Effects Of Cardiopulmonary Bypass On Neutrophil Apoptosis And Related Protein In Pediatric Open-heart Surgery

Objective: Cardiopulmonary bypass (CPB) induces systemic inflammatory response. Polymorphonuclear neutrophil (PMN) plays a central role in this"inflammatory like response"to CPB by releasing oxidants and proteases that damage or kill tissues. Granulocyte apoptosis is a key control process in the clearance of neutrophil from inflamematory sites. Therefore, the desired therapy for truncating the inflammatory response would be to promote apoptosis.The aim of the study was to investigate the changes in neutrophil apoptosis and Fas, Bcl-2 protein expression undergoing open-heart surgery with CPB, and to approach the molecular mechanisms of the change in neutrophil apoptosis after CPB.Methods: Thirty pediatric patients with ventricular septal defect(13male,17female) aged 1-5yrs,weighing 10-20kg under- going open heart surgery with CPB were studied. Their cardiac function was well preserved (NYHA classⅠ-Ⅱ).The surgery was performed under fentanyl anesthesia supplemented with low concentration of isoflurane.Before aortic cannulation, 3mg/kg heparin was administered. The extracorporeal circuit consisted of hollow fiber membrane oxygenators and nonpulsatile occlusive roller pumps(Sarns 8000). The bypass circuit was primed with crystalloid, colloid, packed red blood cells. Full flow on bypass was calculated using a cardiac index of 2.2~2.8L·(min·m2)-1. Systemic perfusion pressure was maintained at a mean of 50~80mmHg. Core temperature was controlled using a heat exchanger in the bypass circuit and monitored with a nasopharyngeal probe. Heparin was reversed at the end of the procedure with protamine.Blood samples were taken after induction of anesthesia(T0), 30 minutes after the initiation CPB(T1), 30 minutes after the termination CPB(T2), 24 hours postoperatively(T3). Neutrophil was isolated by Ficoll-Hypaque density gradient centrifugation. Neutrophil apoptosis and the expression of neutrophil Fas, Bcl-2 protein was detected by Flow Cytometry(FCM).All statistical analyses were performed using the SPSS 10.0 program package. Data were expressed as the mean±standard deviation( x±s). Changes with time within individual group were analyzed by analysis of variances for repeated measurement data. Linear correlation was performed by Pearson correlation coefficient. P < 0.05 was considered to be statistically significant.Results:1. The duration of CPB is 52±17 minutes. he duration of aortic cross-clamping is 28±11 minutes. 2. The percentage of neutrophil apoptosis during CPB was significantly lower, and reached its lowest value at 30 minutes after the termination of CPB(1.79±0.57%), which had statistical significance compared with T0(P<0.05). It returned to the baseline value on 24h postoperatively. The percentage of PMN apoptosis correlated negatively with the duration of CPB (r=-0.648,P<0.05).3. FACS results showed that the expression of Fas on PMN membrane was significantly downregulated during CPB. It also reduced 30 minutes after the initiation CPB, which had statistical significance compared with T0(P<0.05), and began to return on 24h postoperatively. Fas levels correlated positively with PMN apoptosis rate (r=0.546, P<0.05).4. PMN intracellular expression of Bcl-2 was significantly upregulated during CPB. T1,T2,T3 had statistical significance compared with T0 (P<0.05). Bcl-2 levels correlated negatively with PMN apoptosis rate(r=-0.569, P<0.05).Conclusion:1. The neutrophil apoptosis can be delayed in children undergoing open-heart operation with CPB. Inflammatory mediators during CPB prolong the functional lifespan of neutrophil through modulation of apoptosis.2. The inhibition of neutrophil apoptosis has relation with the down regulation expression of Fas.3. The inhibition of neutrophil apoptosis has relation with the up regulation expression of Bcl-2.