beta-Amyloid toxicity in organotypic hippocampal cultures: Protection by EUK-8, a synthetic catalytic free radical scavenger

Oxygen free radicals have been proposed to mediate amyloid peptide (beta AP)-induced neurotoxicity. To test this hypothesis, we evaluated the effects of EUK-8, a synthetic catalytic superoxide and hydrogen peroxide scavenger, on neuronal injury produced by beta AP in organotypic hippocampal slice cultures, Cultures of equivalent postnatal day 35 (defined as mature) and 14 (defined as immature) were exposed to various concentrations of beta AP (1-42 or 1-40) in the absence or presence of 25 mu M EUK-8 for up to 72 hours, Neuronal injury was assessed by lactate dehydrogenase release and semiquantitative analysis of propidium iodide uptake at various times after the initiation of beta AP exposure, Free radical production was inferred from the relative increase in dichlorofluorescein fluorescence, and the degree of lipid peroxidation was determined by assaying thiobarbituric acid-reactive substances, Treatment of mature cultures with beta AP (50-250 mu g/ml) in serum-free conditions resulted in a reproducible pattern of damage, causing a time-dependent increase in neuronal injury accompanied with formation of reactive oxygen species, However, immature cultures were entirely resistant to beta AP-induced neurotoxicity and also demonstrated no dichlorofluorescein fluorescence or increased lipid peroxidation after beta AP treatment, Moreover, mature slices exposed to PAP in the presence of 25 mu M EUK-8 were significantly protected from beta AP-induced neurotoxicity. EUK-8 also completely blocked beta AP-induced free radical accumulation and lipid peroxidation. These results not only support a role for oxygen free radicals in beta AP toxicity but also highlight the therapeutic potential of synthetic radical scavengers in Alzheimer disease.