Studies of reperfusion injury in skeletal muscle: preserved cellular viability after extended periods of warm ischemia.

Four hours of complete normothermic ischemia in the rat hindlimb has been thought to produce extensive and irreversible damage and no possibility of salvage by reperfusion. This study tests the hypothesis that, in contrast to conventional wisdom, the cellular integrity is preserved after 4 hours of complete warm ischemia and control of the initial reperfusion can restore immediate contractility in these limbs. Ninety-two rat hindlimbs were isolated and 26 of the 92 did not undergo ischemia or reperfusion and served as controls. Sixty-six limbs were subjected to 4 hours of complete warm ischemia; of those 34 were assessed after the ischemic period without reperfusion and 32 were reperfused after the ischemic period. Nineteen hindlimbs were reperfused with Krebs-Henseleit buffer at a pressure of 100 mmHg to simulate embolectomy (uncontrolled reperfusion). In 13 legs a modified reperfusate at a pressure of 60 mmHg was used during the initial 30 minutes followed by an additional 30 minutes of reperfusion with 100 mmHg using Krebs-Henseleit buffer (controlled reperfusion). At the end of each experimental protocol, limbs were assessed by the following methods: muscle contraction, water content, volume, high energy phosphate content, muscle pH, effluent pH, mitochondrial function, ultrastructure, flow, and creatinkinase activity in the effluent. Data are expressed as mean +/- SEM. Significant differences were defined as probabilities for each test of p less than 0.05. Four hours of complete warm ischemia resulted in a severe reduction of adenosine triphosphate (4.0 +/- 0.8 vs 27.1 +/- 6.7 mumol/gm protein, p less than 0.001) and no contractions could be stimulated (0.0 +/- 0.0% CC). Muscle pH fell to 6.3 +/- 0.1 (p less than 0.001), and ultrastructural damage occurred (score 3.3 +/- 0.4 vs 0.8 +/- 0.1, p less than 0.002). However, there was only a slight increase in water content of the soleus muscle (78.7 +/- 0.2% vs 74.8 +/- 1.1%, p less than 0.05) without increase in limb volume (103.6 +/- 0.6% CV). In addition mitochondrial function was preserved well: mitochondrial oxidative phosphorylation capacity remained at 94% of control levels, ST3 at 93%, and ADP/O at 100% of control. Most importantly, controlled reperfusion restored immediate contractility in all limbs and was superior in all parameters investigated compared to uncontrolled reperfusion. These data support our inference that necrosis of skeletal muscle does not invariably occur after four hours of complete warm ischemia and suggest that muscle salvage by controlled reperfusion is possible after at least 4 hours of warm ischemia.