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Inhalation of Carbon Monoxide Reduces Skeletal Muscle Injury Following Hind Limb Ischemia/Reperfusion

Rajendra Patel, Hassan Albadawi, Jeanwan Kang, Hyung-Jin Yoo, Faraz F. Hashmi, Glenn M. LaMuraglia, Michael T. Watkins.
Massachusetts General Hospital, Boston, Mass.

OBJECTIVES: Acute limb ischemia/reperfusion (I/R) injury is a prevalent clinical problem associated with significant morbidity and mortality. Therapeutic inhalation with carbon monoxide (CO) has been shown to reduce I/R injury in lung, cardiac and hepatic tissue. The purpose of this study was to determine if the administration of inhaled CO after the onset of ischemia protects against skeletal muscle injury in a model of hind limb I/R.

METHODS: Unilateral hind limb I/R was created using an orthodontic rubber band (ORB) applied to mouse limbs for 1.5 hours followed by 24 hours of reperfusion after removal of the band. Immediately after application of the ORB, mice were placed in sealed chambers equilibrated with gases composed of either 250 ppm CO mixed with room air (n=10) or room air alone (n=13) during ischemia and the initial 6 hours of reperfusion. After 6 hours reperfusion, all mice were placed in room air at ambient temperature and sacrificed after 24 hours. Blood pressure (BP) and heart rate (HR) was recorded by non-invasive monitoring at baseline, 6 and 24 hours reperfusion. The I/R and the uninjured contralateral hind limbs were harvested for biochemical analysis and histologic evidence of tissue injury. Biochemical assays included Adenosine Triphosphate (ATP) and the cytokine Keratinocyte Chemoattractant protein (KC). Statistical analysis included parametric and nonparametric student t tests.

RESULTS: CO treated mice had less skeletal muscle injury compared to room air treated mice (16.8%±2.7 vs. 29.2%±4.1, p=0.03). The CO treated group also had higher tissue ATP levels when compared to the control group (21.9±7.7 vs. 8.0±3.0 % contralateral; p=0.04). Serum and muscle KC levels were markedly reduced in the CO group when compared to the control group (Serum: 125.2±19.8 vs. 382.5±70.9 pg/ml; p<0.001 and tissue: 11.6±1.5 vs. 26.3±3.7 pg/mg total protein; p<0.001). There was no difference in mean BP and HR in both groups.

CONCLUSIONS: Inhalation therapy with CO protects skeletal muscle from I/R injury independent of hemodynamic effects. CO treatment is associated with preserved skeletal muscle ATP, decreased fiber injury, local and systemic levels of proinflammatory cytokines. These findings support the role of CO treatment in acute limb threatening ischemia.

AUTHOR DISCLOSURES: R. Patel, None; H. Albadawi, None; J. Kang, None; H. Yoo, None; F.F. Hashmi, None; G.M. LaMuraglia, None; M.T. Watkins, None.