is to elucidate the mechanisms whereby nitric oxide, disruption in iron homeostasis, and endothelial cell derived superoxide contribute to perinatal hypoxic-ischemic brain injury. We hypothesize that they interact synergistically to promote microvascular injury and cerebral infarction after the primary insult.
Specific aims i nclude: 1) to explore the beneficial effects and mechanisms of action of polyethylene glycol superoxide dismutase (PEG SOD) in preventing or reducing hypoxic-ischemic brain injury; 2) to ascertain the presence and extent to which nitric oxide contributes to hypoxic-ischemic brain damage in the immature rat; 3) to determine the contribution of alterations in cellular and extracellular iron homestasis (iron and iron regulatory proteins) to hypoxic-ischemic brain damage in the immature rat. To accomplish these goals, an established model of focal hypoxic-ischemic brain injury in 7-day postnatal rats will be used. Rat pups are subjected to unilateral common carotid artery occlusion combined with exposure to 8% oxygen. Hemispheric water content at 42 hours recovery and cerebral atrophy at 23 days of recovery will determine injury. Nitric oxide synthase will be inhibited with NG-nitro-L- arginine-methyl ester (LNAME) with appropriate L&D arginine controls. Its neuroprotective ability will be compared with the specific inhibitor of the inducible isoform of nitric oxide synthase, 7-nitro indazole (7- NI). The dose responsive neuroprotective potential of PEG SOD and nitric oxide synthase inhibition (NOSI) will be evaluated in pre and post treatment studies. Using the optimal treatment regime (time and dose), we will determine how endothelial cell derived superoxide and nitric oxide contribute to microvascular and parenchymal brain injury by serial measurements during hypoxia-ischemia and recovery of: regional cerebral blood flow, microvascular occlusion, blood-brain barrier transfer coefficient to 14C aminoisobutyric acid (AIB), hemispheric plasma volume with [3H]inulin, energy metabolism, protein oxidation (carboxyl content), neutrophil accumulation and myeloperoxidase activity, hydroxy radical formation (salicylate trapping). As evidence of nitric oxide injury; nitrates, nitrites, and nitrotyrosine will be measured quantitatively, as well a immunohistochemically. Immunohistochemical techniques will be employed to localize the cellular targets of free radical injury (nitrotyrosine & carbonyl formation) and alterations to iron homeostasis. Specifically, stains will be prepared for iron (Perl's stain) as well as the iron regulatory proteins (transferrin receptor, H&L chain ferritin) and their mRNA message. With digoxigen labelled in situ probes and dual labelled immunohistochemistry colocalization of message with cell type will be achieved. The amount of iron available for free radical formation will be measured (deferoxamine available iron) and compared with total iron content. Results of the proposed research will elucidate the sites and sources of free radical formation and will provide a basis for the timing and targeting of future therapeutic intervention of human infants who have sustained cerebral hypoxia-ischemia during the perinatal period.
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