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  • Title: A simple method for evaluation of superoxide radical production in neural cells under various culture conditions: application to hypoxia.
    Author: Daval JL, Ghersi-Egea JF, Oillet J, Koziel V.
    Journal: J Cereb Blood Flow Metab; 1995 Jan; 15(1):71-7. PubMed ID: 7798340.
    Abstract:
    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 microM 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.
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