Apoptotic cell death makes a minor contribution to reperfusion injury in skeletal muscle in the rat

Objective: to determine if apoptotic cell death contributes to skeletal muscle reperfusion injury. Methods: leg ischaemia was induced in rats with a tourniquet and maintained for 4 h before reperfusion for 24 or 72 h. Apoptosis was assessed by morphology, in situ end labelling of DNA fragments, DNA laddering, expression of p53 mRNA and detection of caspase-3-like proteolytic activity. Results: increased caspase-3-like activity was detected in muscle following ischaemia and zero, 24 h or 72 h of reperfusion. Levels remained relatively low but with a highly significant difference in enzyme activity between the ischaemic and non-ischaemic legs (p<0.0001, Repeated Measures Analysis of Variance). Morphological examination showed considerable oedema, disruption of muscle fibres and infiltration of white cells into tissues. Muscle nuclei did not show any morphological evidence of apoptosis and were negative for DNA fragmentation, while occasional neutrophils contained fragmented DNA. Expression of p53 was not induced by ischaemia and reperfusion and DNA ladders were not detected. Conclusions: the cells undergoing apoptosis were infiltrating neutrophils rather than muscle cells and reperfused muscle was damaged largely by an inflammatory process involving considerable oedema.