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Postconditioning of the Lungs with Inhaled Carbon Monoxide After Cardiopulmonary Bypass in Pigs

Goebel, Ulrich, MD*; Siepe, Matthias, MD; Schwer, Christian I., MD*; Schibilsky, David, MD; Brehm, Kerstin, MD; Priebe, Hans-Joachim, MD*; Schlensak, Christian, MD; Loop, Torsten, MD*

doi: 10.1213/ANE.0b013e318203f591
Cardiovascular Anesthesiology: Research Reports
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BACKGROUND: Administration of inhaled carbon monoxide before organ ischemic injury exerts protective effects in animal models. Because there are no data showing that this also works after an ischemic insult, our objective in this study was to investigate whether inhaled carbon monoxide attenuates cardiopulmonary bypass (CPB)-induced lung injury in a pig model.

METHODS: Animals were randomized to a SHAM group (n = 5), a SHAM group plus inhaled carbon monoxide (n = 5), standard CPB (n = 10), and to CPB plus inhaled carbon monoxide (n = 10). Carbon monoxide (250 ppm) was given for 1 hour after termination of CPB. Lung biopsies were obtained before CPB, immediately after separation from CPB, and for 5 hours after termination of CPB to determine expression of pulmonary heat shock proteins 70 and 90, cytokines, alveolar macrophage infiltration, and fluorogenic caspase-3 activity.

RESULTS: At 5 hours after CPB, administration of inhaled carbon monoxide was associated with reduced pulmonary expression of the inflammatory cytokines tumor necrosis factor (CPB + CO 521 ± 77 vs CPB 821 ± 97 pg · mL−1, P < 0.001) and interleukin-6 (304 ± 81 vs 860 ± 153 pg · mL−1, P < 0.001), increased pulmonary expression of the cytoprotective heat shock protein 70 (CPB + CO 79 ± 14 vs CPB 36 ± 9 ng · mL−1, P < 0.001) and the antiinflammatory cytokine interleukin-10 (CPB + CO 278 ± 40 vs CPB 63 ± 20 pg · mL−1, P < 0.001), and with reduced pulmonary apoptotic protein caspase-3 activity (CPB + CO 0.73 ± 0.11 vs CPB 0.99 ± 0.1 RFU, P < 0.05). Carbon monoxide administration was associated with reduced histological evidence of lung injury and alveolar macrophage infiltration (78 ± 39 vs 145 ± 34 counts per field of vision, P < 0.001).

CONCLUSIONS: These results suggest that administration of low concentrations of carbon monoxide after CPB (“postconditioning”) protects the lung from CPB-related lung injury.

Published ahead of print December 14, 2010

From the Departments of *Anesthesiology and Cardiovascular Surgery, University Medical Centre, Freiburg, Germany.

Supported by the Deutsche Forschungsgemeinschaft (DFG SCHL 604/2-1) and departmental funding.

The authors declare no conflicts of interest.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (www.anesthesia-analgesia.org).

Address correspondence and reprint requests to Torsten Loop, MD, Department of Anesthesiology and Critical Care Medicine, University Medical Centre, Hugstetterstrasse 55, D-79106 Freiburg im Breisgau, Germany. Address e-mail to torsten.loop@uniklinik-freiburg.de.

Accepted October 14, 2010

Published ahead of print December 14, 2010

Cardiopulmonary bypass (CPB) results in a systemic inflammatory response that may result in lung injury contributing to morbidity and mortality after cardiac surgery.15 Several animal studies have demonstrated that carbon monoxide may ameliorate organ injury from a variety of insults including ischemia/reperfusion injury, transplant rejection, sepsis, and autoimmunity if administered before injury.614 Carbon monoxide may exert its protective effects by modulating intracellular signaling pathways and systemic vasoactivity.1419 Whereas the benefits of carbon monoxide preconditioning have been documented in a variety of in vitro and experimental in vivo studies, whether inhalation of low concentrations of this gas can provide organ protection when given after the injury (“postconditioning”) has not been sufficiently studied.6,11,1822

Accordingly, the objective of this study was to investigate whether inhaled carbon monoxide administered after CPB protects the lung from ischemic injury assessed by examination for reduced pulmonary inflammation, apoptosis, and morphology.

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METHODS

German Landrace Hybrid pigs weighing 33 to 34 kg received care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Guide Vol. 25, No. 28, 1996). All procedures were performed after obtaining the approval of the IRB (Regierungspraesidium, Freiburg, Germany) and in accordance with the German Animal Protection Law.

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Experimental Protocol

Healthy pigs were premedicated with IM ketamine (20 mg · kg−1 body weight) and midazolam (0.5 mg · kg−1). Anesthesia was induced with IV propofol (2 mg · kg−1) and fentanyl (2 mg · kg−1) and maintained by IV infusion of fentanyl (10 mg · kg−1 · h−1) and propofol (4–6 mg · kg−1 · h−1). Muscle relaxation was achieved by cisatracurium (0.7–1.0 mg · kg−1 · h−1). The trachea was intubated and the lungs were ventilated at a rate of 14 · min−1, a tidal volume of 8 to 10 mL · kg−1, and a positive end-expiratory pressure of 3.75 mm Hg. Inspired oxygen fraction was maintained at 0.5. Normal saline was infused at 350 mL · h−1. The right carotid artery was cannulated to monitor arterial blood pressure and obtain blood samples for measurements of concentrations of hemoglobin, carboxyhemoglobin and electrolytes, pH, PaCO2, and PaO2. A thermodilution catheter (7F; Arrow, Reading, PA) was introduced into the pulmonary artery via the right internal jugular vein for measurements of cardiac output and central venous, mean pulmonary artery, and pulmonary capillary wedge pressures, and for calculation of systemic and pulmonary vascular resistances. Full hemodynamic monitoring was established before sternotomy. After administration of 300 · IU · kg−1 heparin IV, CPB was instituted as previously described.22,23 The CPB circuit (Stöckert Roller Pump, Munich, Germany), membrane oxygenator, and connecting tubes (Medos 2800, Stolberg, Germany) were primed with isotonic saline solution (500 mL), 6% hydroxyethyl starch (500 mL), mannitol (250 mL), and heparin (300 · IU · kg−1). Nonpulsatile CPB was maintained for 120 minutes at normothermia with a beating heart. Flow rate and pump speed were based on the cardiac output determined immediately before initiation of CPB. The proximal pulmonary artery was cross-clamped to exclude antegrade flow during CPB and declamped immediately before termination of CPB. Hemodilution caused by the large priming volume was treated with up to maximally 20 mg furosemide. Continuous positive airway pressure was maintained at 3.75 mm Hg during CPB. Before weaning from CPB, a 30-second lung recruitment maneuver was performed and standard ventilation (tidal volume 6–8 mL · kg−1, respiratory rate 14 · min−1, and end-expiratory CO2 concentration 35–38 mm Hg) was reestablished. After separation from CPB, 1 mg protamine per 100 U heparin was administered and the animals were observed for 5 hours.

Animals were randomly allocated to 4 groups (Fig. 1) using a computer-generated randomization sequence. Animals in the SHAM group (n = 5) underwent sternotomy only and served as controls. Group SHAM + CO (n = 5) served as carbon monoxide vehicle group. In group CPB (n = 10), CPB was maintained for 2 hours. In group CPB + CO (n = 10), CPB was maintained for 2 hours followed by administration of inhaled carbon monoxide 250 ppm for 1 hour immediately after the termination of CPB. Because the animals of the SHAM groups merely served to document the effects of surgical preparation and carbon monoxide inhalation in the absence of CPB, we included only 5 animals in these groups. End-expiratory carbon monoxide concentration was monitored by a carbon monoxide analyzer (Micro Smokerlyzer, Breath CO Monitor; Bedfont Scientific, Kent, UK). The carboxyhemoglobin blood concentration was measured photometrically. At the end of each experiment, plasma concentrations of various indicators of cardiac, hepatic, and renal function (see below) were determined by the Department of Clinical Chemistry of the University Hospital Freiburg, using standardized assays and methods as previously described.22 The animals were euthanized by intracardiac potassium injection.

Figure 1

Figure 1

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Tissue Biopsy and Analysis

Arterial blood samples (for determination of hemoglobin and electrolyte concentrations, pH, PaCO2, PaO2, and acid base status), lung biopsies, and measured hemodynamic variables were obtained at the following time points (Fig. 1): immediately before CPB (baseline), immediately after termination of CPB (0 hours post-CPB), and every hour for 5 hours after termination of CPB (1, 2, 3, 4, and 5 hours post-CPB). Lung biopsies were obtained from the cranial and caudal portions of the apical lobe, and from the basal lobe. Samples were immediately minced and stored at −80°C for protein analysis.

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Enzyme-Linked Immunosorbent Assay

Protein was extracted and the concentrations were determined using the Bradford Assay (Bio-Rad Laboratories, Munich, Germany). Enzyme-linked immunosorbent assays were performed following the manufacturer's instruction (Quantikine, R&D Systems, Minneapolis, MN and StressXpress, Biomol, Hamburg, Germany). Sensitivity is 2.8 to 5.0 pg · mL−1 for tumor necrosis factor (TNF)-α, 10 pg · mL−1 for interleukin (IL)-6, and 1.8 to 5.5 pg · mL−1 for IL-10. These assays recognize natural TNF-α, IL-6, and IL-10 with no significant cross-reactivity or interference.

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Fluorogenic Caspase-3 Activity Assay

Lung protein extracts (10 μL ≈ 10 μg) were mixed with 90 μL assay buffer (100 mM HEPES, pH 7.5, 2 mM dithiothreitol, and 2 mM phenylmethylsulfonyl fluoride). The respective fluorogenic substrate for caspase-3 (Ac-DEVD-AMC, 1 μL ≈ 60 μM; Alexis Corp. [now Enzo Life Sciences], Lausen, Switzerland) was added, and the fluorescence was measured at 30°C for 30 minutes in a Microplate Spectra Max Gemini XS reader (Molecular Devices, Sunnyvale, CA) at 380/460 nm.

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Lung Histopathology

Lung biopsies were placed overnight in 4% neutral buffered formaldehyde for fixation at room temperature. The tissues were processed into paraffin blocks and sliced into microscopic sections of 4-μm thickness by a microtome. These were placed on slides and stained with hematoxylin and eosin. Histology was assessed by microscopy. For macrophage tracking, a mouse monoclonal macrophage antibody against the calprotectin molecule ([MAC-387], Abcam #ab22506; Abcam, Cambridge, UK) was used in each lung section of the individual animals. Ten fields of vision were randomly allocated for macrophage quantification by 2 blinded investigators (CIS and KB).

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Data Analysis

Data were analyzed using SigmaStat® for Windows (Version 3.1; Systat Software Inc., San Jose, CA). To be able to detect a 50% difference in the pulmonary cytokine expression after carbon monoxide inhalation compared with control animals with an expected standard deviation of 10% and an α error of 0.05 (2-sided hypothesis) at a power of 0.9, the calculated sample size was 8 animals. Based on the previous investigations, we expected that SHAM animals would remain hemodynamically stable over the planned observation period. Thus, to keep the number of euthanized animals at a minimum, we a priori decided to include only 5 animals in the SHAM groups. The results are presented as mean ± SD after normal distribution of data had been verified. One-way analysis of variance for repeated measurements (hemodynamic, organ function, gas exchange variables, and protein analysis) and 2-way analysis of variance were used for within-group and between-group comparisons, respectively. A P value <0.05 was considered statistically significant.

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RESULTS

All animals survived the experiments and were included in the data analysis. Baseline values of PaO2, carboxyhemoglobin concentrations, PaCO2, arterial pH, body temperature, and hemoglobin concentrations were within normal ranges and did not differ among groups (Fig. 2) (Appendix 1, see Supplemental Digital Content 1, http://links.lww.com/AA/A207). Initiation of CPB caused a decrease (P < 0.05) in hemoglobin concentration of approximately 15% in both CPB groups. Respiratory alkalosis developed during CPB, which resolved after CPB (P < 0.05). Immediately after the administration of inhaled carbon monoxide, PaO2 was lower in both carbon monoxide–treated (CPB + CO and SHAM + CO) compared with the noncarbon monoxide–treated groups (SHAM and CPB) (Fig. 2A; P < 0.05). However, by 2 hours after CPB, there were no longer any significant differences in PaO2 among groups (Fig. 2A).

Figure 2

Figure 2

After 1 hour of carbon monoxide inhalation, carboxyhemoglobin concentrations increased approximately 8- to 10-fold from baseline values (Fig. 2B; P < 0.05). By 5 hours after CPB, carboxyhemoglobin concentrations were no different from baseline. In the noncarbon monoxide–treated groups (SHAM and CPB), carboxyhemoglobin concentrations remained within the normal range throughout the experiment (Fig. 2B).

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Systemic Hemodynamics

There were no differences in central venous pressure, pulmonary capillary wedge pressure, cardiac index, and systemic vascular resistance before CPB among groups (Table 1). For the first 2 hours after CPB, heart rate in both CPB groups was higher compared with baseline (P < 0.05). Administration of inhaled carbon monoxide (SHAM + CO and CPB + CO groups) was associated with decreases (P < 0.05) in mean pulmonary artery pressure throughout the experiment compared with SHAM and CPB groups. In the CPB + CO group, during the first 2 hours after the administration of inhaled carbon monoxide, mean arterial blood pressure and pulmonary vascular resistance was lower compared with the time point “before CPB” and before carbon monoxide treatment (P < 0.05).

Table 1

Table 1

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Pulmonary Cytokine Expression

In both SHAM groups, pulmonary protein concentrations of the proinflammatory cytokines TNF-α and IL-6 remained unchanged throughout the experiment (Fig. 3, A and B). In contrast, CPB led to an increase in pulmonary concentrations of both proteins until the end of the experiment. Whereas TNF-α concentration continued to increase in the CPB group, it tended to decrease in the carbon monoxide–treated group (CPB + CO) after inhalation of carbon monoxide (Fig. 3A). As a result, at 3, 4, and 5 hours after CPB, TNF-α concentrations were significantly lower (P < 0.05) in the CPB + CO compared with the CPB group.

Figure 3

Figure 3

Pulmonary protein expression of IL-6 remained unchanged in the SHAM and SHAM + CO groups throughout the observation period of 5 hours. At the termination of CPB, IL-6 protein concentrations were increased and then decreased during the following hour in both CPB groups compared with both SHAM groups (Fig. 3B; P < 0.001). Whereas IL-6 expression in the CPB group increased again, reaching constant high levels during the final 4 h of the observation period, inhalation of carbon monoxide after CPB inhibited pulmonary IL-6 expression permanently (Fig. 3B; P < 0.001 CPB versus CPB + CO).

Pulmonary IL-10 protein concentrations were not different at baseline in the CPB and SHAM animals, and they remained unchanged during the 5-hour observation period (Fig. 3C). In contrast, in both carbon monoxide–treated groups (SHAM + CO and CPB + CO), IL-10 concentrations began to increase by 2 hours and were significantly higher (P < 0.05) compared with the SHAM and CPB groups at 3, 4, and 5 hours (Fig. 3B).

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Pulmonary Heat Shock Protein Expression and Caspase-3 Activity

Pulmonary heat shock protein (HSP)-70 protein expression remained unchanged throughout the experiment in the carbon monoxide–untreated SHAM and the CPB-only group (Fig. 4A). HSP-70 concentrations began to increase in both carbon monoxide–treated groups (SHAM + CO and CPB + CO) by 2 hours and were higher (P < 0.05) compared with the SHAM- and CPB-only groups at 3, 4, and 5 hours (Fig. 4A). Pulmonary HSP-90 protein expression remained unchanged and comparable among groups throughout the experiment (Fig. 4B).

Figure 4

Figure 4

As shown in Figure 4C, pulmonary caspase-3 activity remained unchanged throughout the experiment in both SHAM groups (SHAM and SHAM + CO). By contrast, in the CPB-only group, caspase-3 activity increased compared with both SHAM groups (Fig. 4C; P < 0.05). Whereas caspase-3 activity continued to increase in the CPB-only group, further increase was inhibited after administration of inhaled carbon monoxide in the CPB + CO group and was significantly lower (P < 0.05) compared with the CPB group at 4 and 5 hours after CPB (Fig. 4C).

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Lung Histopathology

Alveolar histology was normal and not different among groups at baseline, as shown in Figure 5. Alveolar histology remained unaffected by time in the 2 SHAM groups (SHAM and SHAM + CO). Immediately after CPB, histological examination revealed alveolar edema, atelectasis, and infiltration. Whereas in the CPB group pathologic alveolar histology tended to worsen during the 5-hour observation period, there was evidence for histological improvement during this time after the administration of inhaled carbon monoxide (CPB + CO).

Figure 5

Figure 5

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Lung Macrophages

Immediately after CPB, pulmonary macrophage counts were comparable in CPB and CBP + CO groups, as shown in Figure 6. Whereas macrophage counts tended to increase over the next 5 hours in the CPB group, macrophage counts tended to decrease in the carbon monoxide–treated group. As a result, at 3 and 5 hours after CPB, macrophage counts were significantly lower (P < 0.05) in the CPB + CO group compared with the CPB group.

Figure 6

Figure 6

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Indicators of Organ Function

As shown in Table 2, in the animals of the SHAM-only group, organ function variables did not change significantly over time. Except for a lower (P < 0.05) serum concentration of lactate dehydrogenase, administration of inhaled carbon monoxide in the SHAM + CO group had no significant effect on any indicator of organ function (Table 2). In the CPB-only group, serum concentrations of pro-B-natriuretic peptide, myoglobin, bilirubin, alkaline phosphatase, creatinine, urea, uric acid, albumin, amylase and lipase, and serum osmolality did not significantly change. By contrast to the SHAM group, serum concentrations of creatine kinase (CK), CK-MB, lactate dehydrogenase, glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, and γ-glutamyl transferase increased (SHAM versus CPB; Table 2, P < 0.05). The administration of inhaled carbon monoxide after termination of CPB had prevented the CPB-associated increases in serum concentrations of CK, CK-MB, lactate dehydrogenase, glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, and γ-glutamyl transferase, which, consequently, were significantly lower compared with the CPB-only group (all P < 0.05, Table 2).

Table 2

Table 2

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DISCUSSION

The main findings of this study are that inhalation of carbon monoxide for 1 hour immediately after CPB resulted in (a) decreased mean pulmonary artery pressure, (b) blunted CPB-associated increases in pulmonary TNF-α and IL-6 protein concentrations, (c) induced pulmonary IL-10 protein expression, (d) pulmonary HSP-70 protein expression, (e) attenuated increase in caspase-3 activity, and (f) blunted CPB-associated histological lung injury and alveolar infiltration with macrophages. These findings indicate that administration of inhaled carbon monoxide immediately after termination of CPB can ameliorate CPB-induced lung injury, at least in part by antiinflammatory and antiapoptotic mechanisms. They thus support our hypothesis that postconditioning with inhaled carbon monoxide is lung protective in our animal model.

The findings of this study are in accordance with our previous findings in the same experimental model of CPB-induced lung injury in which administration of the same low concentration of inhaled carbon monoxide before CPB (i.e., carbon monoxide preconditioning) was associated with antiinflammatory, antiapoptotic, and cytoprotective effects, and with induction of the heat shock response.22,23 Overall, the degree of antiinflammatory and antiapoptotic pulmonary response was more pronounced (80% vs 50%) during preconditioning compared with postconditioning.

The blunted increases in the concentrations of the proinflammatory cytokines TNF-α and IL-6, and the gradual increase in the pulmonary concentration of the antiinflammatory cytokine IL-10 after inhalation of carbon monoxide, reflect an antiinflammatory effect. In contrast to CPB-induced acute increases in TNF-α and IL-6 concentrations, CPB did not cause such increase in IL-10 concentration. However, the intrinsic ability of inhaled carbon monoxide to increase IL-10 protein expression is reflected by the finding of comparable gradual increases in IL-10 protein expression in both the postconditioning (CPB + CO) as well as the control group (SHAM + CO).

Postconditioning with inhaled carbon monoxide after CPB was associated with antiapoptotic effects. Because inhaled carbon monoxide after CPB group and the SHAM group were both associated with increased pulmonary HSP-70 protein expression, it may be possible that the blunted apoptotic responses to CPB after carbon monoxide postconditioning were, at least in part, related to a carbon monoxide–induced heat shock response. The HSPs are important mediators of the pulmonary inflammatory response in lung injury.2426 Carbon monoxide–mediated antiinflammatory and antiapoptotic effects have been shown in heat shock factor knockout mice,24 and, as previously shown by us, inhibition of HSP-70 by quercetin resulted in almost complete reversal of pulmonary antiinflammatory and antiapoptotic effects observed after administration of low-concentration inhaled carbon monoxide before CPB (i.e., preconditioning).27 By contrast to HSP-70 and the data of preconditioning, inhaled carbon monoxide after exposure to CPB did not affect HSP-90α expression. Although, HSP-90 is the most abundant HSP present in eukaryotic cells, its functional role in carbon monoxide– associated organ protection remains to be determined.28 However, because we did not examine the effect of inhibition of the heat shock response by administration of quercetin on carbon monoxide postconditioning, the current data cannot provide the molecular mechanism of the observed protective effect.

Our results also provide histological evidence of carbon monoxide–mediated postconditioning lung protection. Whereas CPB-induced alveolar edema, cell infiltration, and septal destruction tended to worsen over time in the nontreated animals (CPB group), postconditioning with inhaled carbon monoxide (CPB + CO group) prevented further histological deterioration. These qualitative findings are supported by the semiquantitative findings of less alveolar macrophage infiltration in the postconditioned compared with the nonpostconditioned group at 3 and 5 hours after termination of CPB.

Inhaled carbon monoxide was consistently associated with decreases in mean pulmonary artery pressure in the SHAM + CO and the CPB + CO groups and in decreases in pulmonary vascular resistance in the CPB + CO group during the first 3 hours after administration. These findings might be explained by carbon monoxide's potent vasodilatory effects and are consistent with in vivo studies in which inhaled carbon monoxide inhibited hypoxic pulmonary vasoconstriction by a cyclic guanosine monophosphate– independent mechanism.24,29 Lower mean pulmonary artery pressure and pulmonary vascular resistance may have contributed to reduced pulmonary edema after lung injury. A decrease in pulmonary vascular resistance after CPB might possibly lead to better pulmonary perfusion and reduced reperfusion injury.30

Our findings suggest that carbon monoxide postconditioning may possibly protect, to some degree, heart and liver as well. CPB was associated with considerable (up to 10-fold) increases in plasma concentrations of some markers of cardiac and hepatic dysfunction. Five hours after CPB, plasma concentrations of these markers were not only lower in postconditioned (CPB + CO) compared with nonpostconditioned animals (CPB), but they were not different from those in the SHAM group.

When trying to put the findings into proper clinical perspective, a few points need to be taken into consideration. Because carbon monoxide is a potentially toxic gas, translation of these findings into clinical practice has to be done with caution. In humans, carbon monoxide concentrations of >250 ppm had no adverse effects.31 Carboxyhemoglobin concentrations between 15% and 20% seem to be well tolerated in humans and are considered the “biological threshold” above which severe carbon monoxide–mediated injury is likely to occur.32 After inhalation of carbon monoxide in our study, plasma concentrations of all investigated markers of organ function, all evaluated hemodynamic variables (with the exception of a lower mean pulmonary artery pressure), and alveolar histology remained unaffected (SHAM versus SHAM + CO group). These findings only apply to a 1-hour administration of inhaled carbon monoxide at a concentration of 250 ppm.

In summary, using a well-established model of CPB-associated lung injury, we show that administration of low-dose inhaled carbon monoxide immediately after CPB is lung protective as reflected by blunted inflammatory and apoptotic pulmonary responses. Future investigations are required to determine whether postconditioning with inhaled carbon monoxide may constitute an effective modality of ameliorating CPB-induced lung injury.

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DISCLOSURES

Name: Ulrich Goebel, MD.

Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.

Attestation: Ulrich Goebel has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

Name: Matthias Siepe, MD.

Contribution: This author helped design the study, conduct the study, and analyze the data.

Attestation: Matthias Siepe has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Christian I. Schwer, MD.

Contribution: This author helped conduct the study and analyze the data.

Attestation: Christian I. Schwer has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: David Schibilsky, MD.

Contribution: This author helped conduct the study.

Attestation: David Schibilsky has seen the original study data, reviewed the analysis of the data and approved the final manuscript.

Name: Kerstin Brehm, MD.

Contribution: This author helped conduct the study.

Attestation: Kerstin Brehm has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Hans-Joachim Priebe, MD, PhD.

Contribution: This author helped design the study and write the manuscript.

Attestation: Hans-Joachim Priebe has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Christian Schlensak, MD, PhD.

Contribution: This author helped design the study and write the manuscript.

Attestation: Christian Schlensak has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Torsten Loop, MD.

Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.

Attestation: Torsten Loop has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

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