Pathologic assessment of myocardial cell necrosis and apoptosis after ischemia and reperfusion with molecular and morphological markers

The present work illustrates the critical subcellular changes in the rat heart after 10-30 min of left coronary artery (LCA) occlusion and 120 min of reperfusion with a combination of several staining techniques. Triphenyltetrazolium chloride (TTC) to detect non-injured myocytes, horseradish peroxidase (HRP) and terminal deoxynucleotide nick-end labeling (TUNEL) to detect necrotic and apoptotic cells were employed and electron microscopy (EM) was used to validate these changes, After 20 min of LCA occlusion, myocytes began to undergo necrosis whilst after 10 min occlusion, no myocyte underwent irreversible cell injury in the risk area. After 30 min of LCA occlusion and 120 min reperfusion, 36.3, 26.6 and 25% cells were normal, necrotic, and reversibly injured, respectively; the remaining 12.8% cells were apoptotic. Necrotic cells were strongly positive with HRP and negative for TTC and TUNEL. TUNEL-positive or apoptotic cells were slightly HRP-positive, indicating altered cell membrane permeability. Reversibly-injured myocytes were TTC-, HRP- and TUNEL-negative. These changes were more accurately defined in the 100-mu m thick sections than in the traditional slices. It is concluded that: (1) TTC-staining of 100-mu m thick sections is far superior and accurate for the detection of ischemic changes with shorter period of ischemia (10 min); (2) the combination of TTC-staining, HRP reaction and TUNEL, method is excellent for demarcation of early ischemic changes; (3) TTC-negativity in ischemia less than 20 min does not indicate necrosis but only represents reversible changes; (4) the apoptosis is absent in early ischemia of 20 min with or without reperfusion at a time when sufficient ATP is present, and appears only after 30 min of coronary ligation and reperfusion; and (5) the apoptotic cells lose membrane integrity accompanied by decreased glycocalyx thickness and cell swelling as opposed to commonly known characteristics of apoptotic cells. (C) 2000 Academic Press.