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

Abstract

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.

Muscle apoptosis is induced in pressure-induced deep tissue injury

“…It would be interesting to compare the present results to the findings obtained from complete ischemia. Although the experimental protocol of our study was not exactly the same from the study conducted by Hatoko and colleagues (14), our findings generally shared the similarities in the results that apoptotic signaling responded to 6 h of muscle ischemia. By clamping the femoral blood vessels, ischemic condition was produced in rat gastrocnemius muscle (14).…”

“…Although the experimental protocol of our study was not exactly the same from the study conducted by Hatoko and colleagues (14), our findings generally shared the similarities in the results that apoptotic signaling responded to 6 h of muscle ischemia. By clamping the femoral blood vessels, ischemic condition was produced in rat gastrocnemius muscle (14). They reported that the protein level of p53, p21 , and Bax, as determined by Western immunoblotting, was not changed immediately after 6 h of ischemia alone but was gradually increased during the subsequent 12-72 h of reperfusion (14).…”

“…By clamping the femoral blood vessels, ischemic condition was produced in rat gastrocnemius muscle (14). They reported that the protein level of p53, p21 , and Bax, as determined by Western immunoblotting, was not changed immediately after 6 h of ischemia alone but was gradually increased during the subsequent 12-72 h of reperfusion (14).…”

(−)-Epigallocatechin-3-gallate (EGCG) attenuates functional deficits and morphological alterations by diminishing apoptotic gene overexpression in skeletal muscles after sciatic nerve crush injury

“…In ischemia-reperfusion of skeletal muscle using the rat lower limb model, p53 also accumulated with concomitant increase in the level of Bax protein and apoptosis was induced (Hatoko et al 2002). Following 14 days of denervation, the extent of apoptotic DNA fragmentation was increased by 100 % in the gastrocnemius muscle.…”

Biomechanics of Pressure Ulcer in Body Tissues Interacting with External Forces during Locomotion

“…By postulating that the pressure ulcer problem can be adequately described by the applied pressure, the duration of loading, tissue density, tissue modulus of elasticity, and tissue blood flow, it was predicted in a dimensional analysis that the allowable pressure p decreased with duration time t in the manner of p ∝ t -4/3 (87). The hypothesis involving blood flow occlusion was among the earliest hypotheses made to explain the formation of pressure ulcer (89 documented that tissues can survive ischemia for duration much longer than those usually observed in pressure ulcer formation (94).…”