A Simple Method for Evaluation of Superoxide Radical Production in Neural Cells under Various Culture Conditions: Application to Hypoxia

To evaluate the potential deleterious influence of oxygen-derived free radicals following hypoxia in a model of primary culture of neurons obtained from the fetal rat brain, superoxide radicals were measured as a function of time in the extracellular medium. Neuronal cells were grown for 8 days in the presence or absence of serum, then incubated in a buffered Krebs–Ringer solution containing 60 μM acetyl-cytochrome c. The rate of superoxide radical formation was quantified spectrophotometrically by measuring the specific reduction of acetyl-cytochrome c. Under normoxic conditions (95% air-5% CO₂), basal production of superoxide that increased with time was recorded. It was significantly more pronounced in cells grown in serum-free medium. Under both culture conditions, acute hypoxia (95% N₂–5% CO₂) for 6 h increased superoxide radical amounts in the extracellular medium, and they were still enhanced 3 h after reoxygenation. The addition of superoxide dismutase to the incubating medium abolished the detection of superoxide radicals. The present study describes a new reliable method for superoxide radical measurement in cells in vitro and demonstrates hypoxia/reoxygenation-induced overproduction of superoxide in cultured neurons that may account for cell injury.