Maulik N, Yoshida T
Department of Surgery, University of Connecticut School of Medicine, Farmington 06030-1110, USA.
J Cardiovasc Pharmacol. 2000 Nov;36(5):601-8. doi: 10.1097/00005344-200011000-00009.
Apoptosis, a genetically controlled programmed cell death, has been found to play a role in ischemic reperfusion injury in several animal species including rats and rabbits. To examine whether this also is true for other animals, a surgically relevant model was established using an isolated in situ swine heart. Hearts were subjected to 15 min of normothermic regional ischemia by left anterior descending artery (LAD) occlusion followed by 30 min of normothermic cardioplegic arrest and 3 h of reperfusion. Oxygen free radicals have been shown to be the inducers of apoptosis and because reperfusion of ischemic myocardium is associated with the generation of free radicals, an additional group of hearts was preperfused with three different doses (5, 10, and 25 nM) ebselen, a glutathione peroxidase mimic, for 15 min before 15 min of LAD occlusion. Hearts were then subjected to 30 min of normothermic cardioplegic arrest followed by 3 h of reperfusion at normothermia. Control experiments were performed by perfusing the hearts for 4 h at normothermia. Two other groups of hearts were subjected to either 30 or 60 min of LAD occlusion followed by 30 min of cardioplegic arrest without subjecting them to reperfusion. At the end of each experiment, hearts were processed for the evaluation of apoptosis and DNA laddering. The in situ end-labeling (ISEL) technique was used to detect apoptotic cardiomyocyte nuclei while DNA laddering was evaluated by subjecting the DNA obtained from the cardiomyocytes to 1.8% agarose gel electrophoresis followed by photographing under UV illumination. The apoptotic cells appeared only after 90 min of reperfusion, as demonstrated by the intense fluorescence of the immunostained genomic DNA when observed under fluorescence microscopy. None of the ischemic hearts showed any evidence of apoptosis. These results were corroborated with the findings of DNA fragmentation showing increased ladders of DNA bands in the same reperfused hearts. The presence of apoptotic cells and DNA fragmentation in the myocardium was abolished by preperfusing the hearts in the presence of 10 nM ebselen, which also moderated the oxidative stress developed in the heart. Apoptotic cells and DNA ladders were completely absent in the hearts subjected to either 30 or 60 min of LAD occlusion. The results demonstrate that reperfusion of the ischemic heart induces apoptosis, which can be reduced with ebselen by reducing the oxidative stress associated with ischemia/reperfusion.
细胞凋亡是一种由基因控制的程序性细胞死亡，已发现在包括大鼠和兔子在内的几种动物物种的缺血再灌注损伤中发挥作用。为了研究这在其他动物中是否也成立，使用离体原位猪心脏建立了一个与手术相关的模型。通过左前降支动脉（LAD）闭塞使心脏经历15分钟的常温局部缺血，随后进行30分钟的常温心脏停搏和3小时的再灌注。氧自由基已被证明是细胞凋亡的诱导剂，并且由于缺血心肌的再灌注与自由基的产生有关，另一组心脏在LAD闭塞15分钟前用三种不同剂量（5、10和25 nM）的依布硒仑（一种谷胱甘肽过氧化物酶模拟物）预灌注15分钟。然后心脏进行30分钟的常温心脏停搏，随后在常温下再灌注3小时。通过在常温下灌注心脏4小时进行对照实验。另外两组心脏分别经历30或60分钟的LAD闭塞，随后进行30分钟的心脏停搏，不进行再灌注。在每个实验结束时，对心脏进行处理以评估细胞凋亡和DNA梯状条带。原位末端标记（ISEL）技术用于检测凋亡心肌细胞核，而通过将从心肌细胞获得的DNA进行1.8%琼脂糖凝胶电泳，然后在紫外光下拍照来评估DNA梯状条带。如在荧光显微镜下观察到的免疫染色基因组DNA的强烈荧光所示，凋亡细胞仅在再灌注90分钟后出现。没有一个缺血心脏显示出任何细胞凋亡的迹象。这些结果与DNA片段化的发现一致，表明在相同的再灌注心脏中DNA条带的梯状条带增加。通过在10 nM依布硒仑存在下预灌注心脏，心肌中凋亡细胞和DNA片段化的存在被消除，这也减轻了心脏中产生的氧化应激。在经历30或60分钟LAD闭塞的心脏中完全没有凋亡细胞和DNA梯状条带。结果表明，缺血心脏的再灌注诱导细胞凋亡，依布硒仑可以通过减少与缺血/再灌注相关的氧化应激来减少细胞凋亡。
