Superoxide dismutase ameliorates neuronal death from hypoxia in culture

Abstract

Background and Purpose Studies showing efficacy with free radical scavengers have been conflicting, and when protection was demonstrated it was attributed to action at the level of the vascular endothelium. The purpose of this study was to test the hypotheses that neuronal free radical formation plays a role in the ischemic cascade and occurs intracellularly and that free radical scavengers, if taken up intracellularly, will protect against hypoxic damage. Methods A tissue culture model of hypoxia followed by restoration of oxygen was employed, using both superior cervical ganglia and hippocampal neurons. Cells were exposed to an anoxic atmosphere of 95% N2 and 5% CO2 and examined 2 to 24 hours later after restoration of oxygen. Determination of survival was measured by trypan blue exclusion. Nitroblue tetrazolium stain was used to assess free radical formation. Results Pretreatment with free Superoxide dismutase did not decrease cell death after hypoxia as measured by trypan blue exclusion. However, when Superoxide dismutase was taken up intracellularly under depolarizing conditions (55 mmol/L KC1 in the medium), cell death was decreased significantly compared with hypoxic controls (28.7±4.34 versus 40.3±4.33; P