Mechanisms of delayed ischemia/reperfusion evoked ROS generation in the hippocampal CA1 zone of adult mouse brain slices.

ROS overproduction is an important contributor to delayed ischemia/reperfusion induced neuronal injury, but relevant mechanisms remain poorly understood. We used oxygen-glucose deprivation (OGD)/reperfusion in mouse hippocampal slices to investigate ROS production in the CA1 pyramidal cell layer during and after transient ischemia. OGD evoked a 2-stage increase in ROS production: 1st-an abrupt increase in ROS generation starting during OGD followed by a marked slowing; and 2nd-a sharp ROS burst starting ~ 40 min after reperfusion. We further found that a slight mitochondrial hyperpolarization occurs shortly after OGD termination. Consequently, we showed that administration of low dose FCCP or of FTY720 (both of which cause mild, ~ 10%, mitochondrial depolarization), markedly diminished the delayed ROS burst, suggesting that mitochondrial hyperpolarization contributes to ROS production after reperfusion. Zn chelation after OGD withdrawal also substantially decreased the late surge of ROS generation-in line with our prior studies indicating a critical contribution of Zn entry into mitochondria via the mitochondrial Ca uniporter (MCU) to mitochondrial damage after OGD. Thus, reperfusion-induced mitochondria hyperpolarization and mitochondrial Zn accumulation both contribute to mitochondrial ROS overproduction after ischemia. As these events occur after reperfusion, they may be amenable to therapeutic interventions.