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Background and Hypothesis: Neutrophils infiltration following reperfusion is the hallmark of ischemic stroke. Integrin α9β1 is highly expressed on neutrophils, upregulated upon activation, stabilizes adhesion to activated endothelium in synergy with β2 integrin, and plays a role in transmigration. Fibronectin containing alternatively spliced extra domain A (Fn-EDA) is a ligand for integrin α9β1, and is known to be pro-thrombotic and pro-inflammatory. The role of α9β1 in ischemic stroke is not elucidated yet. Following STAIR guidelines, we tested the hypothesis that integrin α9β1/Fn-EDA axis contributes to the pathophysiology of ischemic stroke in comorbid condition of hyperlipidemia.Methods: Susceptibility to brain ischemia/reperfusion injury was evaluated in male and female mice (N=14-16) by transient occlusion of middle cerebral artery (1 hr) followed by 1, 3, and 7 days of reperfusion. Quantitative assessment of stroke outcome was evaluated by measuring infarct volume (MRI), neurological outcome (0-4), and post ischemic thrombo-inflammation (fibrin, neutrophil and inflammatory cytokines) within ischemic region (by Western and immunohistochemistry). Using intravital microscopy, susceptibility to arterial thrombosis was evaluated in FeCl3 and laser injury-induced thrombosis models.Results: Irrespective of gender, myeloid specific α9β1-/- mice on wild-type and hyperlipidemic background (α9fl/flLysMCre and α9fl/flLysMCreApoe-/-) exhibited smaller infarcts and improved neurological outcomes at days 1, 3 and 7 concomitant with enhanced survival rate and decreased post ischemic thrombo-inflammation (P<0.05 vs. α9fl/fl or α9fl/flApoe-/- mice littermates). Intravital microscopy showed that myeloid specific α9β1-/- mice were less susceptible to arterial thrombosis compared to controls (P<0.05). Bone marrow transplantation experiments revealed that Fn-EDA contributes to α9β1 mediated stroke exacerbation. Infusion of a specific inhibitor of α9β1 into Apoe-/- mouse 15 minutes after reperfusion significantly improved stroke outcome.Conclusions: Following STAIR guidelines, we provide genetic and pharmacologic evidence that targeting myeloid specific integrin α9β1 in hyperlipidemic mice improves stroke outcome.