Effect of post-hypoxic reoxygenation on DNA fragmentation in cortical neuronal nuclei of newborn piglets

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, 24 h and 7 days. The Hx groups were exposed to 7% oxygen for 1 h followed by reoxygenation to room air for 6, 12, 24 h 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 (μmol/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 6 h reoxygenation, 4.21 ± 0.12/3.27 ± 0.09 for 12 h reoxygenation and 4.63 ± 0.09/3.75 ± 0.27 for 24 h 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/mm2) 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/mm2) decreased to 2018 ± 249 after 6 h (p < 0.05 versus Hx) but increased to 3408 ± 206, 2782 ± 406 and 3256 ± 302 after 12 24 h and 7 days, respectively. The data show a decrease in DNA fragmentation in the early phase (6 h) of reoxygenation but is comparable to acute hypoxia during the later phases (12, 24 h 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.