Oxygen treatment after perinatal hypoxia-ischemia reduces neuronal damage at short survival times,but worsens injury with long-term survival

Brain injury resulting from hypoxic-ischemic (H/I) damage during the perinatal period is an important cause of neonatal morbidity and mortality. Infants with hypoxia are commonly treated with 100% oxygen, which may further exacerbate injury via free radical formation and DNA damage. PARP-1 is activated in response to DNA damage, but its role in H/I cell death remains uncertain. PARP-1 knock-out is neuroprotective in male, but not female perinatal mice (Hagberg et al., J Neurochem 90: 1068, 2004). It is unknown if there are sex differences in the response to H/I in outbred animals. Dated, pregnant Wistar rats were purchased from Charles River. HI was induced on day P7 by irreversible common carotid artery ligation and subsequent hypoxia. Pups were anesthetized with halothane and the left common carotid artery was electrocauterized. After recovery from anesthesia and surgery, pups returned to their mother for 2–4 hours. Sham-operated animals were anesthetized, and the carotid artery exposed, but not ligated. Pups were then exposed to hypoxia (8% oxygen/balance nitrogen) in a humidified Plexiglas box at 37 C for 2 hours. Hypoxia only (H) pups were returned to room air, while hypoxia/hyperoxia (HH) pups were exposed to 100% O₂ for another 2 hours. Pups were returned to their mother until euthanasia at survival times of 2, 6, 12, 48 and 96 hours, and 6 weeks after hypoxia (n=8 each sex per group per treatment). Brains were perfusion fixed with 4% paraformaldehyde and stained with H&E. The degree of injury was graded on a scale of 0–4. Statistical analysis was performed using Kruskal-Wallis analysis of variance, with post-hoc Mann-Whitney Rank Sum tests. When males and females were combined, H and HH groups at all survival times except 6 weeks H had significantly greater histologic injury scores compared to the corresponding sham groups. At 12 hr survival, the H group had more cortical injury than the HH group (1.94 +/− 1.3 vs 0.94 +/− 0.85, p = 0.02). At 6 weeks, however, the HH group had greater injury scores than the H group for both cortex (2.56 +/− 1.79 vs 1.0 +/− 1.46, p = 0.013), and hippocampus (2.47 +/− 1.9 vs 1.0 +/− 1.46). At other survival times the differences were not significant due to high intragroup variability. When groups were broken down by sex, at 12 hours only females had significant differences (H=2.63 +/− 1.41, HH=0.75 +/− 1.04, p=0.021). The female H group had greater injury than the male H group (2.63 +/− 1.41 vs 1.33 +/− 0.87, p=0.036). At 6 weeks, both HH females and HH males had more cortical injury than the corresponding H groups, but not statistically significant due to high variability within groups (fHH=2.67 +/− 1.73 vs fH=0.89+/−1.45, mHH=2.44 +/− 1.94 vs mH= 1.13 +/− 1.55). There were no differences in cortical or hippocampal injury between males and females at 6 weeks. These results indicate that while treatment with 100% oxygen may improve short-term outcome, it worsens brain injury at 6 weeks survival.