AbstractIschemic stroke is a serious neurological disorder that is associated with dysregulation of the neurovascular unit (NVU) and impairment of the blood–brain barrier (BBB). Paradoxically, reperfusion therapies can aggravate NVU and BBB dysfunction, leading to deleterious consequences in addition to the obvious benefits. Using the recently established EPAM-ia method, we identified osteopontin as a target dysregulated in multiple NVU cell types and demonstrated that osteopontin targeting in the early acute phase post-transient middle cerebral artery occlusion (tMCAO) evolves protective effects. Here, we assessed the time course of osteopontin and CD44 receptor expression in NVU cells and examined cerebroprotective effects of osteopontin targeting in early and late acute phases of ischemic stroke. Expression analysis of osteopontin and CD44 receptor post-tMCAO indicated increased levels of both, from early to late acute phases, which was supported by their co-localization in NVU cells. Combined osteopontin targeting in early and late acute phases with anti-osteopontin antibody resulted in further improvement in BBB recovery and edema reduction compared to targeting only in the early acute phase comprising the reperfusion window. Combined targeting led to reduced infarct volumes, which was not observed for the single early acute phase targeting. The effects of the therapeutic antibody were confirmed both in vitro and in vivo in reducing osteopontin and CD44 expression. Osteopontin targeting at the NVU in early and late acute phases of ischemic stroke improves edema and infarct size in mice, suggesting anti-osteopontin therapy as promising adjunctive treatment to reperfusion therapy.