The importance of free hydroxyl radicals to hypoxia preconditioning

Hypoxia preconditioning states that a sublethal hypoxia period will afford neuroprotection against a second harmful event. In our experiments, we carried out a procedure for the development of hypoxia preconditioning in adult male Wistar rats using hypoxic exposure (9% O(2); 91% N(2)) for 1 h. The protection against pentylenetetrazol (PTZ)-induced seizures was studied. For this, rats were tested by a single injection of PTZ (55 mg/kg i.p.) on days 1-21 after hypoxia exposure. The hypoxia exposure significantly prevented the development of acute PTZ convulsion at different times after hypoxia. The present study was designed to determine the effect of N-t-butyl-alpha-phenylnitrone (PBN), an electron-trapping agent and free radical scavenger, on hypoxia preconditioning against PTZ seizures 7 days after hypoxia exposure. PBN abolished the protective action of hypoxia exposure. The generation of free hydroxyl radicals in the brains of animals exposed to hypoxia was determined in a second experiment. For this purpose, the rats were i. p. pretreated with 30 mg/kg PBN and NaCl, respectively, 20 min before the start of hypoxia exposure. Forty-five minutes later the rats were i.p. injected with 300 mg/kg sodium salicylate and once again exposed to hypoxia for 15 min. Immediately after that the animals were decapitated and the free hydroxyl radicals and the salicylate content were estimated in the whole brain without cerebellum. Hypoxia preconditioned animals pretreated with NaCl showed a significantly higher extent of free hydroxyl radicals in the brain compared with PBN-injected preconditioned animals and with naive and sham exposed controls. The results pointed out that the generation of free reactive oxygen species under hypoxic conditions in the brain is involved in the development of the hypoxic preconditioning phenomenon.