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 mus cle 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 morph ology, 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 relativel y low but with a highly significant difference in enzyme activity between t he ischaemic and non-ischaemic legs (p<0.0001, Repeated Measures Analysis o f Variance). Morphological examination showed considerable oedema, disrupti on of muscle fibres and infiltration of white cells into tissues. Muscle nu clei did not show any morphological evidence of apoptosis and were negative for DNA fragmentation, while occasional neutrophils contained fragmented D NA. Expression of p53 was not induced by ischaemia and reperfusion and DNA ladders were not detected. Conclusions: the cells undergoing apoptosis were infiltrating neutrophils r ather than muscle cells and reperfused muscle was damaged largely by an inf lammatory process involving considerable oedema.