PROJECT SUMMARY Stroke is a leading cause of death and results in permanent disability in up to 30% of survivors. Among them, ischemic stroke accounts for about 87 percent of all cases. Tissue plasminogen activator (tPA) is the only effective treatment for ischemic stroke but has a very limited therapeutic window of 3-4.5h. Undesirably, delayed tPA treatment beyond 3-4.5h disease onset increases risk of blood brain barrier (BBB) disruption and hemorrhagic transformation (HT) that further exacerbates brain injury. In addition, tPA has been shown to aggravate neuroinflammation by enhancing microglia (MG) activation. Thus, there is a critical need to develop a therapeutic approach to attenuate brain inflammation and widen the tPA therapeutic window following ischemic stroke. Interferon beta (IFNβ), a cytokine with immunomodulatory properties, was approved by the FDA for the treatment of relapsing-remitting multiple sclerosis (MS) for more than a decade. It has been well-established that IFNβ suppresses CNS inflammation in MS, thereby suggesting that IFNβ might have a therapeutic potential for the treatment of ischemic stroke. Indeed, our recent findings showed that IFNβ attenuated ischemia-induced brain infarct and lessened neurological deficits in rodent stroke models, thereby demonstrating that IFNβ confers a protective effect against ischemic stroke. However, detailed mechanisms involved in the protective effects of IFNβ on the suppression of ischemia-induced primary and reperfusion-mediated secondary neuroinflammation remain to be elucidated. More importantly, the effect of IFNβ on tPA-mediated neuroinflammation and delayed tPA-induced BBB disruption and HT in the ischemic brain is unknown. We hypothesize that IFNβ ameliorate ischemic brain injury by converting ischemia, reperfusion, and tPA-induced inflammatory MG into anti- inflammatory MG and modulating inflammatory immune cell infiltration. In addition, IFNβ extends the tPA therapeutic window by inhibiting delayed tPA-induced MMP3/9 production, BBB disruption, and HT in the ischemic brain. We propose the following three specific aims to test our hypothesis. In Aim 1, we will test the effect of IFNβ, co-administered with tPA, on ischemic brain injury and on the tPA therapeutic window in ischemic stroke. In Aim 2, we will determine the effect of IFNβ, co-administered with tPA, on MG activation during acute phase and on infiltrating inflammatory immune cell activation during sub-acute phase in ischemic stroke. In Aim 3, we will determine whether IFNβ alleviates delayed tPA-induced BBB disruption and HT in ischemic stroke. The completion of our proposed studies will provide strong evidence that IFNβ can be developed as a novel therapy for ischemic stroke and ultimately lead to a new venue of medical intervention for cerebral ischemia.