DESCRIPTION (provided by applicant): Na+/H+ exchanger isoform 1 (NHE1) is the most abundantly expressed isoform in the central nervous system (CNS) and carries H+ extrusion in exchange with Na+ influx (1:1 ratio) in regulation of cellular pH. In the previous funding periods, using in vitro and in vivo cerebral ischemia models, we have established that excessive stimulation of NHE1 activity led to dysregulation of intracellular Na+ and Ca2+ homeostasis in conjunction of reversal activation of Na+/Ca2+ exchange (NCXrev) in neurons and astrocytes. We also discovered novel findings that NHE1 plays a primary role in H+ extrusion and maintaining the optimal pHi for NADPH oxidase (NOX) function during the "respiratory burst" of the activated microglia. Pharmacological inhibition of NHE1 activity with its potent inhibitor cariporide (HOE 642) or global transgenic knockout protected neurons and astrocytes from ischemic damage in ischemic models. It also abolished production of superoxide (O2-.) and pro-inflammatory cytokines (IL- 1β, TNFα) in the activated microglia. In this renewal proposal, we created Nhe1flox/flox mouse line (Nhe1f/f) with exon 5 of Nhe1 flanked with two flox sites in attempt to selectively ablate NHE1 in neurons, astrocytes, or microglia. Using ischemic stroke model (a transient middle cerebral artery occlusion, tMCAO), we detected neuroprotection and less neurological function deficits in hGfapCreER/+;Nhe1f/f mice treated with tamoxifen. Most importantly, selective knockout of astrocytic Nhe1 in GFAP+ astrocytes prevented reactive astrocyte formation, decreased S100β release, reduced microvessel damage and BBB leakage after ischemia. These novel findings suggest that astrocytic NHE1 protein plays an important role in reactive astrocyte-mediated brain damage. We hypothesize that NHE1 activation in reactive astrocytes not only disrupts astrocytic ionic homeostasis, as we demonstrated previously, but it also alters perivascular astrocytic functions at the blood brain barrier (BBB). n this renewal application, we focus to investigate the possible cellular mechanisms underlying deletion of astrocytic NHE1 protein and its impact on reducing reactive astrogliosis, and BBB damage after ischemic stroke. To date, numerous studies have used GfapCreER/+ approaches to selectively alter astrocytes functions. To our knowledge, GfapCreER/+;Nhe1f/f mouse line is the first to exhibit less reactive astrogliosis in ischemic brains. Better understanding the underlying mechanisms will bring new insights into roles of NHE1 protein in dysregulation and dysfunction of reactive astrocytes in ischemic stroke.