Caspase-dependent and caspase-independent neuronal injury in the developing brain
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.
Göteborgs universitet/University of Gothenburg
Department of Physiology
Avdelningen för fysiologi
Åke Göransson, F2421, Institutionen för fysiologi och farmakologi, Avdelningen för medicinsk fysik, Medicinaregatan 11, kl. 09.00
Date of defence
Zhu, Changlian 1964-