**3.1 Animal experiment in permanent occlusion of coronary artery**

In a rat model of permanent coronary artery ligation, Kajstura et al. investigated the contribution of the apoptosis and necrosis to cardiomyocyte death using TUNEL method and DNA laddering for apoptosis and antimyosin monoclonal antibody labeling (Kajstura, et a., 1996). After 2 h of the left main coronary artery ligation, TUNEL positive myocytes appeared in the central portion of the left ventricular free wall and peaked at 4.5 h. Myosin labeled cells also appeared at 2 h after ligation and significantly increased after 6 h to 2 days. However, at 2 h after coronary ligation, number of apoptotic cells was 2.8 x 106 and number of necrotic cells was 9 x 104. Therefore, early after myocardial infarction, apoptosis is the predominant form of cell death, and 1 to 2 days after infarction, necrosis is the dominant form of cell death followed by low levels of both apoptosis and necrosis at 7 days after infarction. If cells undergoing apoptosis are not cleared before they deplete their intracellular ATP stores that are necessary to maintain plasma membrane integrity, plasma membrane permeability barrier breakdown occurs and cells are converted from apoptosis to necrosis as known as secondary necrosis, results in inflammation.
