Carbon monoxide–saturated preservation solution protects lung grafts from ischemia– reperfusion injury Junichi Kohmoto, MD, Atsunori Nakao, MD, Ryujiro.

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Carbon monoxide–saturated preservation solution protects lung grafts from ischemia– reperfusion injury Junichi Kohmoto, MD, Atsunori Nakao, MD, Ryujiro Sugimoto, MD, Yinna Wang, MD, JiangHua Zhan, MD, Hideo Ueda, PhD, Kenneth R. McCurry, MD The Journal of Thoracic and Cardiovascular Surgery Volume 136, Issue 4, Pages 1067-1075 (October 2008) DOI: 10.1016/j.jtcvs.2008.06.026 Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 1 A, The solubility of carbon monoxide (CO) in University of Wisconsin solution at 4°C after 5 minutes of bubbling with different concentrations of CO gas (n = 3 for each concentration). CO solubility increases in a dose-dependent manner. B, Sequential analysis of CO content in University of Wisconsin solution after 5 minutes of bubbling with 100% CO gas with or without secured, air-tight lids. CO levels were maintained as long as 24 hours when the container of University of Wisconsin solution was sealed without an air phase (filled circles, n = 3 for each time point). However, CO concentrations decreased quickly when CO-bubbled University of Wisconsin solution was exposed to air (open circles, n = 3). The Journal of Thoracic and Cardiovascular Surgery 2008 136, 1067-1075DOI: (10.1016/j.jtcvs.2008.06.026) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 2 A, The pulmonary vein Po2 level of the sham-operated animals was 305.3 ± 13.1 mm Hg. The graft pulmonary vein Po2 level of the control University of Wisconsin (UW) group was markedly reduced (80.6 ± 9.4 mm Hg) at 2 hours after reperfusion. Lung grafts in the 5% CO-UW group showed significantly higher Po2 levels compared with those of the control UW group at 2 hours after reperfusion (5% CO-UW group, 135.9 ± 19.8 mm Hg; ∗P < .05 vs control UW group). B, The Pco2 levels of graft pulmonary veins were not significantly different between groups. The Journal of Thoracic and Cardiovascular Surgery 2008 136, 1067-1075DOI: (10.1016/j.jtcvs.2008.06.026) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 3 Neutrophil infiltration of lung grafts 2 hours after reperfusion. Prolonged cold storage and reperfusion after transplantation resulted in massive infiltration of myeloperoxidase-positive neutrophils in control grafts. Flush and storage of lung grafts in University of Wisconsin (UW) solution bubbled with 5% carbon monoxide (CO) significantly reduced the number of neutrophils seen in the graft (∗P < .05 vs control UW solution). (Original magnification ×400). PMN, Polymorphonuclear neutrophil. The Journal of Thoracic and Cardiovascular Surgery 2008 136, 1067-1075DOI: (10.1016/j.jtcvs.2008.06.026) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 4 ED1+ macrophage recruitment to lung grafts. The number of infiltrating alveolar ED1+ macrophages remarkably increased in control University of Wisconsin (UW) solution at 2 hours after reperfusion. Carbon monoxide (CO)–UW (5%) solution significantly reduced macrophage infiltration (∗P < .05 vs control UW group). (Original magnification ×400). The Journal of Thoracic and Cardiovascular Surgery 2008 136, 1067-1075DOI: (10.1016/j.jtcvs.2008.06.026) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 5 Intragraft mRNA levels of inflammatory mediators. mRNA for the proinflammatory cytokines interleukin 6 (IL-6), interleukin 1β (IL-1β), and tumor necrosis factor α (TNF-α); the stress-induced molecules inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2); and the adhesion molecule intercellular adhesion molecule 1 (ICAM-1) were rapidly upregulated within 2 hours after reperfusion in control animals. 5% Carbon monoxide (CO)–University of Wisconsin (UW) solution significantly reduced expression of these inflammation-related molecules (∗P < .05 vs the control UW group). The Journal of Thoracic and Cardiovascular Surgery 2008 136, 1067-1075DOI: (10.1016/j.jtcvs.2008.06.026) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 6 The effects of a higher concentration of carbon monoxide (CO) on pulmonary graft function and cellular infiltration. A, Graft function, as assessed by means of graft oxygenation, was no different between grafts flushed and stored with 5% CO–University of Wisconsin (UW) solution compared with those flushed with 100% CO-UW solution. Both groups demonstrated improved graft function compared with that seen in control grafts. B, The number of infiltrating neutrophils was significantly reduced when the grafts were stored in UW solution with supplementation of 5% CO gas. However, the effects of 100% CO-UW solution were marginal in preventing neutrophil recruitment (∗P < .05 vs control UW solution, #P < .05 vs 5% CO-UW solution). C, Although 5% CO-UW solution significantly reduced the number of ED1+ macrophages in the grafts, 100% CO-UW solution did not alter macrophage recruitment in the graft lung 2 hours after reperfusion (∗P < .05 vs control UW solution, #P < .05 vs 5% CO-UW solution). The Journal of Thoracic and Cardiovascular Surgery 2008 136, 1067-1075DOI: (10.1016/j.jtcvs.2008.06.026) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions

Figure 7 Real-time RT-PCR analysis for inflammatory mediators 2 hours after reperfusion. The upregulation of mRNA for inflammatory mediators was significantly inhibited when lung grafts were perfused and stored in University of Wisconsin (UW) solution with 5% carbon monoxide (CO). The effects of 100% CO had comparable effects in preventing interleukin 6 (IL-6), inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), and intercellular adhesion molecule 1 (ICAM-1) mRNA upregulation; however, the anti-inflammatory effects of 100% CO were marginal on tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) mRNA levels (∗P < .05 vs control UW solution, #P < .05 vs 5% CO-UW solution). The Journal of Thoracic and Cardiovascular Surgery 2008 136, 1067-1075DOI: (10.1016/j.jtcvs.2008.06.026) Copyright © 2008 The American Association for Thoracic Surgery Terms and Conditions