Mechanisms of hippocampal reoxygenation injury.

Mechanisms of 12 min of hypoxia and subsequent reoxygenation were studied in rat hippocampal slices. General cell injury in reoxygenation was indicated by increased lactate dehydrogenase (LDH). Increase in conjugated dienes (CD) showed that oxygen radical burst induced lipid peroxidation (LPO). ATP increase was also involved in reoxygenation injury, since cyanide, an inhibitor of ATP synthesis, decreased this damage. The results obtained on using inhibitors of oxygen radicals generation, i.e., allopurinol, indomethacin, rotenone, and antimycin A, strongly suggest that the sources of oxygen radicals were the xanthine/xanthine oxidase system, prostaglandin synthesis, and mitochondrial respiratory chain. The involvement of oxygen radicals in oxidative stress was confirmed also by using chain-breaking antioxidants, trolox alpha-tocopherol and stobadine, [(-)-cis-2,8-dimethyl-2,3,4,4a,5,9b-hexahydro-1H-pyrido (4,3b)indole]. Stobadine added at the onset of reoxygenation was most effective, acting in a dose-dependent manner and found to be without effect when applied in hypoxia. Cytochrome-c oxidase was decreased in reoxygenated hippocampal slices treated with stobadine.

Mechanisms of hippocampal reoxygenation injury. Treatment with antioxidants.

The effects of hypoxia of different durations (8, 12 or 15 min) and of subsequent reoxygenation were studied in rat hippocampal slices by measuring enzyme activities related to oxidative stress: superoxide dismutase (SOD), cytochrome c oxidase and lactate dehydrogenase (LDH). Simultaneously the degree of lipid peroxidation was estimated by measuring conjugated dienes (CD). Reoxygenation after 8-min of hypoxia induced general cell injury indicated by increased LDH activity. Reoxygenation after 12-min of hypoxia started lipid peroxidation assessed by an increase in CD, and after 15-min of hypoxia followed by reoxygenation CD were found to be significantly decreased, suggesting lipid degradation. The injury induced by a hypoxia of 12 min and reoxygenation was reduced by SOD and catalase, indicating that oxygen radicals were involved in this process. The oxygen radicals originating from the xanthine/xanthine oxidase system, from the synthesis of prostaglandins, as well as from the mitochondrial respiratory chain, since allopurinol, indomethacin and rotenone decreased while antimycin increased reoxygenation injury. An increase in ATP may also have been involved as cyanide, an inhibitor of ATP synthesis, decreased the reoxygenation injury. The chain-breaking antioxidants trolox, alpha tocopherol and the pyridoindole stobadine were effective in preventing reoxygenation injury, indicating the involvement of lipid peroxidation in this process.

Teratological study of stobadin after single and repeated administration in rats.

The toxic developmental potential of the anti-arrhythmic drug stobadin was assessed after single intravenous or repeated oral doses to pregnant rats. Stobadin was studied in the form of dihydrochloride (DH 1011) at doses of 2 and 6 mg/kg, given in single intravenous injections on days 3, 6, 9, or 12 of gestation. Immediately after injection of the 6-mg/kg dose of DH 1011 to pregnant rats, saccade abdominal respiration, tremor of hindlimbs, and sedative behaviour were observed on each day of medication. No deaths of females occurred in either the control or experimental groups. Slight foetal toxicity was manifested by significantly decreased foetal weight only after treatment on day 3 of gestation at 6 mg/kg and by significantly increased incidence of delayed ossification of the parietal and supraoccipital bone also at 6 mg/kg DH 1011 given on day 12 of gestation. The effect of repeated oral treatment in the form of dipalmitate salt (DP 1031) was studied in doses of 5, 15, and 45 mg/kg from days 2-15 of gestation. Oral exposure to 45 mg/kg DP 1031 resulted in significant reduction of maternal body weight gain and in embryofoetal toxicity, namely, increased preimplantation foetal loss, anomalies of sternebrae, and, after 15 and 45 mg/kg DP 1031, significantly decreased foetal weight and smaller litter size. The relevance of the two routes of stobadin administration for risk extrapolation is discussed.