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Stroke is a major cause of death and long-term disability. Pak1 is implicated in many neurological disorders as it has been suggested to promote neuronal survival. However, its function and mechanisms have not been investigated in stroke. We assessed the hypothesis that Pak1 provides neuroprotection in stroke by stimulating the secretion of brain-derived neurotrophic factor (BDNF) in endothelial cells.Transient focal ischemia was induced by middle cerebral artery occlusion (MCAO, 90 mins) in young WT male mice. Two doses of IPA3, a Pak1 specific inhibitor, were injected (i.p.) at 30 minutes prior to stroke and 1.5 hours after stroke. For mechanism studies, lentivirus (LV) encoding Pak1 or IPA3 was used to overexpress or pharmacologically inhibit Pak1 in mouse primary brain microvascular endothelial culture. Then the cells were subjected to OGD (16h) with 48 h re-oxygenation and conditioned media (Endo-CM) were collected subsequently. Mouse primary cortical neuron culture was treated with Endo-CM after OGD (90 mins) and neuronal death was assessed 24 hours later.Pak1 activation (phosphorylated Pak1) was observed in the ischemic hemispheres 6 and 72 hours after MCAO (p-Pak1: sham 94.5± 4.7% versus 6-hour stroke 192.5 ± 10.4% ; sham 94.5± 4.7% versus 72-hour stroke 254.4 ± 32.3%, n = 4 p/g, p<0.05 for both). Administration of IPA3 increased brain infarct size assessed 24 hours after MCAO stroke. In endothelial cultures, overexpression of Pak1 increased BDNF secretion in Endo-CM after OGD (LV-GFP 22.7 ± 2.8 versus LV-Pak1 48.6± 5.5, n = 4 p/g, p<0.05). Accordingly, inhibition of Pak1 attenuated levels of BDNF in Endo-CM (Vehicle 24.5 ± 1.5 versus IPA3 9.4± 0.7, n = 3 p/g, p<0.05). Finally, treatment with Endo-CM collected from Pak1 overexpressing culture reduced neuronal death after OGD (LV-GFP 36 ± 2% versus LV-Pak1 10± 1%, n = 3 p/g, p<0.05), while Endo-CM from cells treated with Pak1 inhibitor increased neuronal mortality (Vehicle 24± 7% versus IPA3 43± 2%, n = 3 p/g, p<0.05).Taken together, we demonstrated that endothelial Pak1 provides neuroprotection after ischemia. Mechanically, our data revealed that activation of Pak1 stimulates endothelial production of a well-known potent neuroprotectant, BDNF.