心肺复苏后大鼠脑线粒体通透性转换孔开放对线粒体呼吸功能的影响

目的 研究大鼠心肺复苏(CPR)后神经细胞线粒体通透性转换孔(MPTP)开放对线粒体呼吸功能的影响,并探讨其可能机制.方法 建立窒息加冰KC1致大鼠心搏骤停(CA)/CPR动物模型,在自主循环恢复(ROSC)后3、6、12、24、48和72 h断头处死大鼠制备大脑皮质内线粒体匀浆.采用分光光度法测定线粒体MPTP的开放程度,用Clark氧电极法分析线粒体呼吸功能,并电镜下观察线粒体超微结构.结果 CA/CPR后大鼠神经细胞线粒体功能明显受损,线粒体呼吸Ⅲ态(R3)速率下降;ROSC后神经细胞MPTP持续处于开放状态,开放程度并不是瞬间增至最大,而是具有时间依赖性.具体表现为:ROSC后6h内神经细胞MPTP开放程度保持低水平,6 h以后开始迅速大量开放,12 h开放程度达到最大,24 h开放程度略有缩小,表明线粒体开始收缩.至48 h开放程度再次加大.72 h又明显缩小,但未达到正常水平(P均＜0.05).虽然线粒体R3速率下降.但线粒体呼吸Ⅳ态(R4)速率升高,呼吸控制率(RCR)和磷/氧(P/O)比值明显下降;随着ROSC时间延长,RCR和P/O比值持续在低水平状态(P＜0.05或P＜0.01).透射电镜下观察细胞有明显损伤.相关分析表明,MPTP开放与RCR呈明显正相关(r=0.025,P＜0.05).结论 CPR后MPTP开放是加重神经细胞能量代谢障碍的主要原因,在ROSC后早期即12 h以内如能及时应用抑制MPTP开放的策略,可能会使神经细胞的线粒体功能朝着良性方向发展.为神经功能的恢复赢得机会.

Effect of opening of neuronal mitochondrial permeability transition pore on respiratory function after cardiopulmonary resuscitation in rats

Objective To investigate the effect of opening of neuronal mitochondrial permeability transition pore (MPTP) on respiratory function after cardiopulmonary resuscitation (CPR) in rats and its possible mechanism.Methods Cardiac arrest (CA)/CPR rat model was reproduced by asphyxiation and ice-cold KCI followed resuscitation and restoration of spontaneous circulation (ROSC). The rats were sacrificed by decapitation at 3,6,12,24,48 and 72 hours.Isolation of brain cortex neuronal mitochondria was processed.MPTP opening degree was examined by speetrophotometer.Clark oxygen electrode was used to measure mitochondrial respiratory function: the mitochondrial ultra structure was examined with transmission electron microscope (TEM).Results Mitochondrial respiratory function was severely injured after CA/CPR. Mitochondrial respiratory state 1 (R3) was decreased. Neural cell MPTP opened persistently after ROSC.The opening degree of MPTP did not reach the peak instantly,and its change depended on time.It remained at a low level within 6 hours after ROSC,then rapidly opened,reaching the maximal degree at 12 hours,but it became smaller at 24 hours.At 48 hours the degree of opening became larger again,but shrank once more at 72 hours.However,it did not reach the normal level (all P＜0.05).Although R3 was decreased,mitoehondrial respiratory state Ⅳ(R4) was increased,meanwhile the respiratory control rate (RCR) and P/O ratio descended markedly.They maintained at low levels along with the elapse of time (P＜0.05 or P＜0.01).TEM revealed obvious injury to neurons.Correlation analysis showed that the MPTP opening degree and RCR was obviously positively correlated (r=0.025,P＜0.05).Conclusion The opening of MPTP is the main cause of aggravation of energy metabolism disturbence of neural cells after CPR.To take measures to inhibit the opening of MPTP within 12 hours after ROSC may promote improvement of neuronal mitochondrial function,and it might help win the chances for neural function to recover.