Regional Vulnerability to Endogenous and Exogenous Oxidative Stress in Organotypic Hippocampal Culture

Organotypic cultures of the brain provide a unique opportunity to directly examine the regional vulnerability of specific brain regions like the hippocampus. Two well-characterized models of oxidative stress were used to examine the regional vulnerability of the hippocampus. Endogenous oxidative stress was induced by blocking synthesis of the endogenous antioxidant, glutathione with buthionine sulfoximine (BSO). Exogenous oxidative stress was induced with paraquat, an intracellular generator of superoxide. Injury was measured by quantitative fluorescence microscopy using the vital dye propidium iodide. BSO caused dose- and time-dependent injury that took at more than 24 h to develop. Injury began in discrete patches in the culture. In any given culture, each patch increased in size and intensity as incubation continued. The pattern was not clearly correlated with neuronal anatomy and may demonstrate glial vulnerability. Injury caused by BSO could be prevented with the antioxidants trolox or the 21-aminosteroid U-83836E, both of which are vitamin E derivatives. Paraquat also caused dose- and time-dependent injury, but the CA1 region of the hippocampus was most vulnerable. The same pattern of selective CA1 injury was caused by brief exposures to high concentrations and by prolonged exposures to much lower concentrations. Under some conditions, paraquat injury was prevented by iron chelation with deferoxamine or by blockade of either NMDA or AMPA/kainate glutamate receptors. During paraquat exposure, glutathione concentration in the cultures was reduced prior to onset of propidium staining. The observation that the hippocampus has a similar selective regional pattern of vulnerability to paraquat and ischemia suggests that their mechanisms of injury may be related.