Background Neonatal hypoxic-ischemic (HI) injury is mediated by both caspase-dependent and caspapse-independent pathways. Prior studies in our laboratory have shown that deficiency of p53 decreases neonatal HI by 50%, suggesting that p53 is involved in neuronal cell death following neonatal HI. However, the mechanism of p53 action in neonatal HI is unknown.
Objective To determine whether neonatal HI results in changes of subcellular localization of p53 protein or in protein levels of p53-regulated proteins, including mdm2, a p53 inhibitor or in apoptosis-inducing factor (AIF), a caspase-independent regulator of apoptosis.
Methods Postnatal day 7 mice underwent left carotid ligation followed by 45 min exposure to 8% oxygen and then were sacrificed 0, 1, 3, 6, 12, 24, and 48 hrs later. Frozen 10 micron-thick coronal brain sections were stained with cresyl violet or processed for immunohistochemistry using antibodies against cleaved "activated " caspase 3, glial fibrillary acidic protein (GFAP), p53, mdm2, and AIF. Alternatively, nuclear and non-nuclear fractions were prepared from left and right cortex and hippocampal homogenates at the same time points.
Results At time 0, mdm2 immunoreactivity was most prominent in the CA2-CA3 region of the hippocampus but decreased markedly in the ischemic left hippocampus 3-6 hours following neonatal HI and then returned to baseline levels 24 hrs after neonatal HI. In contrast, both p53 and AIF immunostaining was observed within the CA1 region of ischemic hippocampus 6-12 hrs following neonatal HI. Subcellular fractionation experiments showed p53 immunoreactivity peaked 6-12 hrs in non-nuclear fractions of ipsilateral cortex and hippocampus and then decreased by 48 hrs.
Conclusion These studies suggest that p53 and regulators of p53 action are involved in neonatal HI.
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