Effect of DNase I treatment and neutrophil depletion on acute limb ischemia-reperfusion injury in mice.

OBJECTIVE: Extracellular traps (ETs) consisting of DNA-protein complexes formed after tissue injury contribute to the inflammatory and thrombosis cascades, thereby exacerbating injury. Exogenous DNase I has been suggested as a therapeutic strategy to limit injury in the brain and myocardium. These studies were designed to evaluate the effects of exogenous DNase I treatment on skeletal muscle injury after acute hindlimb ischemia-reperfusion (IR) injury in mice and to determine whether neutrophils are a major source of ETs in postischemic muscle tissue. METHODS: C57BL6 mice were subjected to 1.5 hours of tourniquet ischemia and 24 hours of reperfusion with and without human recombinant DNase I treatment. A separate set of mice was subjected to neutrophil depletion (ND), followed by the same intervals of IR. Laser Doppler imaging and tissue harvesting were done at 24 hours for assessment of limb perfusion, muscle fiber injury, adenosine triphosphate (ATP) level, markers of inflammation, thrombosis, and formation of ETs. RESULTS: DNase I treatment significantly reduced detection of ETs in postischemic muscle but did not alter skeletal muscle fiber injury, levels of proinflammatory molecules, or ATP level. DNase I treatment did enhance postischemic hindlimb perfusion, decreased infiltrating inflammatory cells, and reduced the expression of thrombin-antithrombin III. ND resulted in a significant yet small reduction in ETs in the postischemic muscle. ND did not alter skeletal muscle fiber injury, hindlimb perfusion, or ATP levels. CONCLUSIONS: These data suggest that neither DNase I treatment nor ND was protective against IR injury, even though both decreased detection of ETs in skeletal muscle after IR. Neutrophils are not the only source of ETs after IR.

Effect of limb demand ischemia on autophagy and morphology in mice.

BACKGROUND: Obesity is a major risk factor for diabetes and peripheral arterial disease, which frequently leads to lower limb demand ischemia. Skeletal muscle autophagy and mitochondrial biogenesis are important processes for proper oxidative capacity and energy metabolism, which are compromised in diabetes. This study compares autophagy, mitochondrial biogenesis, energy metabolism, and morphology in the hind limbs of obese diabetic mice subjected to demand or sedentary ischemia. MATERIALS AND METHODS: Unilateral hind limb demand ischemia was created in a group of diet-induced obese mice after femoral artery ligation and 4 wk of daily exercise. A parallel group of mice underwent femoral artery ligation but remained sedentary for 4 wk. Hind limb muscles were analyzed for markers of autophagy, mitochondrial biogenesis, adenosine triphosphate, and muscle tissue morphology. RESULTS: At the end of the 4-wk exercise period, demand ischemia increased the autophagy mediator Beclin-1, but it did not alter the autophagy indicator, LC3B-II/I ratio, or markers of mitochondrial biogenesis, optic atrophy/dynamin-related protein. In contrast, exercise significantly increased the level of mitochondrial protein-succinate dehydrogenase subunit-A and reduced adipocyte accumulation and the percentage of centrally nucleated myofibers in the demand ischemia limb. In addition, demand ischemia resulted in decreased uncoupling protein-3 levels without altering muscle adenosine triphosphate or pS473-Akt levels. CONCLUSIONS: Limb demand ischemia markedly decreased adipocyte accumulation and enhanced muscle regeneration in obese mice, but it did not appear to enhance autophagy, mitochondrial biogenesis, energy metabolism, or insulin sensitivity. Future studies aimed at evaluating novel therapies that enhance autophagy and mitochondrial biogenesis in diabetes with peripheral arterial disease are warranted.