Reduced expression of DNA glycosylases in post-hypoxic newborn pigs undergoing therapeutic hypothermia.

Supplementary oxygen during resuscitation of the asphyxiated newborn is associated with increased generation of reactive oxygen species and oxidative stress. It is suspected that hyperoxic reoxygenation may cause increased damage to DNA, resulting in replication errors, and cell death or potential fixation of mutations if unrepaired. Therapeutic hypothermia may attenuate the development of brain damage after asphyxia, but it is not known how post-hypoxic hyperoxia and hypothermia affect accumulation of DNA-damage and DNA repair. Anaesthetised newborn pigs were randomised to control (n=6) or severe global hypoxia (n=46). After 20min of reoxygenation with either room air or 100% O(2), followed by 6.5h of normothermia (deep rectal temperature 39°C) or total body cooling (35°C), oxidative DNA damage (8-hydroxy-2'-deoxyguanosine) in brain, liver and urine, and transcription of DNA repair glycosylases (NEIL1, NEIL3, and OGG1) in brain and liver were measured. Hypoxic pigs displayed increased urinary 8-oxodG levels: mean (SD) 8-oxodG/creatinine was 3.55 (1.46) vs. control 2.02 (0.53), p<0.05, but levels were not affected by hyperoxia or hypothermia. Accumulation of 8-oxodG in the brain and liver did not differ across groups. Post-hypoxic transcription of DNA glycosylases was down-regulated by hypothermia: OGG1 in hippocampus and liver (p<0.01); NEIL1 in hippocampus (p<0.01), cortex and striatum (p<0.05) and liver (p<0.001); and NEIL3 in hippocampus (p<0.01) and cerebellum (p<0.001). Hyperoxia did not affect transcription of glycosylases in the brain. We confirm increased oxidative stress after hypoxia. DNA repair glycosylases were down-regulated by hypothermia but with no effect on accumulation of oxidative damage in genomic DNA.

Resuscitation of hypoxic piglets with 100% O2 increases pulmonary metalloproteinases and IL-8.

We hypothesized that resuscitation with 100% O2 compared with 21% O2 is detrimental to pulmonary tissue. The pulmonary injury was assessed by matrix metalloproteinase (MMP) activity, oxidative stress, IL-8, and histology 2.5 h after resuscitation from a hypoxic state. In pulmonary tissue extracts, MMP activity was analyzed by broad matrix-degrading capacity (total MMP) and zymography. MMP-2 mRNA expression was evaluated by quantitative real-time PCR. Total endogenous antioxidant capacity was measured by the oxygen radical absorbance capacity (ORAC) assay, and IL-8 was analyzed by ELISA technique. In bronchoalveolar lavage (BAL) fluid, MMPs were analyzed by zymography. In pulmonary tissue, pro- and active MMP-2 levels were increased in piglets that were resuscitated with 100% O2 compared with 21% O2. Pro-MMP-9, total MMP activity, and MMP-2 mRNA levels were significantly increased in resuscitated piglets compared with baseline. Net gelatinolytic activity increased in submucosa and blood vessels after 100% O2 and only in the blood vessels after 21% O2. Compared with baseline, ORAC values were considerably lowered in the resuscitated piglets and significantly reduced in the 100% O2 versus 21% O2 group. In BAL fluid, both pro-MMP-9 and pro-MMP-2 increased 2-fold in the 100% O2 group compared with 21% O2. Moreover, IL-8 concentration increased significantly in piglets that were resuscitated with 100% O2 compared with 21% O2, suggesting a marked proinflammatory response in the pulmonary tissue. Altogether, these data strongly suggest that caution must be taken when applying pure O2 to the newborn infant.