Project Summary Stroke results in immense health and economic burden. Current strategies to improve stroke outcome is fibrinolysis of the clot with tissue plasminogen activator and mechanical thrombectomy, which involves reperfusion of the ischemic region. However, accumulating evidence suggests that cerebral reperfusion is often accompanied by oxidative stress, thrombosis, and vascular inflammation, which exacerbates neuronal death in the ischemic penumbra. There is currently no effective intervention available to protect the brain damage following reperfusion therapy. Clinical studies have shown a positive correlation between the massive influx of neutrophils and severity of injury following reperfusion. Integrin alpha9beta1 is highly expressed on neutrophils (relative to monocytes), upregulated upon activation and transmigration, and is known to stabilize neutrophil adhesion to activated endothelium in synergy with alpha2beta1 integrin. The mechanistic role of alpha9beta1 in stroke outcome is not explored yet. Utilizing novel mutant strains, in pilot studies, we found that alpha9beta1 exacerbates stroke outcome by modulating thrombosis and vascular inflammation. Following updated Stroke Therapy Academic Industry Roundtable (STAIR) pre-clinical guidelines and compelling pilot data, we propose to test the innovative hypothesis that integrin alpha9beta1 contributes to ischemic stroke exacerbation in stroke models with comorbidities and therapeutic targeting alpha9beta1 will improve stroke outcome by limiting thrombosis and inflammation. To accomplish this, we will utilize complementary genetic and pharmacological approaches, state-of-the-art intravital microscopy, magnetic resonance imaging, laser speckle imaging and follow current STAIR/RIGOR guidelines. Aging and biological sex are key determinants that influence stroke outcome. We will determine whether targeting alpha9beta1 will improve stroke outcome in the context of biological sex and aging so that the observed effect is generalizable to the broader context. This project may have significant clinical implications since understanding the mechanisms by which neutrophils contribute to reperfusion injury in stroke models with comorbidities may provide new therapeutic interventions to treat ischemic stroke.