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 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.
Daval
J L
JL
INSERM U.272, Université de Nancy I, France.
Ghersi-Egea
J F
JF
Oillet
J
J
Koziel
V
V
D000107:Acetylation; D000818:Animals; D001921:Brain / Q000166:cytology / Q000196:embryology; D015687:Cell Hypoxia; D002478:Cells, Cultured; D003574:Cytochrome c Group / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D005260:Female; D005609:Free Radicals; D009474:Neurons / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013481:Superoxides / Q000378:metabolism*
D003574:Cytochrome c Group; D005609:Free Radicals; D013481:Superoxides