Abstract 3041: Activated Thrombin Increase after Ischemic Stroke

Abstract

Background and Purpose- The role of thrombin in mediating cytotoxicity after intracerebral hemorrhage is well known. Recently, we demonstrated activated thrombin co-localizing with neurons after ischemia using immunohistochemistry (IHC). We sought to quantify activated thrombin protein levels in ischemic brain using protein extraction and Western blots. Methods- Focal ischemia was induced by middle cerebral artery occlusion (MCAo) with a suture for 4 hours in adult Sprague Dawley rats (n=4). Brain regions with severe neurovascular disruption were identified using 2MD dextran linked to fluorescein isothiocyanate (FITC) injected intravenously during surgery. After 30 minutes reperfusion, brains were divided_using FITC whole-brain fluorescence_into ischemic, non-ischemic ipsilateral, and non-ischemic contralateral sections. Protein was extracted from homogenates and thrombin and prothrombin were quantified using Western immunoblots. Integrated density values (IDV) were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) bands. Results- In ischemic brains, we detected severe vascular damage after 4 hours MCAo as significant FITC leakage. Thrombin was elevated (p<0.05 Newman-Keuls) in ischemic core (0.1908 ± 0.05) compared to non-ischemic region contralateral (0.05 ± 0.005) to the MCAo. In addition, non-ischemic tissue was devoid of FITC, but trace amounts of thrombin were detected on Westerns. Using IHC on sections for ischemic regions, prothrombin was detected within damaged blood vessels only, and activated thrombin co-localized with neurons and less so with vessels, astrocytes, or microglia. Conclusion- Elevation of thrombin was found in both ischemic and non-ischemic regions after MCAo. While prothrombin appeared to localize in vessels, activated thrombin was clearly localized to neurons. These findings suggest that after ischemia, prothrombin enters brain via disrupted BBB where it undergoes activation to thrombin then aggregates on neurons. Future experiments are needed to confirm this hypothesis and determine the effect of thrombin on neurons.