Thornell L E, Holmbom B, Eriksson A, Reiz S, Marklund S, Näslund U
Department of Anatomy, University of Umeå, Sweden.
Histochemistry. 1992 Dec;98(6):341-53. doi: 10.1007/BF00271069.
The usefulness of different enzyme and immunohistochemical stains to distinguish reversible and irreversible myocardial cell injury after experimental coronary artery occlusion of varying duration and reperfusion with or without superoxide dismutase as adjunct was investigated. Biopsies or parts of the infarcted and non-infarcted area were rapidly frozen and sectioned in series for enzyme and immunohistochemical evaluation. Sections were stained for the demonstration of phosphorylase, myofibrillar ATPase and mitochondrial oxidative enzymes and also with periodic acid-Schiff, alizarin red S and routine histological stains. Other sections in series were stained with antibodies against fibronectin and the intermediate filament proteins desmin and vimentin. In 49 biopsies a blind quantitative estimation of the area stained for fibronectin, phosphorylase and alizarin red S was performed and evaluated statistically. Phosphorylase, periodic acid-Schiff, fibronectin and alizarin red S allowed delineation of affected myocardium after 30 min of ischaemia followed by reperfusion whereas with the other stains, affected myocardium was readily detectable only after 60 or 90 min of ischaemia followed by reperfusion as well as after 24 h of ischaemia without reperfusion. The immunostaining for fibronectin was very distinct and inversely related to the phosphorylase activity. We show that fibronectin is an excellent marker for damaged cells and that these positively stained myocytes are necrotic as confirmed ultrastructurally. Using alizarin red S as a marker of calcium accumulation in myocytes, a marked discrepancy was observed between the area of fibronectin-containing myocytes and that of myocytes stained by alizarin red S. Calcium accumulation in mitochondria is thus not a prerequisite for myocyte necrosis but does occur only in some of the irreversibly damaged cells. Of special interest is the finding that there was a significant reduction of intracellular calcium in pigs where superoxide dismutase had been used as an adjunct at reperfusion, thus supporting the theory that free radicals do play a role during reperfusion of ischaemic myocardium.
研究了不同的酶和免疫组织化学染色方法在区分实验性冠状动脉闭塞不同时长后再灌注（有无超氧化物歧化酶作为辅助）时可逆和不可逆心肌细胞损伤方面的效用。对梗死区和非梗死区的活检组织或部分组织进行快速冷冻，并连续切片用于酶和免疫组织化学评估。切片用于显示磷酸化酶、肌原纤维ATP酶和线粒体氧化酶，还用高碘酸-希夫试剂、茜素红S和常规组织学染色剂染色。连续的其他切片用抗纤连蛋白以及中间丝蛋白结蛋白和波形蛋白的抗体染色。对49份活检组织进行了纤连蛋白、磷酸化酶和茜素红S染色面积的盲法定量估计并进行统计学评估。磷酸化酶、高碘酸-希夫试剂、纤连蛋白和茜素红S能在缺血30分钟后再灌注时勾勒出受影响的心肌，而使用其他染色剂时，仅在缺血60或90分钟后再灌注以及缺血24小时未再灌注后才能容易地检测到受影响的心肌。纤连蛋白的免疫染色非常明显，且与磷酸化酶活性呈负相关。我们表明纤连蛋白是受损细胞的优良标志物，并且这些阳性染色的心肌细胞经超微结构证实为坏死。用茜素红S作为心肌细胞钙积聚的标志物，观察到含纤连蛋白的心肌细胞区域与茜素红S染色的心肌细胞区域之间存在明显差异。因此，线粒体中的钙积聚不是心肌细胞坏死的先决条件，但仅在一些不可逆受损细胞中发生。特别有趣的是发现，在再灌注时使用超氧化物歧化酶作为辅助的猪中，细胞内钙显著减少，从而支持了自由基在缺血心肌再灌注过程中确实起作用的理论。
