The Effect of Reoxygenation with 21% or 100% Oxygen on Hypercapnic-Hypoxia Induced DNA Damage in Cerebral Cortices of Newborn Piglets † 1024

Previous studies have shown that both hypoxia, and hypoxia followed by reoxygenation with either 21% or 100% oxygen are associated with significant generation of free radicals in the cerebral cortices of newborn piglets. The present study investigates the DNA degradation pattern associated with cell death following cerebral hypercapnic hypoxia. Studies were performed on groups(n=4 each) of anesthetized and ventilated newborn piglets. Group 1 was a room air control, and Groups 2 and 3 were exposed to 1hr of hypercapnic-hypoxia followed by reventilation with either 21% or 100% oxygen for 45 minutes, respectively. Group 4 was made hypercapnic and hypoxic but was not reventilated. Hypercapnic hypoxia was achieved by slow inhalation of CO₂ and a reduction in the FiO₂ to 0.05-0.07. Prior to reventilation, mean values for pH, PaO₂ and PaCO₂ were 7.05, 20 mmHg and 82 mmHg respectively. After the period of reventilation, brain tissue was removed for analysis. ATP and phosphocreatine (PCr) concentrations were determined biochemically as indices of brain tissue energy metabolism. Mean concentrations of ATP in Grp 1, and the 21% (Grp 2) and 100% (Grp 3) reventilated groups were 4.26, 5.18 and 4.40 mmol/ g brain; PCr values were 3.71, 3.65 and 3.18 mmol/g brain, respectively. Brain cell nuclei and DNA were isolated and the pattern of DNA degradation was assessed using 1% agarose gel-electrophoresis. The gel pattern in Group 1 showed a single band of DNA. High-molecular weight DNA appeared as a large smear in Groups 2 and 3. Although the gel pattern in Group 4 revealed DNA degradation predominantly as a continuous smear, discrete bands of DNA fragments were identified in the lower molecular weight range (2,000-100 bp). The presence of a smear as opposed to a ladder-type pattern implies random fragmentation of DNA, possibly mediated by free radicals, and not due to the action of specific endonucleases. We speculate that irrespective of the O₂ concentration used during reoxygenation, the molecular structure of the DNA is altered without being cleaved into small fragments.