Chloride transport inhibitors influence recovery from oxygen–glucose deprivation-induced cellular injury in adult hippocampus

Abstract

Cerebral ischemia in vivo or oxygen–glucose deprivation (OGD) in vitro are characterized by major disturbances in neuronal ionic homeostasis, including significant rises in intracellular Na +, Ca 2+, and Cl − and extracellular K +. Recently, considerable attention has been focused on the cation–chloride cotransporters Na–K–Cl cotransporter isoform I (NKCC-1) and K–Cl cotransporter isoform II (KCC2), as they may play an important role in the disruption of ion gradients and subsequent ischemic damage. In this study, we examined the ability of cation–chloride transport inhibitors to influence the biochemical (i.e. ATP) and histological recovery of neurons in adult hippocampal slices exposed to OGD. In the hippocampus, 7 min of OGD caused a loss of ATP that recovered partially (~50%) during 3 h of reoxygenation. Furosemide, which inhibits the NKCC-1 and KCC2 cotransporters, and bumetanide, a more specific NKCC-1 inhibitor, enhanced ATP recovery when measured 3 h after OGD. Furosemide and bumetanide also attenuated area CA1 neuronal injury after OGD. However, higher concentrations of these compounds appear to have additional non-specific toxic effects, limiting ATP recovery following OGD and promoting neuronal injury. The KCC2 cotransporter inhibitor DIOA and the Cl − ATPase inhibitor ethacrynic acid caused neuronal death even in the absence of OGD and promoted cytochrome c release from isolated mitochondria, indicating non-specific toxicities of these compounds.