Enoxaparin Does Not Ameliorate Limb Ischemia-Reperfusion Injury

Abstract

Since low molecular weight heparin has greater bioavailability and sustained serum levels in vivo than unfractionated heparin, it has been used to supplant unfractionated heparin to achieve therapeutic anticoagulation in humans. These studies were designed to determine whether treatment with enoxaparin could protect murine skeletal muscle from ischemia reperfusion injury. C57BL6 mice were divided into four groups. Sham control animals underwent 90 min of anesthesia alone. All other groups underwent 90 min of unilateral hindlimb ischemia. At the onset of reperfusion, animals received either normal saline (control and saline) or 4 mg/kg of enoxaparin subcutaneously twice daily. Groups were followed for 24 or 48 h reperfusion. Hindlimb skeletal muscle blood flow was measured by laser Doppler, and muscle was removed for histological and protein analysis. Tissue thrombosis was evaluated by thrombin antithrombin III (TAT III), local inflammation by measurement of proinflammatory cytokines (macrophage inflammatory protein-2: MIP-2, monocyte chemoattractant protein-1: MCP-1), and neutrophil infiltration by myeloperoxidase (MPO) using enzyme-linked immunosorbent assay. Plasma levels of Factor Xa were measured during reperfusion to confirm therapeutic levels of anticoagulation. Comparisons were calculated using analysis of variance. At 24 h reperfusion, there was increased expression of MIP-2, MCP-1, MPO, and TAT III in saline and enoxaparin treated mice compared with control (*P < 0.05). By 48 h reperfusion, all parameters measured remained greater than control except for the enoxaparin treated mice whose TAT III levels were significantly less than untreated mice (P < 0.05). Despite documented therapeutic anticoagulation and decreased levels of markers of thrombosis in enoxaparin treated mice, there was no difference in tissue cytokines, inflammatory markers, degree of muscle fiber injury (31% +/- 8% versus 30% +/- 5%) or muscle flow between ischemia-reperfusion groups (2447 +/- 141 versus 2475 +/- 74 flux units) at 48 h reperfusion. Post hoc administration of enoxaparin did not affect local tissue thrombosis, inflammatory markers, or muscle necrosis. This suggests that despite its potent in vivo activity, enoxaparin did not modulate skeletal muscle injury, thrombosis, or inflammatory following ischemia reperfusion. enoxaparin may not be useful in mediating skeletal muscle injury when administered in a clinically relevant scenario.