Caspase-dependent and caspase-independent neuronal injury in the developing brain

Abstract

Aims: To investigate the mechanisms of neuronal cell death after hypoxia-ischemia in the developing brain.Methods: Rats or mice were subjected to left common carotid artery ligation plus hypoxia. Animals were sacrificed at certain time points after hypoxia-ischemia. The brains were collected for immunohistochemistry, western blotting, enzyme activity measurement and brain injury assessment.Results: Caspase-3 was activated after HI, and reached a peak level at 24 h post-HI. Immunoreactivity for active caspase-3 corresponded well with hybridization with an oligonucleotide hairpin probe, a marker of double-stranded DNA breaks with one nucleotide overhang in the 3 end. Calpain activation and, subsequently, Caspase-3 activation occurred after HI. Calpain inhibitors reduced cleavage of pro-caspase-3 into a 29 kDa product and decreased the activation of caspase-3. Calpain activation facilitated further activation of caspase-3. After HI, AIF was lost from mitochondria and the levels increased in nuclei (translocation) and, subsequently, signs of caspase-independent DNA damage occurred. AIF release occurred earlier than that of cytochrome c and correlated with tissue damage. A broad spectrum caspase inhibitor markedly reduced activation of caspase activity after HI, but still did not confer tissue protection. Nitrotyrosine formation preceded AIF release and caspase-3 activation. Nitrotyrosine is an early marker of cellular injury. Combined inhibition of nNOS and iNOS by 2-iminobiotin(a known neuroprotectant)reduced nitrotyrosine formation and caspase-3 activation. Immaturity greatly influences the outcome of HI, but not in a linear fashion. Apoptosis-related mechanisms of neuronal cell death after HI are much more prevalent in the developing brain, including both caspase-dependent and caspase-independent pathways.Conclusions: Several apoptotic mechanisms are activated in neonatal brain after HI. Inhibition of apoptotic mechanisms may be a fruitful neuroprotective strategy. Brain injury and neuronal cell death after hypoxia-ischemia is age-related and prevention and treatment of brain injury need to be adjusted to the developmental level.