This project tests the hypotheses that parenchymal astrocytes (ACs) contribute to the constriction of cerebral penetrating arterioles (PAs) after hypoxia and reoxygenation (H/RO), and that H/RO-induced arteriolar constriction can be ameliorated by transducing nitric oxide synthase (NOS). Ischemia/Reperfusion constricts intracortical cerebral arterioles in vivo which may contribute to post-ischemic oligemia. H/RO causes vasoconstriction in isolated cortical PAs. It is not known how parenchymal Acs exacerbate vasoconstriction in PAs after HtRO. Since PAs are the last regulator of blood flow to the cortical tissue, even a small additional vasoconstriction would greatly increase vascular resistance and thereby contribute to postischemic oligemia observed in vivo. An H/RO-induced impairment in NO production by PAs may also contribute to the net vasoconstriction. Reducing this vasoconstrictor response by NOS transduction may be instrumental in salvaging cerebral neuronal tissue after stroke. Experiments will be performed in vitro. Rat PAs will be isolated, cannulated, and observed by video microscopy. The vessels will be subjected to hypoxia for one hour. (1) We will test if astrocyte contact exacerbates penetrating arteriolar vasoconstriction after H/RO. We will determine if postischemic glucose deprivation exacerbates H/RO-induced constriction. (2) We will determine if and which reactive oxygen species (ROS: O2"""""""", H202, peroxynitrite) is released from ACs to causes the enhanced arteriolar-constriction. ACs exposed to H/RO may release ROS which deactivate NO, but also other vasoconstrictors. The use of specific inhibitors will reveal the participation of the various mediators in H/RO-induced vasoconstriction. (3) We will test if transduction of NOS into PAs ameliorates H/RO-induced constriction. Impairment of endothelial NOS after cerebral ischemia is a possible cause for the observed vessel constriction. We will transduce inducible NOS (iNOS) protein directly into PAs in vitro and in cultured cerebral microvascular cells. The iNOS protein was recently found to be a beneficial tool to treat endothelial dysfunction. Our studies will give us crucial information on possible treatments targeted precisely to prevent post-ischemic intracerebral arteriolar constriction and to improve post-ischemic microvascular perfusion in vivo.
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