Electron microscopic studies: importance of embedding techniques in quantitative evaluation of cardiac mitochondrial...

This study applies the low protein-denaturation embedding technique for mitochondrial preparation to analyze control cardiac tissue, heart muscle subjected to 6 hours of regional ischemia without reperfusion, and myocardial tissue subjected to 4 to 6 hours of ischemia followed by reperfusion either with normal blood, with the heart in the beating, working state, or with substrate-enriched blood cardioplegia during total vented bypass. Parallel specimens of cardiac tissue were analyzed for ultrastructure by the conventional osmium tetroxide fixation method. Results following conventional tissue preparation with osmium tetroxide showed extensive ultrastructural damage in all hearts subjected to ischemia, with no correlation between ultrastructural findings and methods of reperfusion or functional recovery. In contrast, results following tissue preparation by the low protein-denaturation method showed reperfusion with normal blood in working hearts to cause severe mitochondrial damage, cardiac mitochondria that were intact structurally after 6 hours of ischemia and after controlled reperfusion, a logical sequence of mitochondrial structural changes that may lead to irreversibility, and a new method of quantification of such changes.