Carbon Monoxide Inhalation Protects Rat Intestinal Grafts from Ischemia/Reperfusion Injury
2003; Elsevier BV; Volume: 163; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)63515-8
ISSN1525-2191
AutoresAtsunori Nakao, Kei Kimizuka, Donna B. Stolz, João Seda Neto, Takashi Kaizu, Augustine M.K. Choi, Takashi Uchiyama, Brian S. Zuckerbraun, Michael A. Nalesnik, Leo E. Otterbein, Noriko Murase,
Tópico(s)Organ Transplantation Techniques and Outcomes
ResumoCarbon monoxide (CO), a byproduct of heme catalysis by heme oxygenases, has been shown to exert anti-inflammatory effects. This study examines the cytoprotective efficacy of inhaled CO during intestinal cold ischemia/reperfusion injury associated with small intestinal transplantation. Orthotopic syngenic intestinal transplantation was performed in Lewis rats after 6 hours of cold preservation in University of Wisconsin solution. Three groups were examined: normal untreated controls, control intestinal transplant recipients kept in room air, and recipients exposed to CO (250 ppm) for 1 hour before and 24 hours after surgery. In air grafts, mRNA levels for interleukin-6, cyclooxygenase-2, intracellular adhesion molecule (ICAM-1), and inducible nitric oxide synthase rapidly increased after intestinal transplant. Histopathological analysis revealed severe mucosal erosion, villous congestion, and inflammatory infiltrates. CO effectively blocked an early up-regulation of these mediators, showed less severe histopathological changes, and resulted in significantly improved animal survival of 92% from 58% in air-treated controls. CO also significantly reduced mRNA for proapoptotic Bax, while it up-regulated anti-apoptotic Bcl-2. These changes in CO-treated grafts correlated with well-preserved CD31+ vascular endothelial cells, less frequent apoptosis/necrosis in intestinal epithelial and capillary endothelial cells, and improved graft tissue blood circulation. Protective effects of CO in this study were mediated via soluble guanylyl cyclase, because 1H-(1,2,4)oxadiazole (4,3-α) quinoxaline-1-one (soluble guanylyl cyclase inhibitor) completely reversed the beneficial effect conferred by CO. Perioperative CO inhalation at a low concentration resulted in protection against ischemia/reperfusion injury to intestinal grafts with prolonged cold preservation. Carbon monoxide (CO), a byproduct of heme catalysis by heme oxygenases, has been shown to exert anti-inflammatory effects. This study examines the cytoprotective efficacy of inhaled CO during intestinal cold ischemia/reperfusion injury associated with small intestinal transplantation. Orthotopic syngenic intestinal transplantation was performed in Lewis rats after 6 hours of cold preservation in University of Wisconsin solution. Three groups were examined: normal untreated controls, control intestinal transplant recipients kept in room air, and recipients exposed to CO (250 ppm) for 1 hour before and 24 hours after surgery. In air grafts, mRNA levels for interleukin-6, cyclooxygenase-2, intracellular adhesion molecule (ICAM-1), and inducible nitric oxide synthase rapidly increased after intestinal transplant. Histopathological analysis revealed severe mucosal erosion, villous congestion, and inflammatory infiltrates. CO effectively blocked an early up-regulation of these mediators, showed less severe histopathological changes, and resulted in significantly improved animal survival of 92% from 58% in air-treated controls. CO also significantly reduced mRNA for proapoptotic Bax, while it up-regulated anti-apoptotic Bcl-2. These changes in CO-treated grafts correlated with well-preserved CD31+ vascular endothelial cells, less frequent apoptosis/necrosis in intestinal epithelial and capillary endothelial cells, and improved graft tissue blood circulation. Protective effects of CO in this study were mediated via soluble guanylyl cyclase, because 1H-(1,2,4)oxadiazole (4,3-α) quinoxaline-1-one (soluble guanylyl cyclase inhibitor) completely reversed the beneficial effect conferred by CO. Perioperative CO inhalation at a low concentration resulted in protection against ischemia/reperfusion injury to intestinal grafts with prolonged cold preservation. Recent developments in the use of potent immunosuppressive drugs, technical innovations in surgery, and improvements in postoperative care have significantly improved outcomes of small intestinal transplantation (SITx), and the procedure has become the critical therapeutic modality for patients with intestinal failure.1Abu-Elmagd K Reyes J Bond G Mazariegos G Wu T Murase N Sindhi R Martin D Colangelo J Zak M Janson D Ezzelarab M Dvorchik I Parizhskaya M Deutsch M Demetris A Fung J Starzl TE Clinical intestinal transplantation: a decade of experience at a single center.Ann Surg. 2001; 234: 404-416Crossref PubMed Scopus (336) Google Scholar However, intestinal tissue is known to be exceptionally susceptible to ischemia/reperfusion (I/R) injury and intestinal grafts frequently suffer preservation injuries, resulting in prolonged intestinal dysfunction, loss of intestinal barrier function, bacterial translocation, and posttransplant sepsis. Therefore, advances in this field will significantly improve posttransplant patient care and subsequent long-term outcome of SITx. Although the exact mechanisms of intestinal I/R injury remain undefined, multiple factors are shown to be involved in the process. The lack of oxygen during preservation is known to initiate ATP depletion, followed by an alteration of intracellular calcium and sodium concentrations and activation of cytotoxic enzymes (eg, proteases, phospholipases).2Gores GJ Nieminen AL Fleishman KE Dawson TL Herman B Lemasters JJ Extracellular acidosis delays onset of cell death in ATP-depleted hepatocytes.Am J Physiol. 1988; 255: C315-C322PubMed Google Scholar, 3Buderus S Siegmund B Spahr R Krutzfeldt A Piper HM Resistance of endothelial cells to anoxia-reoxygenation in isolated guinea pig hearts.Am J Physiol. 1989; 257: H488-H493PubMed Google Scholar In addition, reperfusion of grafts generates reactive oxygen species and further promotes cell damage.4Freeman BA Crapo JD Biology of disease: free radicals and tissue injury.Lab Invest. 1982; 47: 412-426PubMed Google Scholar As a result, death or loss of the integrity of vascular endothelial cells, consequent disruption of microcirculation, generation and release of potent inflammatory mediators (cytokines, adhesion molecules, platelet activating factors), and neutrophil infiltration are known to be characteristic features associated with I/R injuries.5Clavien PA Harvey PR Strasberg SM Preservation and reperfusion injuries in liver allografts. An overview and synthesis of current studies.Transplantation. 1992; 53: 957-978Crossref PubMed Scopus (794) Google Scholar, 6Jaeschke H Preservation injury: mechanisms, prevention and consequences.J Hepatol. 1996; 25: 774-780Abstract Full Text PDF PubMed Scopus (134) Google Scholar Carbon monoxide (CO), a gaseous molecule, has been well described to be toxic and lethal to living organisms when exposed to high concentrations. However, recent evidence suggests that CO acts as a regulatory molecule in cellular and biological processes. Mammalian cells have the ability to endogenously generate CO primarily via the catalysis of heme by heme oxygenases (HO-1, -2, -3) and, to a much lesser degree, via lipid peroxidation.7Poss KD Tonegawa S Heme oxygenase 1 is required for mammalian iron reutilization.Proc Natl Acad Sci USA. 1997; 94: 10919-10924Crossref PubMed Scopus (882) Google Scholar HO-1, the inducible isoform, has been classified as a stress-inducible protein. It is up-regulated in response to oxidative stress and proinflammatory stimuli in the liver, heart, and kidney, and has been shown to exert potent cytoprotective and anti-apoptotic properties.8Maines MD Mayer RD Ewing JF McCoubrey Jr, WK Induction of kidney heme oxygenase-1 (HSP32) mRNA and protein by ischemia/reperfusion: possible role of heme as both promotor of tissue damage and regulator of HSP32.J Pharmacol Exp Ther. 1993; 264: 457-462PubMed Google Scholar, 9Tacchini L Schiaffonati L Pappalardo C Gatti S Bernelli-Zazzera A Expression of HSP 70, immediate-early response and heme oxygenase genes in ischemic-reperfused rat liver.Lab Invest. 1993; 68: 465-471PubMed Google Scholar, 10Amersi F Buelow R Kato H Ke B Coito AJ Shen XD Zhao D Zaky J Melinek J Lassman CR Kolls JK Alam J Ritter T Volk HD Farmer DG Ghobrial RM Busuttil RW Kupiec-Weglinski JW Upregulation of heme oxygenase-1 protects genetically fat Zucker rat livers from ischemia/reperfusion injury.J Clin Invest. 1999; 104: 1631-1639Crossref PubMed Scopus (467) Google Scholar, 11Hangaishi M Ishizaka N Aizawa T Kurihara Y Taguchi J Nagai R Kimura S Ohno M Induction of heme oxygenase-1 can act protectively against cardiac ischemia/reperfusion in vivo.Biochem Biophys Res Commun. 2000; 279: 582-588Crossref PubMed Scopus (112) Google Scholar, 12Shimizu H Takahashi T Suzuki T Yamasaki A Fujiwara T Odaka Y Hirakawa M Fujita H Akagi R Protective effect of heme oxygenase induction in ischemic acute renal failure.Crit Care Med. 2000; 28: 809-817Crossref PubMed Scopus (168) Google Scholar, 13Kato H Amersi F Buelow R Melinek J Coito AJ Ke B Busuttil RW Kupiec-Weglinski JW Heme oxygenase-1 overexpression protects rat livers from ischemia/reperfusion injury with extended cold preservation.Am J Transplant. 2001; 1: 121-128Crossref PubMed Scopus (170) Google Scholar, 14Soares MP Brouard S Smith RN Bach FH Heme oxygenase-1, a protective gene that prevents the rejection of transplanted organs.Immunol Rev. 2001; 184: 275-285Crossref PubMed Scopus (75) Google Scholar, 15Katori M Anselmo DM Busuttil RW Kupiec-Weglinski JW A novel strategy against ischemia and reperfusion injury: cytoprotection with heme oxygenase system.Transpl Immunol. 2002; 9: 227-233Crossref PubMed Scopus (84) Google Scholar CO, a byproduct of HO-1, has been demonstrated to mediate cytoprotective and anti-inflammatory effects,16Pannen BH Kohler N Hole B Bauer M Clemens MG Geiger KK Protective role of endogenous carbon monoxide in hepatic microcirculatory dysfunction after hemorrhagic shock in rats.J Clin Invest. 1998; 102: 1220-1228Crossref PubMed Scopus (149) Google Scholar, 17Brouard S Otterbein LE Anrather J Tobiasch E Bach FH Choi AM Soares MP Carbon monoxide generated by heme oxygenase 1 suppresses endothelial cell apoptosis.J Exp Med. 2000; 192: 1015-1026Crossref PubMed Scopus (895) Google Scholar, 18Otterbein LE Bach FH Alam J Soares M Tao Lu H Wysk M Davis RJ Flavell RA Choi AM Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway.Nat Med. 2000; 6: 422-428Crossref PubMed Scopus (1883) Google Scholar, 19Kyokane T Norimizu S Taniai H Yamaguchi T Takeoka S Tsuchida E Naito M Nimura Y Ishimura Y Suematsu M Carbon monoxide from heme catabolism protects against hepatobiliary dysfunction in endotoxin-treated rat liver.Gastroenterology. 2001; 120: 1227-1240Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 20Amersi F Shen XD Anselmo D Melinek J Iyer S Southard DJ Katori M Volk HD Busuttil RW Buelow R Kupiec-Weglinski JW Ex vivo exposure to carbon monoxide prevents hepatic ischemia/reperfusion injury through p38 MAP kinase pathway.Hepatology. 2002; 35: 815-823Crossref PubMed Scopus (221) Google Scholar, 21Otterbein LE Carbon monoxide: innovative anti-inflammatory properties of an age-old gas molecule.Antioxid Redox Signal. 2002; 4: 309-319Crossref PubMed Scopus (139) Google Scholar similar to those seen with HO-1. Although much is known regarding the toxicity and lethality of environmental concentrations of CO, little if any progress has been made in our understanding as to how CO mediates its beneficial biological and physiological functions when used in low concentrations. Accordingly, based on the hypothesis that a low concentration of CO would provide protection against I/R injury associated with intestinal transplantation, this study examined the efficacy of CO in the rat intestinal I/R injury model with prolonged cold preservation and transplantation. Inbred male Lewis (LEW, RT1l) rats weighing 200 to 250 g were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN), and maintained in laminar flow cages in a specific pathogen-free animal facility at the University of Pittsburgh. Animals were fed a standard diet and provided water ad libitum. 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 SITx with caval drainage was performed using a previously described technique.22Murase N Demetris AJ Woo J Tanabe M Furuya T Todo S Starzl TE Graft-versus-host disease after brown Norway-to-Lewis and Lewis-to-Brown Norway rat intestinal transplantation under FK506.Transplantation. 1993; 55: 1-7Crossref PubMed Scopus (69) Google Scholar The entire donor small intestine from the ligament of Treitz to the ileocecal valve was isolated, perfused with 10 ml of cold University of Wisconsin solution (Viaspan; Du Pont, Wilmington, DE), and placed in University of Wisconsin solution at 4°C for 6 hours. End-to-side anastomoses between the graft aorta and the recipient infrarenal aorta, and between the graft portal vein and recipient vena cava were performed with 10-0 Novafil suture. The entire recipient intestine was removed and the enteric continuity was restored by proximal and distal end-to-end intestinal anastomoses. The entire recipient surgery usually took ∼60 minutes. All recipient animals in this study were given cefamandole nafate (20 mg once a day, intramuscular injection) for 3 days after transplant surgery. Animals were exposed to CO at a concentration of 250 ppm. Briefly, 1% CO in air was mixed with air (21% oxygen) in a stainless steel mixing cylinder and then directed into a 3.70 ft3Buderus S Siegmund B Spahr R Krutzfeldt A Piper HM Resistance of endothelial cells to anoxia-reoxygenation in isolated guinea pig hearts.Am J Physiol. 1989; 257: H488-H493PubMed Google Scholar glass exposure chamber at a flow rate of 12 L/minute. A CO analyzer (Interscan, Chatsworth, CA) was used to continuously measure CO levels in the chamber, to maintain CO concentration at 250 ppm at all times. Animals were maintained in a CO chamber for the duration of CO exposure with regular diet and water ad libitum. LEW to LEW syngenic SITx with 6 hours University of Wisconsin solution cold preservation was performed without immunosuppression. Three groups of animals were examined: group 1 consisted of unoperated normal LEW, group 2 recipients received intestinal grafts and were maintained in room air, and group 3 recipients of SITx were placed in the CO chamber for 1 hour before and for 24 hours immediately after transplant surgery. In each group, recipient animals were sacrificed at 1, 3, 6, 12, 24, and 48 hours after SITx for blood and graft intestine samples (n = 4 to 6 in each group at each time point). A small number (30%) of animals in group 2 was kept in the chamber without CO (room air) and sacrificed 1 to 48 hours after SITx to evaluate the influence of animal housing and after SITx recovery environment. Because animal housing environment did not cause any difference in obtained results, the air-control group in this study included recipients that were kept inside and outside of the chamber. To avoid any divergence caused by patchy changes of intestinal I/R injury, whole intestinal graft was divided into nine equal-length portions and processed as the following: first and fourth segments were used for mRNA isolation, and second and fifth segments were used for protein isolation. All remaining segments were processed for routine immunohistopathology. Peyer's patches were excluded from graft samples for mRNA and protein extraction. Separate groups of animals were followed for 14 days after SITx to determine the efficacy of CO treatment on intestinal graft/animal survival. Because animal survival entirely depended on the function of transplanted intestinal grafts, animal survival was considered to be identical to graft survival in this model. Additional supplementary groups of animals were prepared to test the role of soluble guanylyl cyclase (sGC) and cyclic 3′,5′-guanosine monophosphate (cGMP) system. A selective inhibitor of sGC, 1H-(1,2,4)oxadiazole (4,3-α) quinoxaline-1-one (ODQ) (20 mg/kg; Sigma, St. Louis, MO), was dissolved in dimethyl sulfoxide and intraperitoneally injected to recipients 30 minutes before placing in a CO chamber.23Schrammel A Behrends S Schmidt K Koesling D Mayer B Characterization of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one as a heme-site inhibitor of nitric oxide-sensitive guanylyl cyclase.Mol Pharmacol. 1996; 50: 1-5PubMed Google Scholar, 24Zingarelli B Hasko G Salzman AL Szabo C Effects of a novel guanylyl cyclase inhibitor on the vascular actions of nitric oxide and peroxynitrite in immunostimulated smooth muscle cells and in endotoxic shock.Crit Care Med. 1999; 27: 1701-1707Crossref PubMed Scopus (43) Google Scholar Control animals received dimethyl sulfoxide without ODQ. Animals were sacrificed at 1 hour after SITx to determine graft blood flow and mRNA levels. To determine blood CO and O2 levels, arterial blood samples (0.2 ml) were taken at various time points after CO inhalation (250 ppm). Carboxyhemoglobin (COHb), methemoglobin (MetHb), and oxygen saturation (SaO2) were measured using OSM3 Hemoximeter (Radiometer Copenhagen, Copenhagen, Denmark). Intestinal microvascular blood flow was monitored at 1 hour after SITx by a laser Doppler flowmeter (BLF 21D; Transonic Systems, Ithaca, NY) on the serosal surface of the graft jejunum and ileum adjacent to the mesenteric border. This measurement was repeated three times each in proximal, middle, and distal portions of the intestinal grafts (nine measurements per animal) by one of the authors (TU) without knowledge of the experimental groups. Blood flows in superior mesenteric artery and marginal artery were also analyzed. Intestinal cytosolic proteins (200 μg) were separated by electrophoresis on 12.5% acrylamide sodium dodecyl sulfate gels and transferred to nitrocellulose membranes (Scleicher & Schuell, Keene, NH). After blocking with 5% nonfat dry milk, membranes were incubated with primary rabbit polyclonal anti-HO-1 antibody (SPA-896, 1:1000; Stressgen, Victoria, Canada) for 1 hour, then with secondary goat anti-rabbit antibody (1:2000; Pierce Chemical, Rockford, IL) for 1 hour. Membranes were developed with the SuperSignal detection systems (Pierce Chemical) and exposed to film. Total RNA was extracted from the intestinal graft using the Trizol reagent (Life Technologies, Inc. Grand Island, NY) according to the manufacturer's instructions. RNA content was measured using 260/280 UV spectrophotometry. An RNase protection assay was performed to determine the involvement of apoptosis-associated molecules with the Riboquant kit (Pharmingen, La Jolla, CA) according to the manufacturer's protocol. Briefly, radiolabeled anti-sense RNA multiple probes were synthesized using an in vitro transcription kit and rat multiprobe template set rAPO-1 (Pharmingen). 32P-labeled probes (8.0 × 105 cpm) and sample RNA (5 μg) were hybridized at 56°C for 16 hours and single-stranded RNAs including anti-sense RNA probes were digested by RNase per the manufacturer's protocol (Pharmingen). The protected RNA duplexes were loaded on a 40% polyacrylamide electrophoresis gel and autoradiography was measured using a PhosphorImager system (Molecular Dynamics, Krefeld, Germany). Radioactivity of mRNA bands was quantified with NIH Image (National Institutes of Health, Bethesda, MD), normalized to GAPDH, and expressed as the ratio of apoptotic gene expression/GAPDH. mRNA expression was quantified by SYBR Green two-step, real-time reverse transcriptase-PCR for plasminogen activator inhibitor-1 (PAI-1), intercellular adhesion molecule (ICAM-1), interleukin (IL)-6, tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), HO-1, and GAPDH. Total RNA pellets were suspended in RNase-free water, followed by removal of potentially contaminating DNA by treatment with DNase I (Life Technologies, Rockville, MD). One μg 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 cDNA. PCR reaction mixture was prepared using SYBR Green PCR Master Mix (PE Applied Biosystems, Foster City, CA). Each sample was analyzed in duplicate using the conditions recommended by the manufacturer. The following primers were used: IL-6 sense primer, 5′-CAAAGCCAGAGTCATTCAAGC-3′, anti-sense primer, 5′-GGTCCTTAGCCACTCCTTCTGT-3′; iNOS sense primer, 5′-GGAGAGATTTTTCACG ACACCC-3′, anti-sense primer, 5′-CCATGCATAATTTGGACTTGCA-3′; COX-2 sense primer, 5′-CTCTGCGATGCTCTTCCGAG-3′, anti-sense primer, 5′-AAGGATTTGCTGCATGGCTG-3′; ICAM-1 sense primer, 5′-CGTGGCGTCCATTTACACCT-3′, anti-sense primer, 5′-TTAGGGCCTCCTCCTGAGC-3′; TNF-α sense primer, 5′-GGTGATCGGTCCCAACAAGGA-3′, anti-sense primer, CACGCTGGCTCAGCCACTC-3′; PAI-1 sense primer, 5′-CCGATGGGCTCGAGTATGA-3′, anti-sense primer, 5′-TTGTCTGATGAGTTCAGCATCCA-3′; HO-1 sense primer, 5-CACAAAGACCAGAGTCCCTCACAG-3 anti-sense primer, 5-AAATTCCCACTGCCACGGT-3; and GAPDH sense primer, 5′-ATGGCACAGTCAAGGCTGAGA-3′, anti-sense primer 5′-CGCTCCTGGAAGATGGTGAT-3′. 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). Using the manufacturer's software, real-time PCR data were blotted as the ΔRn fluorescence signal versus the cycle number. The cycle threshold was defined as the cycle number at which the ΔRn crosses this threshold. The expression of each gene was normalized to GAPDH mRNA content and calculated relative to control using the comparative cycle threshold method.25Schmittgen TD Zakrajsek BA Mills AG Gorn V Singer MJ Reed MW Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods.Anal Biochem. 2000; 285: 194-204Crossref PubMed Scopus (868) Google Scholar Serum IL-6 concentrations were determined using a rat enzyme-linked immunosorbent assay kit (ELISA; R&D, Cambridge, MA). The serum nitrite/nitrate levels, the stable end products of NO metabolism, were measured using a commercially available test kit (Cayman, Ann Arbor, MI). Intestinal graft tissues were taken 1 hour after reperfusion and homogenized. Supernatant of the homogenized intestinal tissues was analyzed for total antioxidant power (Oxford Biomedical Research, Oxford, MI) according to the manufacturer's protocol. The antioxidant level in each sample was determined by the reduction of Cu2+ to Cu+ because of the combined action of all antioxidants present in the sample. Generated Cu+ was detected by the complex formation between Cu+ and bathocuproine (BC), and stable complex was detected at absorption maximum between 480 to 490 nm. The obtained absorbance values were compared to a standard curve obtained using uric acid as the reductant. Intestinal graft samples were fixed in 10% buffered formalin, embedded in paraffin, cut into 4-μm-thick sections, and stained with hematoxylin and eosin. Degrees of I/R injuries in the villi were blindly assessed by a pathologist (MAN), based on the extent of mucosal erosion, villous congestion, and epithelialization in at least 12 sections per animal. Mucosal erosion was defined as loss of surface mucosa exceeding approximately one-half of the villous length. Villous congestion was a low-power assessment of vascular congestion in the upper mucosal/villous region. Epithelialization was defined as the presence of a flattened mucosal epithelial region with or without associated erosion. A semiquantitative grading system was applied to the histological variables based on the approximate percentage of the total sample involved with the individual process. Although the grading system is a reflection of the extent of the change, it generally also correlates with the severity of the change. Grade 0 showed involvement in 75%. Formalin-fixed, paraffin-embedded graft tissue was cut into 5-μm sections for activated caspase-3 stain using the avidin-biotin-peroxidase complex method after antigen retrieval. Endogenous peroxidase activity was blocked with superblock (Scy Tek Laboratories, Logan, UT). Sections were incubated with anti-cleaved caspase-3 polyclonal antibody (1:100; Cell Signaling Technology, Beverly, MA) overnight at 4°C, followed with biotinylated goat anti-rabbit IgG for 1 hour at room temperature (DAKO, Glostrup, Denmark). The immune complex was visualized with 3-amino-9-ethyl carbazole and hematoxylin counterstaining. For CD31 immunofluorescent stain, graft tissues were frozen in OCT (Optimal Cold Temperature; Sakura Finetek, Inc., Torrance, CA) cut into 4-μm sections, and fixed with 2% paraformaldehyde. After blocking with 20% (v/v) normal goat serum, the tissue was stained with mouse anti-rat CD31 (PE-CAM, 1:100; Serotec, Raleigh, NC) for 1 hour, then incubated for 1 hour with Alexa 488 (Molecular Probes, Eugene OR) conjugated goat anti-mouse IgG antibody. F-Actin was visualized by staining with rhodamine-phalloidin (1:250, Molecular Probes) for 30 minutes, then with Hoechst dye (bisBenzimide, 1 μg/100 ml) for 30 seconds to stain nuclear DNA. The sections were washed and coverslipped with Gelvatol, a water-soluble mounting media [23 g polyvinyl alcohol, 50 ml glycerol, 0.01% sodium azide in 100 ml of phosphate-buffered saline (PBS)], and visualized with an Olympus BX51 epifluorescence microscope and digitized with an Olympus color video camera. Intestines were immersion fixed in 2.5% glutaraldehyde overnight at 4°C, washed three times in PBS, then postfixed in aqueous 1% OsO4, 1% K3Fe(CN)6 for 1 hour. After three PBS washes, the tissue was dehydrated through a graded series of 30 to 100% ethanol, 100% propylene oxide, then infiltrated in 1:1 mixture of propylene oxide:Polybed 812 epoxy resin (Polysciences, Warrington, PA) for 1 hour. After several changes of 100% resin throughout 24 hours, tissue was embedded in molds, cured at 37°C overnight, followed by additional hardening at 65°C for 2 more days. Ultrathin (70 nm) sections were collected on 200-mesh copper grids, stained with 2% uranyl acetate in 50% methanol for 10 minutes, followed by 1% lead citrate for 7 minutes. Sections were photographed using a JEOL JEM 1210 transmission electron microscope (JEOL, Peabody, MA) at 80 kV onto electron microscope film (ESTAR thick base; Kodak, Rochester, NY). Electron micrographs were digitized on a flatbed scanner at 400 ppi (Studiostar; Agfa, Ridgefield Park, NJ). Digitized images were assembled into montages using Adobe Photoshop 6.1. The abdominal aorta was cannulated with 20 gauge IV catheter and clamped above the superior mesenteric artery. After blood was flushed from the intestinal vasculature using cold lactate Ringer solution, the vasculature was then perfused with Batson's no. 17 methyl methacrylate prepared according to the manufacturer's directions (Polysciences). Casting solution was allowed to polymerize for 1 hour at 4°C, intestines were removed from the animal and 2-cm sections were collected, cut open, then pinned luminal side up onto dental wax. Polymerization was allowed to continue overnight at 4°C. Tissue was then placed into several changes of 10% NaOH solution at room temperature throughout a 1-week period. When connective tissues were removed and vascular casts appeared clean, samples were washed 10 to 15 times with distilled water throughout a 2-day period. After drying at room temperature overnight, the casts were mounted onto aluminum stubs coated with copper double-stick tape, grounded with silver paint, sputter-coated with 3.5 nm of gold-paladium (108 Auto; Cressington, Cranberry, PA) and viewed at 5 kV on a JEOL 6330 scanning electron microscope (JEOL). Results are expressed as mean ± SD. Statistical analysis was performed using Student's t-test or analysis of variance where appropriate. A probability level of P < 0.05 was considered statistically significant. When animals were kept in the CO chamber, they were observed every 1 to 3 hours. Water and food consumption and body weight gain during and after CO inhalation were not different from those kept in room air. One-hour CO inhalation resulted in the elevation of COHb to 20.5 ± 6.0% from 1.6 ± 0.7% in the room air. When animals were removed from the CO chamber to room air for transplant surgery, COHb levels decreased to 13.7 ± 0.1% by the end of surgery. Thus, COHb levels in recipients were between 13% and 22% throughout the experiment. MetHb levels were <1% at all time points. Blood oxygen saturation was 100.4 ± 2.2 in room air and 101.7 ± 3.9 at the end of 24 hours of CO inhalation. Six hours of cold preservation in University of Wisconsin solution of the intestinal graft induced intestinal dysfunction in untreated recipients; 3 of 12 control animals died within 24 hours and an additional 2 animals died 5 and 7 days after SITx because of bowel obstruction secondarily to intestinal I/R injury. In contrast, all CO-treated animals recovered smoothly from SITx, and only 1 of 12 CO-treated animals died of bowel obstruction on day 8. Overall animal survival for 14 days of follow-up was 58.3% (7 of 12) in air control and 91.7% (11 of 12) in CO-treated group (P < 0.05) (Figure 1). CO has been known to play an important role in regulating vasomotor tone by promoting vasorelaxation through sGC activation.26Sammut IA Foresti R Clark JE Exon DJ Vesely MJ Sarathchandra P Green CJ Motterlini R Carbon monoxide is a major contributor to the regulation of vascular tone in aortas expressing high levels of haeme oxygenase-1.Br J Pharmacol. 1998; 125: 1437-1444Crossref PubMed Scopus (213) Google Scholar, 27Fujita T Toda K Karimova A Yan SF Naka Y Yet SF Pinsky DJ Paradoxical rescue from ischemic lung injury
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