EVALUATION OF SUPEROXIDE RADICAL PRODUCTION IN CULTURED NEURONS. EFFECTS OF HYPOXIA AND HYPEROXIA

Abstract

The formation of oxygen free radicals, especially superoxide and hydroxyl radicals, leads to hypoxic neuronal damage. In an attempt to investigate the infiuence of post-hypoxia reoxygenation in a model of primary culture of neurons from fetal rat brain, the production of superoxide radicals (SR) was assessed over 24h in the culture medium. After 8 days in vitro, culture medium was replaced by 1ml Krebs-Ringer buffer with acetyl-cytochrome c (ACc). A first set of cultures (n = 32) was incubated for 6h in a gas mixture of 95%air-5%CO, (normoxia), and a second set (n = 32) was incubated with 95%N₂-5%CO₂ (hypoxia) which reduced the PO₂ by 77% in the culture medium. Half of the two sets of culture dishes was then placed in normoxic atmosphere, and the remaining dishes were incubated for 3h with 95% O2-5% CO₂ (hyperoxia) which increased the PO₂ by 250%. All cultures were finally returned to normoxia for 15h. The rate of SR formation was quantified spectrophotometrically by measuring the reduction of ACc at 550 nm. In control cultures (normoxia), about 4% and 8% of ACc per 100 μg protein were reduced after 6h and 24h, respectively, indicating a low basal production of SR under standard culture conditions. Hypoxia induced a significant increase in the reduction of ACc (18.7±3.1 %) which was even higher 3h after return to normoxia (44.3 ± 3.5%). Hyperoxia did not enhance the SR production over basal values and neither aggravaied hypoxia-induced production (20.3±5.55% vs 18.7±3.1%). The following day, while there was no apparent cell alteration, hypoxia-induced level of SR remained stable (40.8 ± 1.6%) as well as the SR accumulation induced by the sequence hypoxia-hyperoxia (18.6±3.9%). Thus, it appears that hypoxia induces an over-production of SR in cultured neurons which may contribute to cell damage. Unexpectedly, post-hypoxia hyperoxygenation lends to limit the SR formation in vitro.

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