Arterial blood gas measurements in mice bled to and maintained at a MAP of 20 mm Hg for 30 minutes with and without inhaled CO (250 ppm).

<p>*P<0.05 compared to sham mice.</p><p>^§P<0.05 compared to shock mice.</p><p>Sham mice underwent anesthesia and surgical manipulation without hemorrhage. Mice were bled to a pressure of 20 mm Hg over 15 minutes. CO therapy was started once a pressure of 20 mm Hg was reached.</p

CO protects against organ injury and inflammation in a dose dependent fashion in murine model of hemorrhagic shock and resuscitation.

<p>Lung myeloperoxidase activity (MPO; <b>A.</b>) and serum ALT (<b>B.</b>) at 4 hours after resuscitation in mice demonstrates lung and liver injury, respectively. CO limits this injury in a dose-dependent fashion when treated for 30 minutes (25–500 ppm) starting 90 minutes into hypotension. C. Serum TNF-alpha and IL-6 levels were also increased by hemorrhagic shock and resuscitation at a 4 hour time point, and CO therapy limited these markers of inflammation in a dose dependent fashion. Results are mean±SEM for 8 mice per group. *P<0.05 compared to sham and #P<0.05 compared to shock. ANOVA was utilized for above comparisons.</p

CO therapy protects against mortality in a model of murine hemorrhagic shock and resuscitation.

<p>A severe hemorrhagic shock and resuscitation model resulted in 80% mortality by 36 hours. CO therapy (250 ppm) limited mortality to 20% (P<0.05). Kaplan-Meier method was used to compare survival.</p

Hemorrhagic shock and resuscitation-induced skeletal muscle mitochondrial injury was limited by CO therapy.

<p>A. The change in respiratory control ratio (RCR; state 3:state4) from baseline to 2 hours after resuscitation in the porcine model demonstrated that HS/R led to mitochondrial injury. CO treatment resulted in an overall increase in the mean RCR, representing decreased mitochondrial injury (*P<0.05 compared to HS/R). B. Changes in RCR in murine thigh skeletal muscle from baseline to 2 hours after resuscitation demonstrated mitochondrial injury following HS/R, and inhaled CO protected against this injury (N = 8 mice per group; *P<0.05 compared to baseline; #P<0.05 compared to control treated-HS/R mice). ANOVA was utilized for above comparisons.</p

CO decreases oxygen consumption and limits the development of cellular hypoxia in hepatocytes in vitro.

<p>A. Oxygen consumption rates of primary murine hepatocytes were demonstrated <i>in vitro</i> in normoxic cells or in hepatocytes immediately following 30 minutes of hypoxia. CO treatment (250ppm) occurred during this normoxic or hypoxic periods. Hypoxia decreased oxygen consumption rates (*P<0.01 compared to normoxic cells) and this was further decreased by CO therapy (#P<0.05 compared to hypoxia alone). Results of four independent experiments, with each condition performed in triplicate. <b>B, C.</b> Representative immunocytochemistry and quantitative mean fluorescence of hypoxyprobe staining in hepatocytes under normoxic, normoxic+CO, hypoxic, or hypoxic +CO conditions for 30 minutes. Increased green staining represents increased cellular hypoxia. ANOVA was utilized for above comparisons.</p

CO limits hypoxia-induced oxidant and mitochondrial injury in murine skeletal muscle ex-vivo.

<p>Murine skeletal muscle was freshly collected from mice after perfusion with cold PBS, cut into strips (2 X 2 X 10mm) and exposed ex vivo to hypoxia (1% O₂) for one hour followed by reoxia for up to 4 hours. Some samples were treated with CO (250 ppm) during hypoxia. Measurements of ROS by DCF fluorescence, relative mitochondrial membrane potential by TMRE fluorescence, relative ATP levels, and respiratory control ratio (RCR) were determined at baseline, end of hypoxia, 1 and 4 hours. Hypoxia induced increased DCF fluorescence and a drop in mitochondrial membrane potential; however statistically significant differences in these parameters, as well as reductions in ATP and RCR were not seen until after reoxygenation (*P<0.05 compared to baseline). All parameters had returned to or were trending back to baseline by 4 hours after reoxia. CO treatment limited these hypoxia/reoxia-induced changes (#P<0.05 compared to hypoxia+1 hour reoxia). Each experiment was performed in duplicate and repeated three times. T-tests were used for the above comparisons.</p

CO protects against hemorrhagic shock and resuscitation-induced platelet activation and mitochondrial injury.

<p><b>A</b>. HS/R results in decreased ATP linked respiration (*P<0.05 compared to sham), while CO treatment prevented these changes (#P<0.05 compared to HS/R). <b>B</b>. HS/R had a minimal effect on mitochondrial reserve capacity, while CO treated HS/R pigs demonstrated an increase in this parameter (*P<0.05 compared to sham and HS/R) <b>C</b>. HS/R increased platelet activation by 2.33±0.1 fold over sham pigs at a 2 hour time point as determined by staining for CD62p by FACS (*P<0.05 compared to sham). CO treatment limited this activation to only a 1.64±0.08 increase over sham (#P<0.05 compared to HS/R). n = 7–11 pigs per group in each experiment. ANOVA was utilized for above comparisons.</p

CO has minimal influence on gross cardiovascular parameters in porcine hemorrhagic shock and resuscitation.

<p>Hemodynamic data are shown at time points throughout the experiments [baseline, end of hemorrhage (H1), 30 minutes into hypotension (H30), 60 minutes into hypotension (H60), immediately prior to resuscitation (resusc), at the end of the initial hextend bolus (hextend), 2 hours into the resuscitation (Obs2h), and 4 hours into the resuscitation (Obs4h)]. Data is shown for mean arterial pressure (MAP, <b>A</b>.), heart rate (<b>B</b>.), central venous pressure (CVP, <b>C.</b>), mixed venous saturation (S_VO₂%, <b>D</b>.), and mean pulmonary arterial pressure (<b>E</b>.). Expected changes in hemodynamics are seen in shock and resuscitation, with no significant influences demonstrated in the CO treated pigs. ANOVA was utilized for above comparisons.</p