Abstract 1293 Neonatal Disease Oriented Research: Molecular Events and Brain Injury Poster Symposium, Tuesday, 5/4

Previous studies have shown that Bax and Bcl-2 proteins regulate programmed cell death. Bax, the cell death effector, forms a heterodimer with Bcl-2, the cell death repressor. The ratio of these two nuclear membrane proteins determines cellular susceptibility to apoptotic stimuli. The present study tests the hypothesis that expression of Bax protein increases as a function of decrease in cerebral energy metabolism. Studies were conducted on 10 anesthetized and ventilated, 3-5 days old newborn piglets. The normoxic group (n=3) was exposed to room air. In the hypoxic group, varying degrees of cerebral energy metabolism were achieved by ventilating with decreasing concentrations of O2 (15-4%) for 1 hr (n=7). Cerebral hypoxia was documented biochemically by measuring tissue ATP and phosphocreatine levels. Cerebral cortical neuronal nuclei were isolated and purified using a discontinuous 2.1M sucrose gradient. Bax and Bcl-2 proteins in neuronal nuclear membranes were analyzed by Western blot. The proteins were separated using 12% SDS-PAGE, transblotted onto nitrocellulose membranes and probed using specific antibodies directed against Bax and Bcl-2. Protein bands were detected using enhanced chemiluminescence and analyzed using imaging densitometry (GS-700-imaging densitometer Bio-Rad). The density of proteins was expressed as absorbance (OD × mm2). During graded hypoxia, as PCr decreased from 4.99 to 0.61 µmoles/g brain, the amount of Bax protein increased from 4.30 to 10.98 OD × mm2. Bcl-2 protein expression was not altered during graded hypoxia (6.11 Nx vs 6.02 ± 0.08 Hx). The increased expression of Bax protein correlated inversely with tissue ATP and PCr levels (r = 0.82, r = 0.85 respectively), with expression increasing as tissue high energy phosphates decreased during hypoxia. The results demonstrate that the decrease in cerebral energy metabolism during hypoxia is linearly correlated with an increase in expression of Bax protein. We speculate that the increased expression of Bax and altered ratio of Bax to Bcl-2 protein leads to hypoxia-induced programmed cell death in the newborn brain.

(Funded by NIH-HD-20337)