Research ReportAn in vitro ischemic penumbral mimic perfusate increases NADPH oxidase-mediated superoxide production in cultured hippocampal neurons

The currently accepted scheme for reactive oxygen species production during ischemia/reperfusion injury is characterized by a deleterious mitochondria-derived burst of radical generation during reperfusion; however, recent examination of the penumbra suggests a central role for NADPH-oxidase (Nox)-mediated radical generation during the ischemic period. Therefore, we utilized a novel in vitro model of the penumbra to examine the free radical profile of ischemic murine hippocampal neurons using electron paramagnetic resonance spectroscopy, and also the role of Nox in this generation and in cell fate. We report that free radical production increased ~ 75% at 2 h of ischemia, and this increase was abolished by: (1) scavenging of extracellular free radicals with superoxide dismutase (SOD), (2) a general anion channel antagonist, or (3) the Nox inhibitor apocynin. Similarly, at 24 h of ischemia, [ATP] decreased > 95% and vital dye uptake increased 6-fold relative to controls; whereas apocynin, the Cl− channel antagonist 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), or the free radical scavenger N-acetyl cysteine (NAC) each provided moderate neuroprotection, ameliorating 13-32% of [ATP]-depletion and 19-56% of vital dye uptake at 24 h. Our results support a cytotoxic role for Nox-mediated free radical production from penumbral neurons during the ischemic period.

► We model free radical production in the ischemic penumbra, which is poorly understood. ► We utilized EPR, the gold standard to assay ROS in live cells. ► Deleterious Nox-mediated superoxide is produced by ischemic penumbral neurons. ► Increased free radical generation contributes to ischemic neurotoxicity. ► Cl channel or NADPH oxidase antagonists block this increase and are neuroprotective.