Abstract 1997
Poster Session II, Sunday, 5/2 (poster 226)
Previous studies have shown that hypoxia in the guinea pig fetus results in modification of cerebral cortical nuclear membranes resulting in increased high affinity Ca++-ATPase activity and alteration of calcium influx into the nucleus. The increased intranuclear calcium activates nuclear calcium dependent-endonuclease, which cuts genomic DNA at intranucleosomal cleavage sites, causing specific DNA fragmentation pattern, a characteristic of programmed cell death. The present study tests the hypothesis that the fragmentation of neuronal genomic DNA increases with increase in the degree of cerebral hypoxia. Seven anesthetized and ventilated newborn piglets (3 to 5 days old) were exposed to different degrees of hypoxia (FiO2 ranging from 0.21 - 0.05, n=5) for 60 min. Cerebral hypoxia was documented biochemically by measuring tissue levels of ATP and phosphocreatine (PCr). Cerebral cortical nuclei were isolated and purified using a discontinuous sucrose gradient. DNA was isolated by phenol/chloroform/isoamyl-alcohol extraction method. DNA content was measured by absorbance at 260 nm and purity confirmed by a ratio of >1.7 at 260/280 nm. DNA samples were separated by electrophoresis on 1% agarose gel and stained with ethidium bromide. DNA base-pair fragments were compared to a standard DNA ladder of 1 Kb. The densities of the DNA fragments were assessed using Molecular Analyst (BioRad). In the hypoxic samples, unlike in control samples, multiple low molecular weight fragments ranging in size from 3000 to 8000 base-pairs were observed. Levels of high energy phosphates were compared to the area of each smear for each animal to correlate degree of hypoxia with degree of DNA damage. ATP/PCr/area under the peak of smear were: 5.43/5.0/71.0, 4.08/3.36/437.4, 2.86/2.59/2293.0, 1.67/0.91/2342.0, 1.38/1.1/2635.74, 1.09/0.94/2134.22, 1.05/0.61/2066.16. DNA fragmentation increased when high-energy phosphate levels decreased by >50% compared to control. There was no significant difference in the degree of fragmentation with further decreases in ATP and PCr levels from 50% to 80%. We conclude that there is a critical threshold value of oxidative metabolism below which there are progressive changes in the cortical neuronal cells leading to DNA fragmentation. In contrast to our previous studies demonstrating that neuronal plasma membrane Na+, K+-ATPase activity decreased when cerebral energy levels decreased by > 30% compared to baseline, intranuclear changes (DNA fragmentation) appeared when the level of energy depletion was > 50%. We speculate that while energy dependent biochemical processes at the plasma membrane require high concentrations of ATP (mM), the energy requirement for intranuclear functions is lower (µM), thus a higher critical threshold of hypoxia is necessary to induce intranuclear DNA fragmentation.
