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The clinical significance and applicability of interventions aimed at reducing reperfusion injury in postischemic skeletal muscle remain unproven, since long-term muscle salvage has not been demonstrated by most treatment protocols that attenuate early reperfusion injury. We have shown that reperfusion of ischemic skeletal muscle results in an early and prolonged sequestration of white blood cells and activation of the alternative complement cascade. The purpose of this study was to determine if 40 minutes of reperfusion with blood depleted of white blood cells and complement proteins, followed by 2 days of normal perfusion, would reduce muscle necrosis after 5 hours of ischemia. The isolated paired canine gracilis muscle model was used. The treatment muscle was initially reperfused with arterial blood that had been spun, washed, passed through a leukocyte removal filter, and resuspended in hydroxyethyl starch (greater than 99.9% removal of white blood cells and the complement proteins factor B and C4). The contralateral control muscle was subjected to unaltered reperfusion. Blood flow (ml/min/100 gm) was measured by timed collection of gracilis venous blood. Myeloperoxidase activity (absorbance at 655 nm/min/mg tissue protein) in muscle biopsies was used to monitor white blood cell sequestration. After 48 hours of reperfusion in vivo, necrosis was quantified by nitroblue tetrazolium staining. Initial reperfusion with white blood cell and complement depleted blood significantly reduced muscle necrosis (53% +/- 3% vs 29% +/- 8%, p less than 0.0025, paired t test). Early blood flow was improved, (p = 0.0025, repeated measure-ANOVA), but subsequent white blood cell sequestration was not altered (p = 0.33, repeated measure-ANOVA). This suggests that a significant amount of white blood cell mediated injury occurs during the first 40 minutes of reperfusion. Preventing early complement activation and white blood cell mediated reperfusion injury is an intervention that is feasible during surgery and may result in clinically significant salvage of postischemic skeletal muscle.

Prolonged ischemia to skeletal muscle as occurs after an acute arterial occlusion results in alterations in adenine nucleotide metabolism. Adenosine triphosphate continues to be used for cellular functions, and an ischemia-induced degradation of phosphorylated adenine nucleotides is initiated. In this experiment we demonstrated the time-dependent aspect of adenine nucleotide depletion during ischemia and the production of large quantities of soluble precursors. In addition, we studied the rate of conversion of xanthine dehydrogenase to xanthine oxidase, a potential source of oxygen-free radicals, after controlled periods of total normothermic ischemia (4 hours and 5 hours) and during the reperfusion phase. During ischemia complete depletion of creatine phosphate occurred in both groups, and adenosine triphosphate fell from 22.1 +/- 1.3 to 10.3 +/- 1.4 mumol/gm dry weight after 4 hours and from 21.6 +/- 0.7 to 3.9 +/- 0.8 mumol/gm dry weight after 5 hours (p less than 0.05). During reperfusion, creatine phosphokinase resynthesis occurred in both groups, but adenosine triphosphate levels were not significantly increased (p greater than 0.05). A washout of lipid soluble products of adenine nucleotide metabolism occurred equally in both groups. The relationship between phosphorylated adenine nucleotides as measured by the energy charge potential fell significantly in both groups (p less than 0.05), but after the shorter period of ischemia (4 hours it returned to normal during early reperfusion but did not after 5 hours of ischemia. There was 21% +/- 4% necrosis after 4 hours and 51% +/- 8% after 5 hours of ischemic stress when assessed at 48 hours. In conclusion, the degree of adenine nucleotide degeneration as determined primarily by the length of the ischemic period, may be the most important determinant of the ultimate extent of skeletal muscle ischemic necrosis that results from an acute interruption of circulation.