Tourniquet-induced acute ischemia-reperfusion injury in mouse skeletal muscles: Involvement of superoxide

Abstract

Although arterial limb tourniquet is one of the first-line treatments to prevent exsanguinating hemorrhage in both civilian pre-hospital and battlefield casualty care, prolonged application of a limb tourniquet can lead to serious ischemia-reperfusion injury. However, the underlying pathomechanisms of tourniquet-induced ischemia-reperfusion injury are still poorly understood. Using a murine model of acute limb ischemia-reperfusion, we investigated if acute limb ischemia-reperfusion injury is mediated by superoxide overproduction and mitochondrial dysfunction. Hind limbs of C57/BL6 mice were subjected to 3h ischemia and 4h reperfusion via placement and release of a rubber tourniquet at the greater trochanter. Approximately 40% of the gastrocnemius muscle suffered infarction in this model. Activities of mitochondrial electron transport chain complexes including complex I, II, III, and IV in the gastrocnemius muscle were decreased in the ischemia-reperfusion group compared to sham. Superoxide production was increased while activity of manganese superoxide dismutase (MnSOD, the mitochondria-targeted SOD isoform) was decreased in the ischemia-reperfusion group compared to the sham group. Pretreatment with tempol (a SOD mimetic, 50mg/kg) or co-enzyme Q(10) (50mg/kg) not only decreased the superoxide production, but also reduced the infarct size and normalized mitochondrial dysfunction in the gastrocnemius muscle. Our results suggest that tourniquet-induced skeletal muscle ischemia-reperfusion injuries including infarct size and mitochondrial dysfunction may be mediated via superoxide overproduction and reduced antioxidant activity. In the future, this murine ischemia-reperfusion model can be adapted to mechanistically evaluate anti-ischemic molecules in tourniquet-induced skeletal muscle injury.

Figure 1

Time courses of change in blood flow of gastrocnemius muscle in sham (n=8), tourniquet-induced ischemia-reperfusion (n=8), tempol (50 mg/kg) + ischemia-reperfusion (n=5), and CoQ₁₀ (50 mg/kg) + ischemia-reperfusion (n=6) groups. CoQ₁₀, co-enzyme Q₁₀. Data are mean ± S.E.M. *P<0.05 vs. sham; ^#P<0.05 vs. ischemia-reperfusion group.

Figure 2

The representative (A) and summary (B) data for infarct size in gastrocnemius muscles from sham (n=8), tourniquet-induced ischemia-reperfusion (n=10), tempol + ischemia-reperfusion (n=7), and CoQ₁₀ + ischemia-reperfusion (n=6) groups. Infarct size is presented as the ratio (%) of infracted area to total gastrocnemius muscle. Data are mean ± S.E.M. *P<0.05 vs. sham; ^#P<0.05 vs. ischemia-reperfusion group.

Figure 3

Mitochondrial complex enzyme activities in gastrocnemius muscles from sham (n=10), tourniquet-induced ischemia-reperfusion (n=13), tempol + ischemia-reperfusion (n=7), and CoQ₁₀ + ischemia-reperfusion (n=8) groups. Data are mean ± S.E.M. *P<0.05 vs. sham; ^#P<0.05 vs. ischemia-reperfusion group.

Figure 4

A, superoxide production in gastrocnemius muscles from sham, tourniquet-induced ischemia-reperfusion, tempol + ischemia-reperfusion, and CoQ₁₀ + ischemia-reperfusion groups, as assessed by dihydroethidium fluorescence. B, superoxide production in homogenates of gastrocnemius muscles from sham, tourniquet-induced ischemia-reperfusion, tempol + ischemia-reperfusion, and CoQ₁₀ + ischemia-reperfusion groups, as measured by lucigenin chemiluminescence. MLU: mean light units. Data are mean ± S.E.M., n=7 mice in each group. *P<0.05 vs. sham; ^#P<0.05 vs. ischemia-reperfusion group.

Figure 5

Effects of tempol (1 mM) and CoQ₁₀ (100 µM) on rotenone (5 µM)- and antimycin A (5 µM)-enhanced superoxide production in gastrocnemius muscle homogenates from sham mice (n=5), as measured by lucigenin chemiluminescence. Data are mean ± S.E.M. *P<0.05 vs. control; ^#P<0.05 vs. rotenone or antimycin A.

Figure 6

Protein expression (A) and activity (B) of MnSOD in gastrocnemius muscle from sham, tourniquet-induced ischemia-reperfusion, tempol + ischemia-reperfusion, and CoQ₁₀ + ischemia-reperfusion groups. Data are mean ± S.E.M., n=6 mice in each group. *P<0.05 vs. sham; ^#P<0.05 vs. ischemia-reperfusion group.