Neutral proteases are increased in ischemic tissue, and may contribute to reperfusion injury in stroke. Matrix-degrading metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA) open the blood-brain barrier (BBB). Temporary middle cerebral artery occlusion (MCAO) causes a biphasic increase in capillary permeability, which may be related to the inflammatory release of MMPs and uPA. Inhibition of the metalloproteinases reduces the initial opening of the BBB and brain edema at 24 hours. Therefore, this proposal is to use molecular methods to determine the role of neutral proteases in reperfusion injury. HYPOTHESES: 1) In ischemia/reperfusion injury, cerebral capillary vulnerability is mediated by MMPs and serine proteases. 2) MMPs are formed endogenously in astrocytes, microglia, and endothelial cells and brought to the site of inflammation by leukocytes. 3) Agents that reduce proteolysis may reduce the inflammatory response in reperfusion injury.
AIMS AND METHODS: 1) Spontaneously hypertensive rats will have MCAO by the suture method. Capillary permeability and brain edema will be measured at various times after reperfusion. Zymography and quantitative rt-PCR will be done on reperfused tissue to measure MMPs and serine proteases. 2) Immunohistochemistry will be used to study the time course and spatial localization of MMPs. 3) Antibodies to rat neutrophils will be used to determine their contribution to BBB injury and edema. The effect of MMP inhibitors on capillary permeability, brain edema, and infarct size will be measured. SIGNIFICANCE: Thrombolytic therapy has increased the risk of reperfusion injury. Reducing proteolysis may decrease this risk and extend the therapeutic window for reperfusion therapy.
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