Effect of post-hypoxic reoxygenation on DNA fragmentation in cortical neuronal nuclei of newborn piglets.
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Previous studies have shown an increased fragmentation of genomic DNA following hypoxia in cortical neuronal nuclei of newborn piglets. The present study tests the hypothesis that DNA fragmentation following hypoxia persists during reoxygenation in cortical neuronal nuclei of newborn piglets. To test this hypothesis, DNA fragmentation was assessed in 36 newborn piglets divided into six groups: normoxic (Nx), hypoxic (Hx) and hypoxic/reoxygenated for 6, 12, 24h and 7 days. The Hx groups were exposed to 7% oxygen for 1h followed by reoxygenation to room air for 6, 12, 24h and 7 days. Cerebral tissue hypoxia was confirmed biochemically by ATP and phosphocreatine (PCr) levels. Nuclei were isolated and purified using discontinuous sucrose gradient. DNA was isolated by phenol/chloroform/isoamyl-alcohol extraction method. ATP/PCr (micromol/g brain) were 4.11+/-0.15/3.67+/-0.30 for Nx, 1.31+/-0.68/0.74+/-0.30 for Hx, 3.81+/-0.11/3.24+/-0.14 for 6h reoxygenation, 4.21+/-0.12/3.27+/-0.09 for 12h reoxygenation and 4.63+/-0.09/3.75+/-0.27 for 24h reoxygenation and 4.31+/-0.12/3.70+/-0.21 for 7 days reoxygenation. There was a significant difference in the ATP and PCr values between Nx and Hx groups (p<0.05) and between Hx and hypoxic reoxygenated groups (p<0.05). DNA fragments (OD/mm(2)) increased from 1776+/-267 in the Nx group to 3211+/-285 in the Hx group (p<0.05). In the reoxygenation groups, DNA fragments (OD/mm(2)) decreased to 2018+/-249 after 6h (p<0.05 versus Hx) but increased to 3408+/-206, 2782+/-406 and 3256+/-302 after 12, 24h and 7 days, respectively. The data show a decrease in DNA fragmentation in the early phase (6h) of reoxygenation but is comparable to acute hypoxia during the later phases (12, 24h and 7 days) of reoxygenation. We propose that the biphasic pattern of DNA fragmentation during reoxygenation occurs by an initial oxidative DNA injury followed by an enzymatic cleavage of DNA by endonucleases activation.
