The broad objective of this proposal is to demonstrate that the collateral vascular response to experimental thrombotic stroke of the middle cerebral artery (MCA) territory in the rat can be controlled by stimulating or inhibiting the synthesis of endothelial-derived relaxing factor (EDRF). If this assertion is correct, the volume of MCA territory infarct should be maximal and consistent if EDRF synthesis is suppressed (specific aim 1), and minimized (but likely inconsistent) if EDRF synthesis is enhanced (specific aim 2). Observation of infarct consistency in a normally well- collateralized but not carotid artery-ligated (Wistar) rat would be unprecedented, and important for evaluation of anti-ischemic drugs. Observation of infarct mitigation by EDRF stimulation would suggest that EDRF-induced activation of collateral circulation be used clinically to reduce infarct volume, even if the MCA (or other intracranial artery) remains occluded. In specific aim 3, infarct volume and consistency will be monitored in rats initially subjected to EDRF inhibition and MCA thrombosis (for time periods of up to 3 hours) after complete restoration of anterograde flow by lysis of the MCA thrombi together with EDRF enhancement. Under these apparently optimal reflow conditions, however, the theoretical possibility of reperfusion injury should also be maximized. This paradox will be evaluated histopathologically and biochemically (in terms of peroxidized lipid conjugated dienes) for this aim, and for the first two also (specific aim 4), thus facilitating assessment of the long- hypothesized contribution of (presumably) oxygen radical-mediated lipid peroxidation to the initiation of reperfusion injury. In our methodology the MCA thrombi are formed in specific arterial segments in response to photochemically induced endothelial injury mediated by an intravenously injected dye in conjunction with a focussed laser beam of the appropriate wavelength. Thrombi formed in response to rose bengal injection and irradiation with an argon/dye laser beam at 562 nm can be lysed by hementin (from the leech Haementeria ghilianii). Inhibition of EDRF synthesis is achieved by intravenous infusion of NG-nitro-1-arginine methyl ester hydrochloride (1-NAME), while enhancement of EDRF synthesis is achieved with infusion of either 1-arginine hydrochloride (ARG) or N(alpha)-benzoyl-l-arginine ethyl ester hydrochloride (BAEE). Conjugated diene content is analyzed spectroscopically in total lipid extracts from small (less than 1 mg) cortical punch biopsies. Under the conditions of aim 3 the detection of lipid peroxidation in time during reperfusion should be much more consistent compared to previous efforts.
NIH ExPorter
NIH ExPorter