Abstract 235
Poster Session III, Monday, 5/3 (poster 120)
Previous studies have shown that DNA fragmentation is a biochemical hallmark of programmed cell death induced by endonucleases during normal processes of DNA repair or under hypoxic DNA damaging conditions. The present study tests the hypothesis that post-hypoxic maternal treatment with MgSO4 will attenuate hypoxia-induced DNA fragmentation in the fetal guinea pig cerebral cortex. Eighteen pregnant guinea pigs at term (60 days) were studied. Fetuses from normoxic mothers (Nx, n=6) were compared to those from hypoxic (Hx, n=6) and MgSO4-treated hypoxic (post-HxMg, n=6) animals. Hypoxia was induced by exposure to a FiO2 of 8% for 1 hr. Following hypoxia the treated group received a 300 mg/kg MgSO4 bolus dose, and then 100 mg/kg/hr doses for 4 hr prior to recovery. Fetal brain tissue was obtained following 24 hr of in utero recovery in all groups. Fetal cerebral hypoxia was documented biochemically by measuring ATP and phosphocreatine tissue levels. Cerebral cortical nuclei were isolated and purified using a discontinuous sucrose gradient. DNA was isolated by phenol / chloroform /isoamyl-alcohol extraction method and content was measured by absorbance at 260 nm. DNA (0.5 µg) was electrophoresed on a 1% agarose gel and stained with ethidium bromide. DNA base-pair fragments were compared to a standard DNA ladder of 1 Kb. The density of the DNA fragments was analyzed by molecular analyst Gel Doc 1000 (Biorad). In Hx samples DNA damage was observed in a "smear" pattern, indicating fragments ranging in sizes from 300 to 12000 basepairs (mean amplitude of peak area: 830.36 ± 244). In nuclei of the post-HxMg group DNA pattern indicated significantly fewer fragments than in hypoxia (Hx) mainly in the 8000-10000 basepairs range (mean amplitude of peak area: 362 ± 225), however the amount of fragmentation detected was still greater than control (Nx) levels (mean amplitude of peak area: 138 ± 38). The degree of DNA damage among the three groups was significantly different (p=0.012, One Way Anova). The data demonstrate that MgSO4 administration post in utero hypoxia attenuated hypoxia-induced DNA cleavage at 24 hr after the hypoxic insult. We speculate that even following hypoxia, administration of Mg++ results in blockade of the NMDA receptor, thus preventing the NMDA receptor-mediated increase in intracellular calcium which leads to oxygen free-radical generation and randomized DNA fragmentation, thereby attenuating the sequelae of excitotoxicity. (Funded by NIH HD 20337)
