A novel dual staining method for identification of apoptotic cells reveals a modest apoptotic response in infarcted mouse myocardium.

Confocal scanning laser microscopy was used to investigate the myocardium of control C57BL/6 and plasminogen activator inhibitor 1 knockout (PAI-1KO) mice 3 days following persistent ligation of the left descending coronary artery. Paraffin sections taken from infarcted areas of the left ventricle were stained with antibodies recognizing cardiomyocytes, neutrophils, macrophages and apoptotic cells. In both animal groups, a strong neutrophil response was noted in the infarcted myocardium, with a large proportion of these cells also displaying staining for anti-alpha-sarcomeric actin in the PAI-1KO animals. Abundant macrophages were also identified in the infarcted regions of both animal groups, forming demonstrable streams at the border region in the C57BL/6 control animals. Surprisingly, only sparse cells from both animal groups were labeled with the apoptotic markers anti-cleaved caspase 3 antibody and anti-single stranded DNA antibody (following formamide treatment). A dual immunostaining protocol was developed to localize both of these apoptotic markers in the same cell. Again, only scattered cells were found displaying both markers in the zones of infarction, suggesting that 3 days of persistent ischemia results in a robust necrotic response, but only a very minor apoptotic response in this mouse model.