A detailed analysis of hydrogen peroxide-induced cell death in primary neuronal culture

Abstract A variety of neurodegenerative disease states have been associated with oxidative damage or stress. Such stress is thought to be mediated by excessive exposure of cells to reactive oxygen species such as free radicals, which can be generated following cell lysis, oxidative burst (as part of the immune response) or by the presence of an excess of free transition metals. Since the neuronal death observed in neurodegenerative disease may be related to free radical damage, we were interested in developing a model system to investigate the mechanisms by which reactive oxygen species may damage or kill neurons. To this end, we have recently reported that brief exposure of cultured cortical neurons to H 2 O 2 can induce neuronal death that proceeds via an apoptotic cell suicide pathway. The studies reported here investigate H 2 O 2 -induced cell death in more detail. Our data suggest that exposure of cultured cortical neurons to H 2 O 2 can induce apoptotic cell death within 3 h, as assessed by cell viability, morphological and ultrastructural measures. In addition, experiments presented show that exposure to high concentrations of H 2 O 2 (100 μM) causes increases in intracellular free calcium within 3 h, suggesting that increased intracellular calcium may be associated with some aspects of H 2 O 2 -induced cell death. However, at intermediate concentrations of H 2 O 2 (30 μM), intracellular calcium remained stable during a 3 h exposure, during which time membrane blebbing was observed in ultrastructural studies. This suggests that some aspects of apoptotic cell death induced by H 2 O 2 may not be associated 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.