Difference of molecular response to ischemia-reperfusion of rat skeletal muscle as a function of ischemic time:: Study of the expression of p53, p21WAF-1, Bax protein, and apoptosis

The authors investigated the expression of p53, p21(WAF-1) Bax protein, and apoptosis to elucidate the cellular response to ischemia-reperfusion of skeletal muscle using the rat lower limb model. The rat left lower limb was dissected in the inguinal region, isolating the bony femoral muscles, and the femoral vessels were clamped to produce an ischemic condition. After 3 or 6 hours, the clamps were removed and the gastrocnemius muscle was resected at various times up to 72 hours after reperfusion. Five specimens of the muscle were obtained at each time point from 5 rats. When any rat died during the study, additional rats were used until 5 specimens could be obtained from 5 rats at each time point. The expression of three proteins was detected by Western blot analysis. The apoptotic cells were detected using terminal deoxytransferase-mediated dUDP (deoxyuridine[-5']diphosphate) nick-end labeling assay. Histopathological study showed severe interstitial edema and leukocyte infiltration at 6 hours of ischemia compared with 3 hours of ischemia. Moreover, at 6 hours of ischemia, muscle fiber fragmentation was observed at 72 hours after reperfusion whereas no fragmentation was found at 3 hours of ischemia. At 3 hours of ischemia, p53 and p21(WAF-1) accumulated after reperfusion, and there was a time lag in the time of onset of elevation and the peak time point between these two proteins. The level of Bax protein did not elevate and the rate of apoptotic cells did not increase. At 6 hours of ischemia, p53 and p21(WAF-1) also accumulated, but the kinetics of p21(WAF-1) were similar to that of p53 in the time of onset of elevation and the peak time point after reperfusion. In addition, the level of Bax protein increased and apoptosis was induced. These results demonstrated that p53 and p21(WAF-1) accumulated after 3 and 6 hours of ischemia of skeletal muscle during reperfusion. Moreover, it was demonstrated that the kinetics of induced p53, p21(WAF-1), and Bax protein differ between 3 hours and 6 hours of ischemia, and it is speculated that this difference plays an important role in determining the consequence of the cell exposed to ischemia.