Apoptotic neuronal death following deep hypothermic circulatory arrest in piglets.

Abstract

Deep hypothermic circulatory arrest (DHCA), as used in infant heart surgery, carries a risk of brain injury. In a piglet DHCA model, neocortical neurons appear to undergo apoptotic death. Caspases, cytochrome c, tumor necrosis factor (TNF), and Fas play a role in apoptosis in many ischemic models. This study examined the expression of these factors in a DHCA piglet model. Thirty-nine anesthetized piglets were studied. After cardiopulmonary bypass (CPB) cooling of the brain temperature to 19 degrees C, DHCA was induced for 90 min, followed by CPB rewarming. After separation from CPB, piglets were killed at 1, 4, 8, 24, and 72 h and 1 week. Caspase-8 and -3 activity, and concentrations of TNF-alpha, Fas, Fas-ligand, cytochrome c, and adenosine triphosphate (ATP) were measured in the neocortex by enzymatic assay and Western blot analysis. Caspase-8 and -3 activity and cell death were examined histologically. Significance was set at P < 0.05. In neocortex, damaged neurons were not observed in control (no CPB), rarely observed in CPB (no DHCA), and rarely observed in the DHCA 1-h, 4-h, and 1-week reperfusion groups. However, they were seen frequently in the DHCA 8-, 24-, and 72-h reperfusion groups. Although neuronal death was widespread 8-72 h after DHCA, cortical ATP concentrations remained unchanged from control. Both caspase-3 and -8 activities were significantly increased at 8 h after DHCA, and caspase-3 concentration remained elevated for as long as 72 h. Caspase-3 and -8 activity was also observed in damaged neocortical neurons. Cytosolic cytochrome c and Fas were significantly expressed at 1 h and 4 h after DHCA, respectively. Fas-ligand and TNF-alpha were not observed in any group. After DHCA, induction of apoptosis in the neocortex occurs within a few hours of reperfusion and continues for several days. Increased Fas, cytochrome c, and caspase concentrations, coupled with normal brain ATP concentrations and apoptotic histologic appearance, are consistent with the occurrence of apoptotic cell death.