Biphasic changes in the levels of poly(ADP-ribose) polymerase-1 and caspase 3 in the immature brain following hypoxia–ischemia

Abstract

Poly(ADP-ribose) polymerase-1 (PARP-1) is a DNA repair-associated enzyme that has multiple roles in cell death. This study examined the involvement of PARP-1 in ischemic brain injury in the 7-day old rat, 0.5–48h after unilateral carotid artery ligation and 2h of 7.8% oxygen. This experimental paradigm produced a mild to moderate injury; 40–67% of animals in the ligated groups had histological evidence of neuronal death. Ipsilateral cortical injury was seen at all survival times, while mild contralateral cortical injury was seen only at the 1h survival time. Hippocampal injury was delayed relative to the cortex and did not show a biphasic pattern. Immunohistochemical staining for PARP showed bilateral increased staining as early as 1h post-hypoxia. PARP staining at early time periods was most intense in layer V of cortex, but did not demonstrate a pattern of cell clusters or columns. Ipsilateral PARP-1 levels quantified by western blotting showed a biphasic pattern of elevation with peaks at 0.5 and 12h post-hypoxia. Contralateral PARP-1 levels were also elevated at 0.5 and 24h. PARP activity as determined by immunoreactivity for poly(ADP-ribose) (PAR) was increased ipsilaterally at 0.5, 2 and 12h survival times. Cortical caspase 3-activity was increased ipsilaterally at 6, 12, and 24h and contralaterally at 0.5, 1, 2 and 6h post-hypoxia.There are three main findings in this study. First, changes in the distribution and amount of cell death correlate well with measured PARP-1 levels after hypoxia–ischemia, and both display biphasic characteristics. Second, there are significant early, transient morphological and biochemical changes in the contralateral cortex after neonatal hypoxia–ischemia due to unilateral permanent occlusion of a carotid artery followed by 2h of systemic hypoxia. Third, variability in the responses of individual pups to hypoxia–ischemia suggests the presence of unidentified confounding factors.