Polymorphonuclear neutrophils (PMNs) may contribute to reperfusion injury in the neonatal brain. A prerequisite ford PMN-mediated injury is adherence to the endothelium. In our newly developed piglet asphyxia model, we have documented adherence of PMNs to cerebral venular endothelium concomitant with delayed hypoperfusion and superoxide free radical production from endogenous xanthine oxidase activity. We will test the hypothesis that adenosine, oxygen free radicals, nitric oxide, and PMN, and endothelial adhesion molecules modulate interactions between PMN and cerebrovascular endothelium during postasphyxic reperfusion in the newborn brain. In particular, we anticipate these modulators exhibit single and combined effects on PMN adherence to endothelial cells during reperfusion. Studies outlined in this proposal will examine the mechanisms and consequences of these interactions in vivo and in vitro. We plan to: 1) Examine the role of adenosine in PMN adherence to endothelium. We will test the hypothesis that adenosine inhibits PMN adherence to cerebrovascular endothelial cells during postasphyxic reperfusion. 2) Investigate whether oxygen free radicals and nitric oxide are involved in PMN- endothelial cell interactions. We will test the hypothesis that oxygen radicals and nitric oxide modulate, in a singular or combined fashion, PMN adherence to cerebrovascular endothelium during reperfusion. 3.) Identify the particular adhesion molecules involved ill PMN-endothelial cell interactions. We will test the hypothesis that the adhesion molecules CD18,ICAM-1, and P-selectin mediate PMN adherence to cerebrovascular endothelium during reperfusion. PMN rolling and adherence to cerebral venules, local blood flow and vascular permeability will be measured in piglets by in vivo videomicroscopy through closed cranial windows. Adherence of isolated PMN to cultured piglet cerebrovascular endothelial cells, oxygen radical production. and endothelial cell injury will be quantified in vitro. The mechanistic involvement of adenosine, free radicals, nitric oxide, and specific adhesion molecules in each system will be revealed by pharmacologic manipulation and immuno-neutralization with monoclonal antibodies. Identification of the precise mechanisms underlying PMN-endothelial cell interactions during reperfusion wide developing therapeutic modalities for treating cerebral in asphyxiated neonates.
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