Activated protein C reduces tissue hypoxia, inflammation, and apoptosis in traumatized skeletal muscle during endotoxemia

Objective: Extensive surgical trauma leads to activation of the coagulation cascade and is often complicated by systemic inflammation and infection. Activated protein C, a natural coagulatory inhibitor, was recently shown to reduce mortality in septic patients. We herein report on the actions of activated protein C on skeletal muscle injury in experimental endotoxemia. Design: Prospective controlled animal study. Setting: University animal research facility. Subjects: Male Sprague-Dawley rats. Interventions: Closed soft tissue trauma was applied on the left hind limb of pentobarbital-anesthetized rats. Six hours later endotoxemia was induced by intraperitoneal injection of Escherichia coli lipopolysaccharide. An equivalent volume of physiologic saline was given in controls. At the same time point, treatment of animals was started by continuous intravenous application of activated protein C (24 mu g/kg.hr) or vehicle solution over 18 hrs. Twenty-four hours after trauma, the extensor digitorum longus muscle was microsurgically exposed and analyzed by means of high-resolution multifluorescence microscopy. Measurements and Main Results: Endotoxemia aggravated traumatized muscle injury, as evidenced by reduced nutritive perfusion, increased tissue hypoxia, enhanced leukocyte-endothelial cell interaction, and apoptotic myocyte cells (249 +/- 17 cm/cm(2) vs. 298 +/- 22 cm/cm(2); reduced nicotinamide adenine dinucleotide [NADH], 149 +/- 15 arbitrary units [AU] vs. 130 +/- 13 AU; 417 +/- 79 cells/mm(2) vs. 344 +/- 77 cells/mm(2) and 62 +/- 9 cells/mm(2) vs. 31 +/- 5 cells/mm(2)). Therapeutic intervention with activated protein C 6 hrs after trama protected nutritive perfusion and tissue oxygenation (341 +/- 24 cm/cm(2) and 115 +/- 8 AU) and reduced inflammatory leukocyte adherence (185 +/- 60 cells/mm(2)) and cellular apoptosis (15 +/- 4 cells/mm(2)). Of note, the protection of traumatized muscle tissue by activated protein C was also maintained during endotoxemia, as indicated by a functional capillary density of 379 +/- 10 cm/cm(2), a NADH-fluorescence of 102 +/- 6 AU, a leukocyte adherence of 82 +/- 12 cells/mm(2), and a myocyte apoptosis of 28 +/- 4 cells/mm(2). Conclusions: Microcirculatory injury of traumatized skeletal muscle tissue is enhanced by intravenous endotoxin application in this model of soft tissue trauma. Activated protein C ameliorates microcirculatory dysfunction and tissue injury, in particular in traumatized animals during endotoxemia.