A DETAILED ANALYSIS OF HYDROGEN PEROXIDE-INDUCED CELL-DEATH IN PRIMARY NEURONAL CULTURE

A variety of neurodegenerative disease states have been associated wit h oxidative damage or stress. Such stress is thought to be mediated by excessive exposure of cells to reactive oxygen species such as free r adicals, which can be generated following cell lysis, oxidative burst (as part of the immune response) or by the presence of an excess of fr ee transition metals. Since the neuronal death observed in neurodegene rative diseases may be related to free radical damage, we were interes ted in developing a model system to investigate the mechanisms by whic h reactive oxygen species map damage or kill neurons. To this end, we have recently reported that brief exposure of cultured cortical neuron s to H2O2 can induce neuronal death that proceeds via an apoptotic cel l suicide pathway. The studies reported here investigate H2O2-induced cell death in more detail. Our data suggest that exposure of cultured cortical neurons to H2O2 can induce apoptotic cell death within 3 h, a s assessed by cell viability, morphological and ultrastructural measur es. In addition, experiments presented show that exposure to high conc entrations of H2O2 (100 mu M) causes increases in intracellular free c alcium within 3 h, suggesting that increased intracellular calcium may be associated with some aspects of H2O2-induced cell death. However, at intermediate concentrations of H2O2 (30 mu M), intracellular calciu m remained stable during a 3 h exposure, during which time membrane bl ebbing was observed in ultrastructural studies. This suggests that som e aspects of apoptotic cell death induced by H2O2 may not be associate d with increased intracellular free calcium. Thus, this model appears valuable for studies of the mechanism(s) by which oxidative injury may induce apoptotic cell death and damage to neurons in the CNS.