Effects of chronic ethanol exposure on oxidation and NMDA-stimulated neuronal death in primary cortical neuronal cultures

Excessive oxidative radical formation has been implicated in a number of ne urodegenerative processes, including glutamate-N-methyl- D-aspartate (NMDA) -mediated excitotoxicity, Previous studies have suggested that the formatio n of reactive oxygen species (ROS) during NMDA exposure is involved in trig gering the excitotoxic cascade. Chronic exposure of primary neuronal cultur es to ethanol has been shown to potentiate NMDA-mediated processes, such as nitric oxide formation and excitotoxicity, It was the objective of this st udy to investigate the role of NMDA and ROS formation in the development of NMDA supersensitivity after chronic ethanol exposure. The fluorescent dyes dichlorofluorescein diacetate (H2DCF) and propidium iodide, which have bee n shown to be reliable markers for the detection of cellular oxidation and cell death, respectively, in neuronal culture preparations, were used to ex amine the relationship between ROS production and NMDA-mediated neuronal de ath after chronic ethanol administration, Cultures treated chronically with ethanol (100 mM) for 96 hr displayed significantly less H2DCF oxidation wh en exposed to various concentrations of FeSO4 for 25 min. However, this dec rease in intracellular oxidation did not have any apparent inhibitory effec ts on the amount of cell death observed 12 hr after the 25-min exposure to FeSO4. When NMDA-FeSO4-mediated oxidation was examined in cultures treated chronically with ethanol, dose-dependent increases in H2DCF oxidation were observed, but only in control-treated cultures. This blunting of intracellu lar H2DCF oxidation did not attenuate the potentiation of NMDA-mediated exc itotoxicity observed after chronic ethanol exposure. These results suggest that the observed supersensitivity to NMDA is not due to increases in intra cellular ROS formation and that chronic ethanol may induce neuronal factors that reduce ROS formation, but do not protect against normal death.