- Email: [email protected]
reperfusion injury of transplanted rat lung grafts
Low-dose carbon monoxide inhalation prevents ischemia/reperfusion injury of transplanted rat lung grafts Junichi Kohmoto, MD,a,b,c Atsunori Nakao, MD,a,c Takashi Kaizu, MD,a,c Allan Tsung, MD,a Atsushi Ikeda, MD,a,c Koji Tomiyama, MD,a,c Timothy R. Billiar, MD,a Augustine M.K. Choi, MD,d Noriko Murase, MD,a,c and Kenneth R. McCurry, MDa,b,c Pittsburgh, Pa
Background. Carbon monoxide (CO), a byproduct of heme catalysis by heme oxygenases, has been shown to provide protection against ischemia/reperfusion (I/R) injury. We examined the cytoprotective effect of CO at a low concentration on cold I/R injury of transplanted lung grafts. Methods. Orthotopic left lung transplantation was performed in syngenic Lewis to Lewis rat combination. Grafts were preserved in University of Wisconsin solution at 4°C for 6 hours. Donors and/or recipients were exposed to CO (250 ppm) in air for 1 hour before surgery and then continuously post-transplantation. Results. Blood oxygen partial pressure of graft pulmonary veins in the CO-treated group versus the air-treated group was significantly higher. The increase of messenger RNA of inflammatory mediators such as interleukin-6, tumor necrosis factor-␣, inducible nitric oxide synthase, and cycloooxygenase-2 was markedly inhibited in the CO-treated group. The expression of phosphorylated-extracellular signalregulated protein kinase 1/2 was significantly reduced in the CO-treated group. CO treatment reduced the number of infiltrating macrophages into the lung grafts. Vascular endothelial cells detected by CD31 stain were well preserved in CO-treated grafts, while those in air-treated grafts were faint and interrupted. Conclusions. These results demonstrate that exogenous low-dose CO treatment of donors and recipients can prevent lung I/R injury and significantly improve function of lung grafts after extended cold preservation and transplantation. (Surgery 2006;140:179-85.) From the Department of Surgery,a Heart, Lung, and Esophageal Surgery Institute,b Thomas E. Starzl Transplantation Institute,c and the Department of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine,d University of Pittsburgh
Lung transplantation (LTx) has evolved over the last 2 decades to become the therapy of choice for many patients with advanced pulmonary disease. Current cold preservation strategies for lung grafts remain imperfect, however, placing recipiPresented at the 67th Annual Meeting of the Society of University Surgeons, First Annual Academic Surgical Congress, February 7-11, 2006, San Diego, California. Supported by National Institutes of Health grant HL076265 (K.R.M.) and Gas Enabled Medical Innovations (GEMI) fund (A.N.). Reprint requests: Kenneth R. McCurry, MD, FACS, Dept of Surgery, University of Pittsburgh, 200 Lothrop St, Ste C-700, Pittsburgh, PA 15213. E-mail: [email protected] 0039-6060/$ - see front matter © 2006 Mosby, Inc. All rights reserved. doi:10.1016/j.surg.2006.03.004
ents at risk for life-threatening graft dysfunction resulting from ischemia/reperfusion (I/R) injury.1 Recipients who survive a severe I/R injury to their graft frequently have a prolonged hospitalization and also have a higher incidence of both acute and chronic rejection. In addition, the lung donor pool has been expanded by using lungs from non-ideal donors and from donation after cardiac death (DCD) donors to overcome the problem of organ shortage. Such lung grafts may be more susceptible to cold I/R injury. Thus, a therapeutic strategy to reduce I/R injury after LTx could have a significant impact on clinical outcomes after lung transplantation and potentially expand the available donor pool further. Carbon monoxide (CO), the gaseous catalytic byproduct of heme degradation by heme oxygenases (HOs), has been suggested to be a signaling SURGERY 179
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molecule that exerts significant cytoprotection via its vasodilating, anti-inflammatory, and antiapoptotic properties.2 Our laboratories have been investigating the ability of CO to ameliorate I/R injury in organ transplantation. We have shown that recipient CO inhalation at low concentrations reduces I/R injury in various organ transplantation models in rats including the small intestine, kidney, and liver.3-7 In the present study, we hypothesized that CO inhalation at a low concentration would ameliorate I/R injury in the transplanted lung, resulting in improved graft function. We evaluated this hypothesis using a rat orthotopic lung transplantation model. MATERIAL AND METHODS Animals. Inbred male Lewis (LEW, RT1) rats weighing 250 to 300 g were purchased from Harlan Sprague Dawley, Inc (Indianapolis, Ind), and maintained in laminar flow cages in a specific pathogenfree animal facility at the University of Pittsburgh. All procedures in this experiment were performed according to the guidelines of the Council on Animal Care at the University of Pittsburgh and the National Research Council’s Guide for the Humane Care and Use of Laboratory Animals. Orthotopic LTx. Orthotopic left LTx was performed as described previously.8 Briefly, after donor rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (45 mg/kg) and heparinization (300 units), the lungs were flushed with 20 mL of cold (4°C) University of Wisconsin solution (Viaspan; Du Pont, Wilmington, Del) via the main pulmonary artery. Recipient rats were intubated and ventilated with a mixture of 100% oxygen and 1% isoflurane (Isoflo; Abbott Laboratories, North Chicago, Ill). After the native left lung was removed, the lung graft was transplanted orthotopically by anastomosing the pulmonary vein (PV), artery, and bronchus of the recipient by using the cuff technique, as described.9 After the chest was closed, a pleural drainage tube was removed after aspiration. The recipients were extubated within 10 minutes after surgery when adequate recovery from anesthesia and spontaneous breathing were observed. CO inhalation. CO (1%) in air was mixed with air (21% oxygen) in a stainless steel mixing cylinder and then directed into a 3.70-ft3 glass exposure chamber at a flow rate of 12 L/min. A CO analyzer (Interscan, Chatsworth, Calif) was used to continuously measure CO levels in the chamber, to maintain CO concentration at 250 ppm at all times. Experimental groups. Syngenic LEW to LEW rat LTx with 6 hours of cold preservation served as the
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control (group 1). The animals in group 1 were maintained in room air either in the identical chamber as CO exposure or in the regular laminar flow cage. In groups 2 and 3, the donors were exposed to CO (250 ppm) for 1 and 18 hour before harvesting, respectively. In group 4, the recipients were exposed for 1 hour before and then continuously beginning immediately after transplant surgery. In group 5, both donors and recipients were exposed for 1 hour before and then continuously beginning immediately after transplant surgery. Sham control animals consisted of LEW rats that were intubated and ventilated under the same conditions as transplant recipients but did not undergo an operation. Recipients were sacrificed at 1, 2, 4, 6, and 24 hours after LTx to obtain blood and lung graft samples. Lung graft function. The lung graft function was assessed with blood gases (iSTAT Portable Clinical Analyzer; iSTAT Corp, East Windsor, NJ), on a FiO2 of 1.0, drawn from the PV of the transplanted lung. Histopathology and immunohistochemistry. Lung graft tissues were fixed in 10% formalin, embedded in paraffin, sectioned into 6 m thickness, and stained with hematoxylin-eosin (H&E). The macrophages were stained by immunohistochemistry for ED1 as described previously.6,10 Positively stained cells were counted in 20 high-power fields (⫻400) per each section and expressed as the number of cells per 0.1 mm2 in a blind manner. Immunofluorescent analysis for CD31. For CD31 immunofluorescent stain, graft tissues were frozen in OCT (Optimal Cold Temperature; Sakura Finetek, Inc, Torrance, Calif) compound and cut into 6-m sections. The tissue was stained with mouse antirat CD31 (PE-CAM, 1:100; Serotec, Raleigh, NC) as described previously.5 Real-time reverse transcription-polymerase chain reaction. Intragraft messenger RNA (mRNA) levels for interleukin (IL)-6, tumor necrosis factor (TNF)-␣, cycloxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) were assessed by SYBR Green 2-step, real-time reverse transcription-polymerase chain reaction. Total RNA was extracted from the lung tissues with the use of the TRIzol reagent (Life Technologies, Inc, Grand Island, NY) according to the manufacturer’s instructions. One microgram of total RNA from each sample was used for reverse transcription with an oligo dT (Life Technologies) and a Superscript II (Life Technologies) to generate first-strand complementary DNA. The polymerase chain reaction mixture was prepared with the use of SYBR Green PCR Master Mix (PE Applied Biosystems, Foster City,
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Table I. Primer sequences Target genes IL-6 TNF-␣ COX-2 iNOS GAPDH
Primer Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
Sequence 5=-CAAAGCCAGAGTCATTCAAGC-3= 5=-GGTCCTTAGCCACTCCTTCTGT-3= 5=-GGTGATCGGTCCCAACAAGGA-3= 5=-CACGCTGGCTCAGCCACTC-3= 5=-CTCTGCGATGCTCTTCCGAG-3= 5=-AAGGATTTGCTGCATGGCTG-3= 5=-GGAGAGATTTTTCACGACACCC-3= 5=-CCATGCATAATTTGGACTTGCA-3= 5=-ATGGCACAGTCAAGGCTGAGA-3= 5=-CGCTCCTGGAAGATGGTGAT-3=
COX-2, Cycloxygenase-2; GAPDH, glyceraldehydes 3-phosphate dehydrogenase; IL-6, interleukin 6; iNOS, inducible nitric oxide synthase; TNF-␣, tumor necrosis factor ␣.
Calif), with the primers being designed according to the literature or published sequences (Table I). Thermal cycling conditions were 10 minutes at 95°C to activate the Amplitaq Gold DNA polymerase, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute on an ABI PRISM 7000 Sequence Detection System (PE Applied Biosystems). The expression of each gene was normalized to the mRNA content of glyceraldehyde-3-phosphate dehydrogenase and calculated relative to normal control. Western blot analysis. Western blot analysis was performed with the use of 50 g of cytoplasmic protein from each graft lung tissue as described previously.11 After the blocking of nonspecific binding with nonfat dry milk, membranes were incubated overnight with antiphosphorylated extracellular signal–regulated kinase (ERK) 1/2 (sc7383) and antitotal ERK1/2 (sc-94, Santa Cruz Biotechnology, Santa Cruz, Calif). After incubation with secondary goat antirabbit or antimouse antibody (1:10,000; Pierce Chemical, Rockford, Ill), the membranes were developed with the SuperSignal detection systems (Pierce Chemical) and exposed to film. Data analysis. Results are expressed as means ⫾ SEM. Statistical analysis was performed with the use of the Student t test or analysis of variance where appropriate. A probability level of P less than .05 was considered statistically significant. RESULTS Lung graft function. Graft function was determined by the blood gas analysis of the isolated graft PV. The oxygen partial pressure (pO2) values of graft PV in sham-operated animals were 297 ⫾ 17.7 mm Hg under mechanical ventilation (100% oxygen). Prolonged cold storage and reperfusion im-
paired pulmonary graft functions and resulted in the significant decrease of pO2 levels of the graft PV to 75.0 ⫾ 11.3 mm Hg at 2 hours after reperfusion. The effects of donor pretreatment of CO for either 1 or 18 hours were marginal (1 hour pretreatment; 107 ⫾ 12.5 mm Hg; 18 hours pretreatment; 119.2 ⫾ 14.5 mm Hg), without statistically significant improvement of graft function, compared with air-treated animals. However, both recipient treatment and donor/recipient treatment significantly improved pO2 levels of the graft PV (recipient-treatment, 167.5 ⫾ 28.3 mm Hg; donor/recipient-treatment, 173.3 ⫾ 22.1 mm Hg; P ⬍ .05 vs air control) (Fig 1, A). The partial pressure of carbon dioxide (pCO2) values of graft PVs were comparable, ranging from 27.8 ⫾ 2.8 mm Hg to 36.4 ⫾ 2.4 mm Hg, and there were no statistical differences in each group at 2 hours post-transplantation (Fig 1, B). Inflammatory mediators. Sequential analysis for mRNA levels of inflammatory cytokines (IL-6 and TNF-␣) and stress-induced molecule (iNOS and COX-2) showed a prompt increase of these mediators peaking at 1 to 2 hours after reperfusion (data not shown). Therefore, we analyzed mRNA expression using samples taken 2 hours after reperfusion. The effects of donor pretreatment were marginal with either 1 or 18 hours exposure before harvesting. There was a tendency of suppression of these mediators in the only recipient treatment. However, only donor/recipient treatment with CO inhalation significantly reduced the peak for these inflammatory mediators up to 40%⬃60% of those seen in air-treated grafts (Fig 2). On the basis of these results, further studies used CO-pretreatment of the donor for 1 hour and recipient exposure to CO for 1 hour before and
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Fig 1. Blood gas analysis of graft pulmonary vein. A, pO2 levels. Graft function was significantly insulted 2 hours after I/R injury. Donor pretreatment for 1 hour (CO-D, n ⫽ 4) did not improve graft functions. However, either recipient only (CO-R, n ⫽ 4) or only both donor (1 hour) and recipient (CO-D⫹R, n ⫽ 7) significantly increased graft pulmonary vein pO2 levels, compared with air-controls (n ⫽ 7). B, pCO2 levels. Regardless of the CO exposure protocol, pCO2 levels of graft pulmonary vein 2 hours after reperfusion were comparable without statistical difference among the group. CO, Carbon monoxide; D, donor; pCO2 , partial pressure of carbon dioxide in pulmonary vein blood; N.S., not significant; pO2 , oxygen in partial pressure in pulmonary vein blood; R, recipient.
continuously for 24 hours after reperfusion throughout the remaining experiments. Macrophage infiltration. ED1⫹ macrophages represent the predominant cells present during I/R injury in many organ systems.10 The number of ED1⫹ macrophages in air-control grafts increased to 56.1 ⫾ 1.6/0.1 mm2 from those in sham-operated grafts (22.8 ⫾ 1.0/0.1 mm2) 24 hours after reperfusion. CO inhalation significantly reduced
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Fig 2. Sequential mRNA expression in lung grafts. mRNA for inflammatory cytokines and stress-induced molecule of IL-6, TNF-␣, iNOS and COX-2 were upregulated during I/R injury in air-treated group 2 hours after reperfusion. Only recipient treatment significantly suppressed iNOS and COX-2 mRNA expression. CO treatment of both donor and recipient showed more potent protection against mRNA expression of these inflammatory mediators. (*P ⬍ .05 vs air-treated group; n ⫽ 4-7). D, Donor; IL-6, interleukin 6; iNOS, inducible nitric oxide synthase; R, recipient; TNF-␣, tumor necrosis factor ␣.
the number of ED1⫹ macrophage infiltration to 29.6 ⫾ 0.8/0.1 mm2 (Fig 3). Vascular endothelial cell injury (CD31 staining). Vascular endothelial cells have been known to be a main target of I/R injury, leading to the disruption of microcirculation in the transplanted organs.12 The morphologic analysis of vascular endothelial changes after I/R injury were performed by the fluorescent immunohistochemical stain for pan-endothelial cell marker (CD31). CD31 stains in airtreated grafts were very faint and interrupted 1 hour after reperfusion, suggesting that I/R injury in our model caused remarkable injury to graft vascular endothelial cells. However, vascular endothelial cells stained by CD31 in CO-treated graft were well preserved and almost identical to those in the sham operation (Fig 4). Phosphorylated ERK1/2 expression. Since mitogen-activated protein kinases (MAPKs) are known to be important signaling cascades involved in inflammation, we examined the effect of CO on ac-
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Fig 3. ED1⫹ macrophage infiltration. The number of infiltrating alveolar ED1⫹ macrophages remarkably increased in air-control grafts 24 hours after reperfusion. CO significantly reduced macrophage infiltration. (*P ⬍ .05 vs air-treated group, n ⫽ 4). CO, Carbon monoxide.
Fig 4. CD31 expression of vascular endothelial cells. A, Immunofluorescent stain of CD31 on the vascular endothelial cells in lungs from sham-operated animals. B, CD31 expression was faint and interrupted in air-treated lung grafts after cold I/R injury at 1 hour after reperfusion. C, CD31-positive vascular endothelial cells were preserved in CO-treated lung grafts at 1 hour after reperfusion. Green, CD31; blue, nuclei. Original magnifications: x600. CO, Carbon monoxide.
tivation of MAPKs using cytosolic protein of the lung grafts. Phosphorylation of ERK1/2 occurred rapidly 1 hour after reperfusion, followed by a decrease of the protein expression of phosphorylated ERK1/2 after 6 hours. CO treatment significantly reduced activation of ERK1/2 early after reperfusion (Fig 5). DISCUSSION Although CO is known to be toxic at high concentrations because of its ability to interfere with oxygen delivery, mammalian cells endogenously generate CO primarily via the catalysis of heme by HOs. Recently, several lines of evidence have indicated that the constitutive HO reaction and gener-
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Fig 5. Phosphorylated ERK expression in the lung graft. Representative pictures of Western blots from four independent experiments for phosphorylated ERK1/2 in the lung grafts. Strong expressions of phosphorylated ERK1/2 were seen at 1 hour and 2 hours after reperfusion. CO-treated group significantly suppressed phosphorylation of ERK1/2, compared with air-treated grafts. CO, Carbon monoxide; ERK1/2, extracellular signal-regulated protein kinase 1/2.
ation of CO serve as a key mechanism to maintain the integrity of the physiologic function of organs. Furthermore, HO-1, an inducible form of the HOs, potentially confers protection against oxidative stress through the generation of byproducts of heme catalysis. Accordingly, CO has been considered as a molecule that has cytoprotective properties, supporting a new paradigm that administration of CO at low concentrations may have therapeutic benefits. In this study, we have demonstrated that, in the presence of exogenously administered, CO lung grafts were significantly devoid of I/R injury after cold ischemia and warm reperfusion, resulting in improved graft function. Much previous work has demonstrated that pulmonary I/R injury results from a cascade of events with many interconnected factors, including upregulation of proinflammatory genes, inflammatory infiltrates, complement activation, impaired microcirculation, increased procoagulant activity, T-cell activation, perturbations in endothelial cell function, and apoptotic cell death.13 Alveolar macrophages are activated in the early phase of reperfusion after LTx and play important roles in I/R lung injury. Alveolar macrophages secrete proinflammatory cytokines that prompt neutrophil activation, migration, and extravasation to the alveolar interstitial space with subsequent production of reactive oxygen species. Indeed it has been suggested that the early phase of LTx reperfusion injury is mediated by donor (resident) pulmonary macrophages, while late injury is induced by recipient circulating neutrophils.14 While our study does not suggest an isolated mechanism for the protective effect observed with CO, our findings that CO treatment decreased expression of the proinflammatory mediators TNF-␣, iNOS, COX-2 and IL-6, as well as decreased macrophage infiltration, suggest that the protective effect observed is
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mediated, at least in part, by downregulation of these inflammatory mediators associated with inflammatory cellular infiltration. CO has also been shown in other models of cold and warm organ I/R to promote improved blood flow through a guanylyl cyclase– dependent mechanism5,15 as well as to inhibit endothelial cell apoptosis in vitro.16 Significantly, apoptosis of parenchymal and vascular cells has been shown recently to be associated with lung I/R injury after human LTx.17 In addition, in an experimental rat model, inhibition of caspases resulted in attenuation of lung I/R injury.18 It will be important and interesting to investigate these potential mechanisms of CO cytoprotection further in our system. Inflammatory processes in I/R injury are regulated and activated through many signaling cascades by numerous transcription factors. One of the important pathways is the phosphorylation of MAPKs (ie, serine/threonine kinase), which occupy a central position in mediating cell survival and cell death.19 Three major subgroups of the MAPK family identified to date include ERK, c-Jun N-terminal kinase (JNK), and p38 MAPK. The involvement of MAPKs has been reported in several experimental studies examining the cytoprotective function of CO.20 In vitro studies using mouse endothelial cells as well as a mouse warm I/R lung injury model have demonstrated that CO exerts its antiapoptotic effects through p38.21 In the current study, CO’s protective effect on cold I/R injury of the lung was associated with inhibition of ERK phosphorylation. Further studies will be necessary to determine the relative involvement of ERK and p38 in our cold I/R system. Our laboratories have previously reported that recipient CO exposure 1 hr before and 24 hours after transplantation confers protection from I/R injury after small intestine,3-5 kidney6 and liver transplantation.7 In this study, we further determined if donor pretreatment alone would provide protection to lungs undergoing I/R. Previous work has demonstrated that overexpression of HO-1 in the donor organ, as well as a single treatment with methylene chloride to induce CO, could enhance allograft survival and prevent I/R injury.22,23 Also recently, Wang et al reported that CO donor treatment (20 hours) alone could lead to long term survival of the islet grafts.24 Furthermore there is a clinical evidence that the transplantation of organs from a limited number of CO-poisoned donors has resulted in good clinical outcomes.25 Thus, donor pretreatment alone with CO may potentially prevent I/R injury of lung allografts thus simplifying potential clinical application. However, interest-
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ingly, the effect of donor pretreatment with CO for either 1 or 18 hours was marginal in our study and did not achieve the statistical difference compared to air-treated controls, although there was a slight tendency to attenuate graft arterial oxygenation 2 hours after reperfusion. While we do not have a clear explanation for the difference observed from the cited studies, the magnitude of insult or organ specificity may be contribute. CO may be a good candidate for treatment to prevent I/R injury in lung transplant patients. In a clinical setting in ventilated patients, gaseous CO can be directly delivered to the lung graft both during the operation and in the early postoperative period. However as is well known, CO avidly binds to hemoglobin and forms carboxyhemoglobin (COHb) with an affinity 240 times higher than that of oxygen, resulting in interference with the oxygen-carrying capacity of the blood and consequent tissue hypoxia. Although, in the current study, CO did not affect animal behavior or water/food consumption during and after the continuous inhalation for as long as 24 hours following up, further studies will obviously be needed for clinical application. Additionally, as the severity of clinical symptoms correlates with COHb levels in the blood, which can be quickly and easily measured from blood gas analysis, careful monitoring of COHb during CO exposure is necessary. CONCLUSION We have demonstrated that exogenous low dose CO inhalation of donors and recipients can prevent lung I/R injury and significantly improve function of lung grafts after extended cold preservation and transplantation. REFERENCES 1. King RC, Binns OA, Rodriguez F, et al. Reperfusion injury significantly impacts clinical outcome after pulmonary transplantation. Ann Thorac Surg 2000;69:1681-5. 2. Ryter SW, Otterbein LE, Morse D, Choi AM. Heme oxygenase/carbon monoxide signaling pathways: regulation and functional significance. Mol Cell Biochem 2002; 234-235:249 – 63. 3. Nakao A, Kimizuka K, Stolz DB, et al. Protective effect of carbon monoxide inhalation for cold-preserved small intestinal grafts. Surgery 2003;134:285-92. 4. Nakao A, Moore BA, Murase N, et al. Immunomodulatory effects of inhaled carbon monoxide on rat syngeneic small bowel graft motility. Gut 2003;52:1278-85. 5. Nakao A, Kimizuka K, Stolz DB, et al. Carbon monoxide inhalation protects rat intestinal grafts from ischemia/ reperfusion injury. Am J Pathol 2003;163:1587-98. 6. Neto JS, Nakao A, Kimizuka K, et al. Protection of transplant-induced renal ischemia-reperfusion injury with car-
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bon monoxide. Am J Physiol Renal Physiol 2004; 287:F979-89. Kaizu T, Nakao A, Tsung A, et al. Carbon monoxide inhalation ameliorates cold ischemia/reperfusion injury after rat liver transplantation. Surgery 2005;138:229-35. Mizuta T, Kawaguchi A, Nakahara K, Kawashima Y. Simplified rat lung transplantation using a cuff technique. J Thorac Cardiovasc Surg 1989;97:578-81. Reis A, Giaid A, Serrick C, Shennib H. Improved outcome of rat lung transplantation with modification of the nonsuture external cuff technique. J Heart Lung Transplant 1995; 14:274-9. Nakao A, Neto JS, Kanno S, et al. Protection against ischemia/reperfusion injury in cardiac and renal transplantation with carbon monoxide, biliverdin and both. Am J Transplant 2005;5:282-91. Takahashi Y, Ganster RW, Gambotto A, et al. Role of NFkappaB on liver cold ischemia-reperfusion injury. Am J Physiol Gastrointest Liver Physiol 2002;283:G1175-84. Eckenhoff RG, Dodia C, Tan Z, Fisher AB. Oxygen-dependent reperfusion injury in the isolated rat lung. J Appl Physiol 1992;72:1454-60. de Perrot M, Young K, Imai Y, et al. Recipient T cells mediate reperfusion injury after lung transplantation in the rat. J Immunol 2003;171:4995-5002. Fiser SM, Tribble CG, Long SM, et al. Lung transplant reperfusion injury involves pulmonary macrophages and circulating leukocytes in a biphasic response. J Thorac Cardiovasc Surg 2001;121:1069-75. Fujita T, Toda K, Karimova A, et al. Paradoxical rescue from ischemic lung injury by inhaled carbon monoxide driven by derepression of fibrinolysis. Nat Med 2001;7:598-604. Zhang X, Shan P, Otterbein LE, et al. Carbon monoxide inhibition of apoptosis during ischemia-reperfusion lung injury is dependent on the p38 mitogen-activated protein
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kinase pathway and involves caspase 3. J Biol Chem 2003;278:1248-58. Fischer S, Cassivi SD, Xavier AM, et al. Cell death in human lung transplantation: apoptosis induction in human lungs during ischemia and after transplantation. Ann Surg 2000;231:424-31. Quadri SM, Segall L, de Perrot M, et al. Caspase inhibition improves ischemia-reperfusion injury after lung transplantation. Am J Transplant 2005;5:292-9. Widmann C, Gibson S, Jarpe MB, Johnson GL. Mitogenactivated protein kinase: conservation of a three-kinase module from yeast to human. Physiol Rev 1999;79:143-80. Otterbein LE, Bach FH, Alam J, et al. Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway. Nat Med 2000;6:422-8. Zhang X, Shan P, Alam J, Davis RJ, Flavell RA, Lee PJ. Carbon monoxide modulates Fas/Fas ligand, caspases, and Bcl-2 family proteins via the p38alpha mitogen-activated protein kinase pathway during ischemia-reperfusion lung injury. J Biol Chem 2003;278:22061-70. Tullius SG, Nieminen-Kelha M, Buelow R, et al. Inhibition of ischemia/reperfusion injury and chronic graft deterioration by a single-donor treatment with cobalt-protoporphyrin for the induction of heme oxygenase-1. Transplantation 2002;74:591-8. Martins PN, Reuzel-Selke A, Jurisch A, et al. Induction of carbon monoxide in the donor reduces graft immunogenicity and chronic graft deterioration. Transplant Proc 2005;37:379-81. Wang H, Lee SS, Gao W, et al. Donor treatment with carbon monoxide can yield islet allograft survival and tolerance. Diabetes 2005;54:1400-6. Luckraz H, Tsui SS, Parameshwar J, Wallwork J, Large SR. Improved outcome with organs from carbon monoxide poisoned donors for intrathoracic transplantation. Ann Thorac Surg 2001;72:709-13.
Background. Carbon monoxide (CO), a byproduct of heme catalysis by heme oxygenases, has been shown to provide protection against ischemia/reperfusion (I/R) injury. We examined the cytoprotective effect of CO at a low concentration on cold I/R injury of transplanted lung grafts. Methods. Orthotopic left lung transplantation was performed in syngenic Lewis to Lewis rat combination. Grafts were preserved in University of Wisconsin solution at 4°C for 6 hours. Donors and/or recipients were exposed to CO (250 ppm) in air for 1 hour before surgery and then continuously post-transplantation. Results. Blood oxygen partial pressure of graft pulmonary veins in the CO-treated group versus the air-treated group was significantly higher. The increase of messenger RNA of inflammatory mediators such as interleukin-6, tumor necrosis factor-␣, inducible nitric oxide synthase, and cycloooxygenase-2 was markedly inhibited in the CO-treated group. The expression of phosphorylated-extracellular signalregulated protein kinase 1/2 was significantly reduced in the CO-treated group. CO treatment reduced the number of infiltrating macrophages into the lung grafts. Vascular endothelial cells detected by CD31 stain were well preserved in CO-treated grafts, while those in air-treated grafts were faint and interrupted. Conclusions. These results demonstrate that exogenous low-dose CO treatment of donors and recipients can prevent lung I/R injury and significantly improve function of lung grafts after extended cold preservation and transplantation. (Surgery 2006;140:179-85.) From the Department of Surgery,a Heart, Lung, and Esophageal Surgery Institute,b Thomas E. Starzl Transplantation Institute,c and the Department of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine,d University of Pittsburgh
Lung transplantation (LTx) has evolved over the last 2 decades to become the therapy of choice for many patients with advanced pulmonary disease. Current cold preservation strategies for lung grafts remain imperfect, however, placing recipiPresented at the 67th Annual Meeting of the Society of University Surgeons, First Annual Academic Surgical Congress, February 7-11, 2006, San Diego, California. Supported by National Institutes of Health grant HL076265 (K.R.M.) and Gas Enabled Medical Innovations (GEMI) fund (A.N.). Reprint requests: Kenneth R. McCurry, MD, FACS, Dept of Surgery, University of Pittsburgh, 200 Lothrop St, Ste C-700, Pittsburgh, PA 15213. E-mail: [email protected] 0039-6060/$ - see front matter © 2006 Mosby, Inc. All rights reserved. doi:10.1016/j.surg.2006.03.004
ents at risk for life-threatening graft dysfunction resulting from ischemia/reperfusion (I/R) injury.1 Recipients who survive a severe I/R injury to their graft frequently have a prolonged hospitalization and also have a higher incidence of both acute and chronic rejection. In addition, the lung donor pool has been expanded by using lungs from non-ideal donors and from donation after cardiac death (DCD) donors to overcome the problem of organ shortage. Such lung grafts may be more susceptible to cold I/R injury. Thus, a therapeutic strategy to reduce I/R injury after LTx could have a significant impact on clinical outcomes after lung transplantation and potentially expand the available donor pool further. Carbon monoxide (CO), the gaseous catalytic byproduct of heme degradation by heme oxygenases (HOs), has been suggested to be a signaling SURGERY 179
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molecule that exerts significant cytoprotection via its vasodilating, anti-inflammatory, and antiapoptotic properties.2 Our laboratories have been investigating the ability of CO to ameliorate I/R injury in organ transplantation. We have shown that recipient CO inhalation at low concentrations reduces I/R injury in various organ transplantation models in rats including the small intestine, kidney, and liver.3-7 In the present study, we hypothesized that CO inhalation at a low concentration would ameliorate I/R injury in the transplanted lung, resulting in improved graft function. We evaluated this hypothesis using a rat orthotopic lung transplantation model. MATERIAL AND METHODS Animals. Inbred male Lewis (LEW, RT1) rats weighing 250 to 300 g were purchased from Harlan Sprague Dawley, Inc (Indianapolis, Ind), and maintained in laminar flow cages in a specific pathogenfree animal facility at the University of Pittsburgh. All procedures in this experiment were performed according to the guidelines of the Council on Animal Care at the University of Pittsburgh and the National Research Council’s Guide for the Humane Care and Use of Laboratory Animals. Orthotopic LTx. Orthotopic left LTx was performed as described previously.8 Briefly, after donor rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (45 mg/kg) and heparinization (300 units), the lungs were flushed with 20 mL of cold (4°C) University of Wisconsin solution (Viaspan; Du Pont, Wilmington, Del) via the main pulmonary artery. Recipient rats were intubated and ventilated with a mixture of 100% oxygen and 1% isoflurane (Isoflo; Abbott Laboratories, North Chicago, Ill). After the native left lung was removed, the lung graft was transplanted orthotopically by anastomosing the pulmonary vein (PV), artery, and bronchus of the recipient by using the cuff technique, as described.9 After the chest was closed, a pleural drainage tube was removed after aspiration. The recipients were extubated within 10 minutes after surgery when adequate recovery from anesthesia and spontaneous breathing were observed. CO inhalation. CO (1%) in air was mixed with air (21% oxygen) in a stainless steel mixing cylinder and then directed into a 3.70-ft3 glass exposure chamber at a flow rate of 12 L/min. A CO analyzer (Interscan, Chatsworth, Calif) was used to continuously measure CO levels in the chamber, to maintain CO concentration at 250 ppm at all times. Experimental groups. Syngenic LEW to LEW rat LTx with 6 hours of cold preservation served as the
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control (group 1). The animals in group 1 were maintained in room air either in the identical chamber as CO exposure or in the regular laminar flow cage. In groups 2 and 3, the donors were exposed to CO (250 ppm) for 1 and 18 hour before harvesting, respectively. In group 4, the recipients were exposed for 1 hour before and then continuously beginning immediately after transplant surgery. In group 5, both donors and recipients were exposed for 1 hour before and then continuously beginning immediately after transplant surgery. Sham control animals consisted of LEW rats that were intubated and ventilated under the same conditions as transplant recipients but did not undergo an operation. Recipients were sacrificed at 1, 2, 4, 6, and 24 hours after LTx to obtain blood and lung graft samples. Lung graft function. The lung graft function was assessed with blood gases (iSTAT Portable Clinical Analyzer; iSTAT Corp, East Windsor, NJ), on a FiO2 of 1.0, drawn from the PV of the transplanted lung. Histopathology and immunohistochemistry. Lung graft tissues were fixed in 10% formalin, embedded in paraffin, sectioned into 6 m thickness, and stained with hematoxylin-eosin (H&E). The macrophages were stained by immunohistochemistry for ED1 as described previously.6,10 Positively stained cells were counted in 20 high-power fields (⫻400) per each section and expressed as the number of cells per 0.1 mm2 in a blind manner. Immunofluorescent analysis for CD31. For CD31 immunofluorescent stain, graft tissues were frozen in OCT (Optimal Cold Temperature; Sakura Finetek, Inc, Torrance, Calif) compound and cut into 6-m sections. The tissue was stained with mouse antirat CD31 (PE-CAM, 1:100; Serotec, Raleigh, NC) as described previously.5 Real-time reverse transcription-polymerase chain reaction. Intragraft messenger RNA (mRNA) levels for interleukin (IL)-6, tumor necrosis factor (TNF)-␣, cycloxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) were assessed by SYBR Green 2-step, real-time reverse transcription-polymerase chain reaction. Total RNA was extracted from the lung tissues with the use of the TRIzol reagent (Life Technologies, Inc, Grand Island, NY) according to the manufacturer’s instructions. One microgram of total RNA from each sample was used for reverse transcription with an oligo dT (Life Technologies) and a Superscript II (Life Technologies) to generate first-strand complementary DNA. The polymerase chain reaction mixture was prepared with the use of SYBR Green PCR Master Mix (PE Applied Biosystems, Foster City,
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Table I. Primer sequences Target genes IL-6 TNF-␣ COX-2 iNOS GAPDH
Primer Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
Sequence 5=-CAAAGCCAGAGTCATTCAAGC-3= 5=-GGTCCTTAGCCACTCCTTCTGT-3= 5=-GGTGATCGGTCCCAACAAGGA-3= 5=-CACGCTGGCTCAGCCACTC-3= 5=-CTCTGCGATGCTCTTCCGAG-3= 5=-AAGGATTTGCTGCATGGCTG-3= 5=-GGAGAGATTTTTCACGACACCC-3= 5=-CCATGCATAATTTGGACTTGCA-3= 5=-ATGGCACAGTCAAGGCTGAGA-3= 5=-CGCTCCTGGAAGATGGTGAT-3=
COX-2, Cycloxygenase-2; GAPDH, glyceraldehydes 3-phosphate dehydrogenase; IL-6, interleukin 6; iNOS, inducible nitric oxide synthase; TNF-␣, tumor necrosis factor ␣.
Calif), with the primers being designed according to the literature or published sequences (Table I). Thermal cycling conditions were 10 minutes at 95°C to activate the Amplitaq Gold DNA polymerase, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute on an ABI PRISM 7000 Sequence Detection System (PE Applied Biosystems). The expression of each gene was normalized to the mRNA content of glyceraldehyde-3-phosphate dehydrogenase and calculated relative to normal control. Western blot analysis. Western blot analysis was performed with the use of 50 g of cytoplasmic protein from each graft lung tissue as described previously.11 After the blocking of nonspecific binding with nonfat dry milk, membranes were incubated overnight with antiphosphorylated extracellular signal–regulated kinase (ERK) 1/2 (sc7383) and antitotal ERK1/2 (sc-94, Santa Cruz Biotechnology, Santa Cruz, Calif). After incubation with secondary goat antirabbit or antimouse antibody (1:10,000; Pierce Chemical, Rockford, Ill), the membranes were developed with the SuperSignal detection systems (Pierce Chemical) and exposed to film. Data analysis. Results are expressed as means ⫾ SEM. Statistical analysis was performed with the use of the Student t test or analysis of variance where appropriate. A probability level of P less than .05 was considered statistically significant. RESULTS Lung graft function. Graft function was determined by the blood gas analysis of the isolated graft PV. The oxygen partial pressure (pO2) values of graft PV in sham-operated animals were 297 ⫾ 17.7 mm Hg under mechanical ventilation (100% oxygen). Prolonged cold storage and reperfusion im-
paired pulmonary graft functions and resulted in the significant decrease of pO2 levels of the graft PV to 75.0 ⫾ 11.3 mm Hg at 2 hours after reperfusion. The effects of donor pretreatment of CO for either 1 or 18 hours were marginal (1 hour pretreatment; 107 ⫾ 12.5 mm Hg; 18 hours pretreatment; 119.2 ⫾ 14.5 mm Hg), without statistically significant improvement of graft function, compared with air-treated animals. However, both recipient treatment and donor/recipient treatment significantly improved pO2 levels of the graft PV (recipient-treatment, 167.5 ⫾ 28.3 mm Hg; donor/recipient-treatment, 173.3 ⫾ 22.1 mm Hg; P ⬍ .05 vs air control) (Fig 1, A). The partial pressure of carbon dioxide (pCO2) values of graft PVs were comparable, ranging from 27.8 ⫾ 2.8 mm Hg to 36.4 ⫾ 2.4 mm Hg, and there were no statistical differences in each group at 2 hours post-transplantation (Fig 1, B). Inflammatory mediators. Sequential analysis for mRNA levels of inflammatory cytokines (IL-6 and TNF-␣) and stress-induced molecule (iNOS and COX-2) showed a prompt increase of these mediators peaking at 1 to 2 hours after reperfusion (data not shown). Therefore, we analyzed mRNA expression using samples taken 2 hours after reperfusion. The effects of donor pretreatment were marginal with either 1 or 18 hours exposure before harvesting. There was a tendency of suppression of these mediators in the only recipient treatment. However, only donor/recipient treatment with CO inhalation significantly reduced the peak for these inflammatory mediators up to 40%⬃60% of those seen in air-treated grafts (Fig 2). On the basis of these results, further studies used CO-pretreatment of the donor for 1 hour and recipient exposure to CO for 1 hour before and
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Fig 1. Blood gas analysis of graft pulmonary vein. A, pO2 levels. Graft function was significantly insulted 2 hours after I/R injury. Donor pretreatment for 1 hour (CO-D, n ⫽ 4) did not improve graft functions. However, either recipient only (CO-R, n ⫽ 4) or only both donor (1 hour) and recipient (CO-D⫹R, n ⫽ 7) significantly increased graft pulmonary vein pO2 levels, compared with air-controls (n ⫽ 7). B, pCO2 levels. Regardless of the CO exposure protocol, pCO2 levels of graft pulmonary vein 2 hours after reperfusion were comparable without statistical difference among the group. CO, Carbon monoxide; D, donor; pCO2 , partial pressure of carbon dioxide in pulmonary vein blood; N.S., not significant; pO2 , oxygen in partial pressure in pulmonary vein blood; R, recipient.
continuously for 24 hours after reperfusion throughout the remaining experiments. Macrophage infiltration. ED1⫹ macrophages represent the predominant cells present during I/R injury in many organ systems.10 The number of ED1⫹ macrophages in air-control grafts increased to 56.1 ⫾ 1.6/0.1 mm2 from those in sham-operated grafts (22.8 ⫾ 1.0/0.1 mm2) 24 hours after reperfusion. CO inhalation significantly reduced
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Fig 2. Sequential mRNA expression in lung grafts. mRNA for inflammatory cytokines and stress-induced molecule of IL-6, TNF-␣, iNOS and COX-2 were upregulated during I/R injury in air-treated group 2 hours after reperfusion. Only recipient treatment significantly suppressed iNOS and COX-2 mRNA expression. CO treatment of both donor and recipient showed more potent protection against mRNA expression of these inflammatory mediators. (*P ⬍ .05 vs air-treated group; n ⫽ 4-7). D, Donor; IL-6, interleukin 6; iNOS, inducible nitric oxide synthase; R, recipient; TNF-␣, tumor necrosis factor ␣.
the number of ED1⫹ macrophage infiltration to 29.6 ⫾ 0.8/0.1 mm2 (Fig 3). Vascular endothelial cell injury (CD31 staining). Vascular endothelial cells have been known to be a main target of I/R injury, leading to the disruption of microcirculation in the transplanted organs.12 The morphologic analysis of vascular endothelial changes after I/R injury were performed by the fluorescent immunohistochemical stain for pan-endothelial cell marker (CD31). CD31 stains in airtreated grafts were very faint and interrupted 1 hour after reperfusion, suggesting that I/R injury in our model caused remarkable injury to graft vascular endothelial cells. However, vascular endothelial cells stained by CD31 in CO-treated graft were well preserved and almost identical to those in the sham operation (Fig 4). Phosphorylated ERK1/2 expression. Since mitogen-activated protein kinases (MAPKs) are known to be important signaling cascades involved in inflammation, we examined the effect of CO on ac-
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Fig 3. ED1⫹ macrophage infiltration. The number of infiltrating alveolar ED1⫹ macrophages remarkably increased in air-control grafts 24 hours after reperfusion. CO significantly reduced macrophage infiltration. (*P ⬍ .05 vs air-treated group, n ⫽ 4). CO, Carbon monoxide.
Fig 4. CD31 expression of vascular endothelial cells. A, Immunofluorescent stain of CD31 on the vascular endothelial cells in lungs from sham-operated animals. B, CD31 expression was faint and interrupted in air-treated lung grafts after cold I/R injury at 1 hour after reperfusion. C, CD31-positive vascular endothelial cells were preserved in CO-treated lung grafts at 1 hour after reperfusion. Green, CD31; blue, nuclei. Original magnifications: x600. CO, Carbon monoxide.
tivation of MAPKs using cytosolic protein of the lung grafts. Phosphorylation of ERK1/2 occurred rapidly 1 hour after reperfusion, followed by a decrease of the protein expression of phosphorylated ERK1/2 after 6 hours. CO treatment significantly reduced activation of ERK1/2 early after reperfusion (Fig 5). DISCUSSION Although CO is known to be toxic at high concentrations because of its ability to interfere with oxygen delivery, mammalian cells endogenously generate CO primarily via the catalysis of heme by HOs. Recently, several lines of evidence have indicated that the constitutive HO reaction and gener-
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Fig 5. Phosphorylated ERK expression in the lung graft. Representative pictures of Western blots from four independent experiments for phosphorylated ERK1/2 in the lung grafts. Strong expressions of phosphorylated ERK1/2 were seen at 1 hour and 2 hours after reperfusion. CO-treated group significantly suppressed phosphorylation of ERK1/2, compared with air-treated grafts. CO, Carbon monoxide; ERK1/2, extracellular signal-regulated protein kinase 1/2.
ation of CO serve as a key mechanism to maintain the integrity of the physiologic function of organs. Furthermore, HO-1, an inducible form of the HOs, potentially confers protection against oxidative stress through the generation of byproducts of heme catalysis. Accordingly, CO has been considered as a molecule that has cytoprotective properties, supporting a new paradigm that administration of CO at low concentrations may have therapeutic benefits. In this study, we have demonstrated that, in the presence of exogenously administered, CO lung grafts were significantly devoid of I/R injury after cold ischemia and warm reperfusion, resulting in improved graft function. Much previous work has demonstrated that pulmonary I/R injury results from a cascade of events with many interconnected factors, including upregulation of proinflammatory genes, inflammatory infiltrates, complement activation, impaired microcirculation, increased procoagulant activity, T-cell activation, perturbations in endothelial cell function, and apoptotic cell death.13 Alveolar macrophages are activated in the early phase of reperfusion after LTx and play important roles in I/R lung injury. Alveolar macrophages secrete proinflammatory cytokines that prompt neutrophil activation, migration, and extravasation to the alveolar interstitial space with subsequent production of reactive oxygen species. Indeed it has been suggested that the early phase of LTx reperfusion injury is mediated by donor (resident) pulmonary macrophages, while late injury is induced by recipient circulating neutrophils.14 While our study does not suggest an isolated mechanism for the protective effect observed with CO, our findings that CO treatment decreased expression of the proinflammatory mediators TNF-␣, iNOS, COX-2 and IL-6, as well as decreased macrophage infiltration, suggest that the protective effect observed is
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mediated, at least in part, by downregulation of these inflammatory mediators associated with inflammatory cellular infiltration. CO has also been shown in other models of cold and warm organ I/R to promote improved blood flow through a guanylyl cyclase– dependent mechanism5,15 as well as to inhibit endothelial cell apoptosis in vitro.16 Significantly, apoptosis of parenchymal and vascular cells has been shown recently to be associated with lung I/R injury after human LTx.17 In addition, in an experimental rat model, inhibition of caspases resulted in attenuation of lung I/R injury.18 It will be important and interesting to investigate these potential mechanisms of CO cytoprotection further in our system. Inflammatory processes in I/R injury are regulated and activated through many signaling cascades by numerous transcription factors. One of the important pathways is the phosphorylation of MAPKs (ie, serine/threonine kinase), which occupy a central position in mediating cell survival and cell death.19 Three major subgroups of the MAPK family identified to date include ERK, c-Jun N-terminal kinase (JNK), and p38 MAPK. The involvement of MAPKs has been reported in several experimental studies examining the cytoprotective function of CO.20 In vitro studies using mouse endothelial cells as well as a mouse warm I/R lung injury model have demonstrated that CO exerts its antiapoptotic effects through p38.21 In the current study, CO’s protective effect on cold I/R injury of the lung was associated with inhibition of ERK phosphorylation. Further studies will be necessary to determine the relative involvement of ERK and p38 in our cold I/R system. Our laboratories have previously reported that recipient CO exposure 1 hr before and 24 hours after transplantation confers protection from I/R injury after small intestine,3-5 kidney6 and liver transplantation.7 In this study, we further determined if donor pretreatment alone would provide protection to lungs undergoing I/R. Previous work has demonstrated that overexpression of HO-1 in the donor organ, as well as a single treatment with methylene chloride to induce CO, could enhance allograft survival and prevent I/R injury.22,23 Also recently, Wang et al reported that CO donor treatment (20 hours) alone could lead to long term survival of the islet grafts.24 Furthermore there is a clinical evidence that the transplantation of organs from a limited number of CO-poisoned donors has resulted in good clinical outcomes.25 Thus, donor pretreatment alone with CO may potentially prevent I/R injury of lung allografts thus simplifying potential clinical application. However, interest-
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ingly, the effect of donor pretreatment with CO for either 1 or 18 hours was marginal in our study and did not achieve the statistical difference compared to air-treated controls, although there was a slight tendency to attenuate graft arterial oxygenation 2 hours after reperfusion. While we do not have a clear explanation for the difference observed from the cited studies, the magnitude of insult or organ specificity may be contribute. CO may be a good candidate for treatment to prevent I/R injury in lung transplant patients. In a clinical setting in ventilated patients, gaseous CO can be directly delivered to the lung graft both during the operation and in the early postoperative period. However as is well known, CO avidly binds to hemoglobin and forms carboxyhemoglobin (COHb) with an affinity 240 times higher than that of oxygen, resulting in interference with the oxygen-carrying capacity of the blood and consequent tissue hypoxia. Although, in the current study, CO did not affect animal behavior or water/food consumption during and after the continuous inhalation for as long as 24 hours following up, further studies will obviously be needed for clinical application. Additionally, as the severity of clinical symptoms correlates with COHb levels in the blood, which can be quickly and easily measured from blood gas analysis, careful monitoring of COHb during CO exposure is necessary. CONCLUSION We have demonstrated that exogenous low dose CO inhalation of donors and recipients can prevent lung I/R injury and significantly improve function of lung grafts after extended cold preservation and transplantation. REFERENCES 1. King RC, Binns OA, Rodriguez F, et al. Reperfusion injury significantly impacts clinical outcome after pulmonary transplantation. Ann Thorac Surg 2000;69:1681-5. 2. Ryter SW, Otterbein LE, Morse D, Choi AM. Heme oxygenase/carbon monoxide signaling pathways: regulation and functional significance. Mol Cell Biochem 2002; 234-235:249 – 63. 3. Nakao A, Kimizuka K, Stolz DB, et al. Protective effect of carbon monoxide inhalation for cold-preserved small intestinal grafts. Surgery 2003;134:285-92. 4. Nakao A, Moore BA, Murase N, et al. Immunomodulatory effects of inhaled carbon monoxide on rat syngeneic small bowel graft motility. Gut 2003;52:1278-85. 5. Nakao A, Kimizuka K, Stolz DB, et al. Carbon monoxide inhalation protects rat intestinal grafts from ischemia/ reperfusion injury. Am J Pathol 2003;163:1587-98. 6. Neto JS, Nakao A, Kimizuka K, et al. Protection of transplant-induced renal ischemia-reperfusion injury with car-
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