Cold Ischemia-reperfusion Research Articles

Polyethylene glycols: An effective strategy for limiting liver ischemia reperfusion injury.

Liver ischemia-reperfusion injury (IRI) is an inherent feature of liver surgery and liver transplantation in which damage to a hypoxic organ (ischemia) is exacerbated following the return of oxygen delivery (reperfusion). IRI is a major cause of primary non-function after transplantation and may lead to graft rejection, regardless of immunological considerations. The immediate response involves the disruption of cellular mitochondrial oxidative phosphorylation and the accumulation of metabolic intermediates during the ischemic period, and oxidative stress during blood flow restoration. Moreover, a complex cascade of inflammatory mediators is generated during reperfusion, contributing to the extension of the damage and finally to organ failure. A variety of pharmacological interventions (antioxidants, anti-cytokines, etc.) have been proposed to alleviate graft injury but their usefulness is limited by the local and specific action of the drugs and by their potential undesirable toxic effects. Polyethylene glycols (PEGs), which are non-toxic water-soluble compounds approved by the FDA, have been widely used as a vehicle or a base in food, cosmetics and pharmaceuticals, and also as adjuvants for ameliorating drug pharmacokinetics. Some PEGs are also currently used as additives in organ preservation solutions prior to transplantation in order to limit the damage associated with cold ischemia reperfusion. More recently, the administration of PEGs of different molecular weights by intravenous injection has emerged as a new therapeutic tool to protect liver grafts from IRI. In this review, we summarize the current knowledge concerning the use of PEGs as a useful target for limiting liver IRI.

Tiprotec preserves endothelial function after cold ischemia and warm reperfusion: comparison between Saline, Custodiol and Tiprotec

Background: Coronary artery bypass surgery provides excellent patency rates, however the early/late graft failure reduces the long-term benefit of myocardial revascularization.

Hydrogen Sulfide Treatment Mitigates Renal Allograft Ischemia-Reperfusion Injury during Cold Storage and Improves Early Transplant Kidney Function and Survival Following Allogeneic Renal Transplantation

Ischemia-reperfusion injury is unavoidable during organ transplantation. Prolonged ischemia-reperfusion injury is detrimental to short-term and long-term graft function and survival. H2S is a recently characterized, endogenously produced gaseous molecule with important physiological roles that has been shown to be cytoprotective during tissue ischemia-reperfusion injury. The current study aimed to determine whether H2S could mitigate cold renal ischemia-reperfusion injury in the clinically relevant context of allogeneic renal transplantation. Following bilateral native nephrectomy Lewis rats underwent renal transplantation with kidneys from Brown Norway donor rats that were flushed with cold (4C) standard University of Wisconsin preservation solution (University of Wisconsin preservation solution group) or cold University of Wisconsin preservation solution plus 150 μM NaHS (H2S group) solution. Kidneys were stored for 6 hours at 4C in the same solution. Recipient animals were monitored for 14 days or until sacrifice using metabolic cages to assess various parameters of renal graft function. H2S treatment improved early allograft survival and function, and decreased early levels of necrosis, apoptosis and Kim-1 compared to University of Wisconsin preservation solution alone. H2S treatment did not affect allograft rejection. Rather, it modulated the early allograft transcriptome to decrease the expression of renal injury, coagulation and cellular stress response genes, and increase the expression of cellular proliferation and Ifn-γ induced genes compared to University of Wisconsin preservation solution alone. To our knowledge our findings are the first to show that H2S protects donor kidneys against cold ischemia-reperfusion injury in the context of allogeneic renal transplantation. This potentially represents a novel cost-effective therapeutic solution to mitigate ischemia-reperfusion injury and improve the clinical outcomes of renal transplantation.

TiProtec preserves endothelial function in a rat model

BackgroundCoronary artery bypass surgery provides excellent patency rates; however, the early and/or late graft failure reduces the long-term benefit of myocardial revascularization. We investigated the effectiveness of generally used saline, Custodiol solutions and a new solution (TiProtec) at preserving endothelium after cold ischemia and warm reperfusion injury. Materials and methodsAortic transplantations were performed in Lewis rats. Aortic arches were stored in saline, Custodiol, and TiProtec solutions for 2 h then were transplanted into the abdominal aorta. Two, 24 hours and 1 week after transplantation, the implanted grafts were harvested. Endothelium-dependent and -independent vasorelaxations were investigated in organ bath. DNA strand breaks were assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-method, messenger RNA expressions by quantitative real-time polymerase chain reaction, and the expression of CD-31 and alpha smooth muscle actin by immunochemistry. ResultsSeverely impaired endothelial function and integrity of implanted aortic grafts were shown after 2 h in the saline, Custodiol group (maximal vasorelaxation to acetylcholine: control: 91 ± 2%, saline: 26 ± 5%, Custodiol: 24 ± 5%, CD-31-positive area control: 96 ± 2%, saline: 35 ± 13% Custodiol: 54 ± 5%, P < 0.05, respectively); however, a preserved endothelial function was observed in the TiProtec group when compared with the saline and Custodiol group (maximal vasorelaxation: 46 ± 7%, CD-31-positive area: 54 ± 10%, P < 0.05). After 1 wk, endothelial function was partially recovered in all groups; however, it was significantly better in the TiProtec group (maximal vasorelaxation to acetylcholine: saline: 42 ± 3%, Custodiol: 48 ± 3%, TiProtec: 56 ± 3%, CD-31-positive area: saline: 56 ± 5%, Custodiol: 54 ± 4%; TiProtec: 83 ± 6%, P < 0.05, respectively). In addition, messenger RNA levels of Bax, B-cell lymphoma-2, endothelial NOS, vascular endothelial growth factor 2, and caspase-3 were significantly altered in both groups. ConclusionsTiProtec appears to be superior for the preservation of endothelial- and smooth muscle cells of bypass graft after cold storage and warm reperfusion in our murine model.

Protective Effect of Intravenous High Molecular Weight Polyethylene Glycol on Fatty Liver Preservation.

Ischemia reperfusion injury (IRI) leads to significant tissue damage in liver surgery. Polyethylene glycols (PEGs) are water soluble nontoxic polymers that have proved their effectiveness against IRI. The objective of our study was to investigate the potential protective effects of intravenous administration of a high molecular weight PEG of 35 kDa (PEG 35) in steatotic livers subjected to cold ischemia reperfusion. In this study, we used isolated perfused rat liver model to assess the effects of PEG 35 intravenous administration after prolonged cold ischemia (24 h, 4°C) and after reperfusion (2 h, 37°C). Liver injury was measured by transaminases levels and mitochondrial damage was determined by confocal microscopy assessing mitochondrial polarization (after cold storage) and by measuring glutamate dehydrogenase activity (after reperfusion). Also, cell signaling pathways involved in the physiopathology of IRI were assessed by western blot technique. Our results show that intravenous administration of PEG 35 at 10 mg/kg ameliorated liver injury and protected the mitochondria. Moreover, PEG 35 administration induced a significant phosphorylation of prosurvival protein kinase B (Akt) and activation of cytoprotective factors e-NOS and AMPK. In conclusion, intravenous PEG 35 efficiently protects steatotic livers exposed to cold IRI.

Isoflurane does not protect from brain death-associated aggravation of cold hepatic ischemia-reperfusion injury.

Previous studies have shown that brain death aggravates cold ischemia-reperfusion injury in liver transplantation. Isoflurane, a volatile anesthetic, has been indicated to reduce warm hepatic ischemia-reperfusion injury. Herein, we studied in Sprague-Dawley rats whether isoflurane is capable of ameliorating brain death-associated aggravation of cold hepatic ischemia-reperfusion injury. Brain-dead animals were treated for 30 min with isoflurane (MAC 1.5%; n=8). Animals without isoflurane treatment served as controls (n=8). Another 13 animals without induction of brain death served as sham controls. After a 4-h period portal venous blood perfusion, hepatic microcirculation and bile flow were determined. Livers were recovered and stored for 24 h in 4°C cold HTK solution, followed by reperfusion with 37°C Krebs-Henseleit-buffer for 60 min. Liver enzymes in the effluent and bile flow were analyzed. Hepatocellular morphology was determined by histology and immunohistochemistry. Brain death reduced portal venous blood perfusion and bile flow, induced heme oxygenase-1 (HO-1), and resulted in hepatocellular damage. Isoflurane treatment did not prevent the reduction of portal venous blood perfusion or bile flow or the induction of HO-1. Accordingly, isoflurane was not capable of reducing the hepatocellular injury. Isoflurane does not protect from brain death-associated aggravation of cold hepatic ischemia-reperfusion injury.

Infusion of mesenchymal stem cells protects lung transplants from cold ischemia-reperfusion injury in mice.

Cold ischemia-reperfusion injury (IRI) is a major cause of graft failure in lung transplantation. Despite therapeutic benefits of mesenchymal stem cells (MSCs) in attenuating acute lung injury, their protection of lung transplants from cold IRI remains elusive. The present study was to test the efficacy of MSCs in the prevention of cold IRI using a novel murine model of orthotopic lung transplantation. Donor lungs from C57BL/6 mice were exposed to 6 h of cold ischemia before transplanted to syngeneic recipients. MSCs were isolated from the bone marrows of C57BL/6 mice for recipient treatment. Gas exchange was determined by the measurement of blood oxygenation, and lung injury and inflammation were assessed by histological analyses. Intravenously delivered MSC migration/trafficking to the lung grafts occurred within 4-hours post-transplantation. As compared to untreated controls, the graft arterial blood oxygenation (PaO2/FiO2) capacity was significantly improved in MSC-treated recipients as early as 4 h post-reperfusion and such improvement continued over time. By 72 h, oxygenation reached normal level that was not seen in controls. MSCs treatment conferred significant protection of the grafts from cold IRI and cell apoptosis, which is correlated with less cellular infiltration, a decrease in proinflammatory cytokines (TNF-α, IL-6) and toll-like receptor 4, and an increase in anti-inflammatory TSG-6 generation. MSCs provide significant protection against cold IRI in lung transplants, and thus may be a promising strategy to improve outcomes after lung transplantation.

Over-expression of microRNA-499 within donor heart reduces cardiac ischemia-reperfusion injury

Objective To explore the role of over-expression of microRNA-499 (miR-499) within donor heart against ischemia-reperfusion injury.Methods Polymerase chain reaction (PCR) was used to identify the miR-499 transgenic mice (Tg).Donor hearts were obtained from wild-type C57BL/6 (WT) and Tg mice and stored in 4 ℃ saline for 16 h.The release of creatine kinase (CK) and lactate dehydrase (LDH) were detected.Both stored donor hearts were transplanted heterotopieally into syngeneie WT recipient mice.The recovery of graft function indicated by its contraction or beating,was assessed by direct palpation and inspection.The transplanted hearts were harvested at 24 h,then histopathology was observed by haematoxylin-eosin staining.Results Tg mice showed positive stripe,which highly expressed miR-499 gene.Donor hearts from Tg mice significantly reduced the release of CK and LDH as compared to WT donors (3 667.0±194.7) vs.(5 043.0±523.8) U/L; (684.2 ±19.1) vs.(1 068.0±150.0) U/L,respectively) and exhibited lower degree of perivascular inflammation,leukocyte infiltration and myocyte necrosis 24 h after transplantation.The scores of graft function for WT heart grafts were significantly lower than those for Tg heart grafts at the same time-points (0.9 vs.2.3 at 15 min and 2.1 vs.3.4 at 24 h) (P ＜ 0.05).Conclusion Over-expression of miR-499 within donor heart can attenuate inflammatory injury and myocardial cell apoptosis caused by cold ischemia and hot reperfusion,benefit for heart function recovery and have a protective role in heart transplantation. Key words: MicroRNA 499; Ischemia-reperfusion injury; Heart transplantation

CD39 Is Critical for Inducing Tolerance in Murine Allogeneic Liver Transplantation.

Background: ATP is abundant in the cell cytosol and important for cell metabolism. Once cells die or are injured, ATP is released into the extracellular space and activates immune cells as a DAMP (damage-associated molecule pattern). CD39 regulates immune activation by hydrolysis of extracellular (e)ATP. We have found that conventional mouse liver myeloid(m) DC express comparatively high levels of surface CD39 and that this contributes to protection of the liver from cold ischemia reperfusion injury after syngeneic liver transplantation (LTx). We hypothesized that CD39 might play an important role in “spontaneous” transplant tolerance after allogeneic mouse LTx. METHODS: 10-12 week-old male B6 WT or CD39 KO (B6 background) livers were transplanted orthotopically into normal 10-12 week-old male C3H mice. Graft rejection was assessed by host survival and histology and also by serum ALT on day 4 after LTx. Incidences of regulatory T cells (Treg) and IFN-γ+ CD8+ T cells in the graft were quantified by flow cytometry at day 4 post LTx. Anti-donor T cell proliferative responses were evaluated by CFSE-MLR. RESULTS: Whereas all the WT B6 liver transplanted C3H mice survived more than 100 days, animals that received CD39KO livers all died within 40 days (MST: WT: >100d vs CD39 KO 8d; p<0.01). Serum ALT at day 4 post LTx was significantly higher in mice given CD39KO livers than in those with WT livers (WT: 480±226 IU/l vs CD39KO 860±484IU/l; p<0.05). The frequency of Treg in liver graft non-parenchymal cells after LTx was significantly lower in CD39KO livers than in WT livers (WT: 3.4±0.5% vs CD39KO 2.6±0.2%; p<0.05). Moreover IFN-γ production by CD8+ liver T cells was significantly higher in CD39KO allografts than in WT allografts (WT: 38.8±1.7% vs CD39KO 46.6±2.0%; p<0.05). Splenic T cells from CD39KO liver transplant recipients exhibited greater proliferation against donor splenocytes than those from WT liver allografted mice (WT: 27.2±5.8% vs CD39KO 36.3±5.7%; p<0.05). CONCLUSION: CD39 deficiency in liver grafts induces stronger anti-donor T cell responses and breaks “spontaneous” tolerance after mouse liver Tx. CD39 may be an important molecule for promoting liver allograft tolerance.

New Insights in Fatty Liver Preservation: A Role for Carbonic Anhydrase II.

Purpose: During liver graft preservation, the switch to anaerobic metabolism provokes accumulation of lactic acid and leads to lower pH. Studies on the relevance of acidic pH are controversial. It can either activate proteases and induce cell death or protect cell against apoptosis. Carbonic anhydrases (CA) are ubiquitous metalloenzymes involved in many physiological and pathological processes, including pH and CO[/sub]2[/sub] homeostasis. However, the role of CA in liver graft preservation has not been investigated. The aim of this work was to evaluate the effects of CAII by its addition to the IGL-1 preservation solution or when it was inhibited by acetazolamide (AZ). A close CAII relationship with well known protective factors (AKT, MAP kinases) and apoptosis prevention was established Experimental: Steatotic livers from obese male Zucker rats (9 weeks aged) were classified as follows: Group1: Livers preserved for 24h (4°C) in IGL-1 solution and then subjected to “ex vivo” perfusion (2h at 37°C); Group2: same as Group1 but IGL-1 supplemented with CAII (10 μg/ml) and Group 3: Same as Group 1 but livers were pretreated with AZ (i.v. 30mg/Kg) before liver preservation. Liver injury (transaminases) and function (bile production) were measured. Akt, GSK3β, Caspase 3, Caspase 9 and MAPKs (p38, ERK and JNK) were also determined by western blot techniques. Results and Discussion: Fatty livers preserved in IGL-1+CAII showed lower injury and better function when compared to IGL-1 alone. IGL-1+CAII solution induced a significant phosphorylation of Akt and significant decreases in MAPKs (ERK, p38 and JNK) after 2h-reperfusion. This was consistent with a major decrease of liver apoptosis parameters (caspase 3, caspase 9, and GSK3β). Surprisingly, the AZ pretreatment protected efficiently steatotic liver grafts, as revealed by lower transaminases, higher bile production and decreases in MAPK levels. AZ showed differential protective mechanism of liver as it fails to activate Akt. Also, it reduced vascular resistance which was not observed with CAII addition. Conclusion: Data reported here, demonstrated that CAII is a potential pharmacological target for preserving liver grafts against cold ischemia reperfusion injury.

A Novel Preservation Solution Prolonged Cold Storage in Mouse Cardiac Grafts.

Background: We developed a novel non-potassium preservation solution contains L-cysteine, glycine, ascorbic acid/ascorbic acid-2-phosphate magnesium to prolonged mouse cardiac grafts cold storage. Methods: B6 mice underwent heterotopic heart transplantation and the animals were derived into 3 groups: recipients with non-preserved grafts (control group), recipients with grafts preserved in HTK solution for 24 and 48 hours (HTK-group), and recipients with grafts preserved in the new solution, called Solution II for 24 and 48 hours (S-II group). Recipient serum and grafts were harvest for assay. Results: In both 24hr- and 48hr-preservation series (24h- and 48h-series), time for restoration of heartbeat after reperfusion in S-II group was significantly shorter than in HTK group (HTK-24h: 306.2±39.6sec, S-II-24h: 110.5±20.3sec, p<0.01; HTK-48h: 1537.5±454.7sec, S-II-48h: 640±286sec, p<0.01). In 48h-series, Release of serum LDH, CK in S-II-group was significantly lower to compare with HTK group. The graft survival of HTK-48h group is 0 day (n=9), but S-II group is 9d, 17d, >100d×2. Graft ATP level of S-II-48h was significantly higher than HTK-48h. In both 24 and 48h-series, S-II significantly inhibited graft apoptosis. Moreover, immunohistochemistry staining revealed that the CD68-positive macrophage infiltration in S-II group was significantly lower compared with HTK group. In 24h-series, graft 8-OHdG positive cells (%) were significantly lower in S-II compared with HTK. Consistent with histologic findings, serum 8-OHdG levels were significantly inhibited in S-II group. Furthermore, the mRNA expression of the IL-6, HO-1, iNOS, CD11b in S-II-48h group, were significantly lower, wherever Nrf2, NF-κB, Arg-1, HIF-1α, TGF-β were significantly higher than HTK-48h group (p<0.01). Conclusion: Hence our data indicated that the novel preservation solution impairs graft macrophage infiltration, inhibits inflammatory cytokines and myocardial apoptosis which results in attenuating mouse cardiac grafts cold ischemia-reperfusion injury and prolonging cold storage.

A novel in situ model of liver cold ischemia–reperfusion in rats

BackgroundOrthotopic liver transplantation (OLT) is being used for studying cold ischemia reperfusion (I/R)-induced injury in experimental animals, but the technique is complicated and it does not accurately reflect the pathophysiology. Here, we report a novel model, termed “in situ liver cold ischemia (ISLCI)”, in Wistar rats. MethodsISLCI was achieved in rats by establishing a portal-jugular shunt and a cannula shunt in inferior vena cava, and the liver was continuously perfused with lactate Ringer's solution at a speed of 150 mL/h through the portal vein for 60 min. Portal venous pressure, serum levels of total bilirubin, alkaline phosphatase, alanine aminotransferase and γ-glutamyl transpeptidase (GGT), and hepatic histopathology were examined, and compared with rats undergoing OLT, in which the donor liver was subjected to a 60 min cold ischemia. ResultsLivers from ISLCI and OLT rats showed histopathologic changes characteristic of I/R-induced injury when examined on days 1 and 7, with complete recovery 14 d after reperfusion. Compared with OLT rats, ISLCI rats had significantly lower levels of portal venous pressure 1 and 10 min after porta hepatis clamping. They suffered a milder degree of I/R-induced hepatic injury, reflected by significantly lower levels of GGT, alanine aminotransferase, and alkaline phosphatase on day 1, and a significant lower level of GGT and a lower histopathologic score on day 7 after reperfusion. ConclusionsOur preliminary results indicate that the ISLCI model is reliable and technically easier, and is superior to OLT for studying cold I/R injury.

Effects of fructose-1, 6-diphosphate pretreatment on lung injury induced by hepatic cold ischemia-reperfusion in rats

Objective To evaluate the effects of fructose-1 ,6-diphosphate （FDP） pretreatment on lung injury induced by hepatic cold liver ischemia-reperfusion in rats .Methods Eighteen healthy male Sprague-Dawley rats were randomly divided into 3 groups （n= 6 each） using a random number table ：sham operation group （S group） ,hepatic cold liver ischemia-reperfusion model group （M group） ,and FDP pretreatment group （FP group） . The animals were anesthetized with intraperitoneal chloral hydrate and kept spontaneous breathing .Laparotomy was performed ,and the related blood vessels were only isolated in group S .Hepatic cold ischemia-reperfusion was induced in M and FP groups .In FP group ,FDP 250 mg/kg was injected via the caudal vein at 15 min before skin incision .At 6 h of reperfusion ,the bronchoalveolar lavage fluid （BALF） was collected to detect the levels of tumor necrosis factor-α（TNF-α） ,interleukin-10 （IL-10） and nitric oxide （NO） by ELISA .Lungs were removed for microscopic examination of the pathological changes by light microscopy .Real-time PCR was used to detect the expression of iNOS mRNA .Results Compared with S group , the levels of TNF-α and NO in BALF were significantly increased , the expression of iNOS mRNA was up-regulated , and the level of IL-10 in BALF was decreased in M and FP groups （ P〈0.05 ） .Compared with M group ,the levels of TNF-αand NO in BALF were significantly decreased ,the expression of iNOS mRNA was down-regulated ,and the level of IL-10 in BALF was＆nbsp;increased in FP group （ P〈0.05 ） .The pathological changes of lungs were significantly attenuated in FP group as compared with M group .Conclusion FDP pretreatment can obviously attenuate lung injury induced by hepatic cold ischemia-reperfusion in rats ,and inhibition of iNOS expression ,reduction of NO synthesis ,and decrease in inflammatory responses are involved in the mechanism . Key words: Fructose; Liver; Lung; Reperfusion injury

Preconditioning renoprotective effect of isoflurane in a rat model of virtual renal transplant

BackgroundThe development of warm–cold ischemia–reperfusion (IR) injury of the kidney grafts is inevitable during renal transplantation. However, there is currently no definite renoprotective strategy available in the protection of the graft tissue. In the present study, we compared the renal protection of preconditioning isoflurane with N-acetylcysteine (NAC) in a novel rat model of warm–cold renal IR injury. Materials and methodsAdult Sprague–Dawley rats were randomly assigned to receive inhaled isoflurane (1.5% for 2 h), NAC (1 g/kg, intra-arterial injection) or placebo before the induction of brief warm ischemia (10 min) followed by cold ischemia (45 min) periods. Plasma levels of creatinine and tissue inflammatory reaction in the kidney were analyzed 72 h after reperfusion. ResultsElevated plasma level of creatinine and urea indicated the development of acute renal injury secondary to IR injury. The creatinine levels were reduced in animals pretreated with inhaled isoflurane and NAC, and the level was more significantly decreased in the isoflurane-treated group. Preconditioning with volatile isoflurane also significantly suppressed the tissue myeloperoxidase activity and expression of the inducible nitric oxide synthase. Immunostaining confirmed that myeloperoxidase expression was most significantly attenuated in the glomerulus and peritubular capillaries of rats pre-exposed to isoflurane. ConclusionsWe present the first study demonstrating that the administration of volatile isoflurane before induction of experimental warm–cold renal IR injury provides preconditioning renoprotective effect, which is superior to the treatment with NAC. The beneficial renoprotective effect of isoflurane is most likely mediated by attenuation of proinflammatory reaction in the injured kidney.

Effects of rapamycin pretreatment on lung injury caused by hepatic cold ischemia-reperfusion injury in rats

Objective To evaluate the effects of rapamycin pretreatment on lung injury caused by hepatic cold ischemia-reperfusion (I/R) injury in rats.Methods Twenty-four healthy male Sprague-Dawley rats,aged 8-10 weeks,weighing 220-250 g,were randomly divided into 3 groups (n =8 each):sham operation group (S group),lung injury caused by hepatic cold I/R injury group (I/R group),and rapamycin pretreatment group (RP group).Laparotomy was performed,and hepatic lobes and related blood vessels were only isolated in group S.Hepatic cold I/R was induced in I/R and RP groups.Rapamycin 10 mg· kg-1 ·d-1 was injected through a gastric tube into stomach for 3 consecutive days before surgery in RP group.The rats were sacrificed at 12 h of reperfusion,and lungs were removed to detect the levels of malondialdehyde (MDA),superoxide dismutase (SOD)and nitric oxide (NO),iNOS mRNA expression (by RT-PCR),tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) expression (by immuno-histochemistry) in lung tissues and for examination of the pathological changes of lungs with light microscope.Results Compared with S group,the levels of MDA,NO,iNOS mRNA,TNF-α and IL-6 were significantly increased and the SOD activity was decreased in I/R and RP groups (P ＜0.05).The SOD activity was significantly higher,and the levels of MDA,NO,iNOS mRNA,TNF-α and IL-6 wee lower in group RP than in I/R group (P ＜ 0.05).The pathological changes of lungs were significantly attenuated in RP group as compared with I/R group.Conclusion Rapamycin pretreatment can reduce lung injury caused by hepatic cold I/R injury in rats,and inhibition of iNOS expression,reduction of NO product and decrease in inflammatory responses are involved in the mechanism. Key words: Sirolimus; Liver; Respiratory distress syndrome, adult; Reperfusion injury

Bortezomib enhances fatty liver preservation in Institut George Lopez-1 solution through adenosine monophosphate activated protein kinase and Akt/mTOR pathways

The aim of this study is to investigate the protective mechanisms induced by bortezomib added to Institut George Lopez (IGL)-1 preservation solution to protect steatotic livers against cold ischaemia reperfusion injury and to examine whether these mechanisms occur through the activation of adenosine monophosphate activated protein kinase (AMPK), Akt/mTOR pathways. Steatotic livers from obese rats were preserved for 24 h (at 4 °C) in IGL-1 solution with or without bortezomib (100 nM) or pretreated with AMPK inhibitor adenine 9-α-D-arabinofuranoside and preserved in IGL-1 + bortezomib. Livers were then perfused for 2 h at 37 °C. Liver injury (alanine aminotransferase/aspartate aminotransferase) and function (bile production and vascular resistance) were measured. Also, Akt/mTOR, phosphorylated AMPK (pAMPK) and apoptosis were determined by Western blot analyses. Bortezomib addition to IGL-1 solution significantly reduced steatotic liver injury, improved graft function and decreased liver apoptosis. These benefits were diminished by the pretreatment of obese rats with AMPK inhibitor Ara. Western blot analyses showed a significant increase in pAMPK after ischaemia and reperfusion. We also observed a significant phosphorylation of Akt in IGL-1 +bortezomib group that, in turn, induced the phosphorylation of mTOR and glycogen synthase kinase 3β. Bortezomib, at low and non toxic concentration, is a promising additive to IGL-1 solution for steatotic liver preservation. Its protective effect is due to the activation of AMPK and Akt/mTOR pathways.

Relationship between the histological appearance of the portal vein and development of ischemic-type biliary lesions after liver transplantation

Ischemic-type biliary lesions (ITBLs) are a major cause of morbidity after liver transplantation (LT). Their assumed underlying pathophysiological mechanism is ischemia/reperfusion injury of the biliary tree, in which the portal circulation has been proposed recently to have a role. The aim of this study was to investigate whether early histological changes, particularly in the portal vein, predispose patients to ITBLs. A case-control study of 22 LT recipients was performed through a retrospective assessment of more than 30 histological parameters in 44 intraoperative liver biopsy samples taken after cold ischemia (time 0) and portal reperfusion (time 1). Eleven grafts developed ITBLs requiring retransplantation (the ITBL group), and 11 matched controls had normally functioning grafts 11 years after LT on average (the non-ITBL group). Additionally, 11 liver biopsy samples from hemihepatectomies performed for metastases of colorectal cancer (CRC) were assessed similarly. Analyses showed no significant histological differences at time 0 between the ITBL and non-ITBL groups. However, the time 1 biopsy samples from the ITBL group showed smaller portal vein branches (PVBs) significantly more often than the samples from the non-ITBL group, which also showed persisting paraportal collateral vessels. Larger PVBs and paraportal collateral vessels were also found in the CRC group. A morphometric analysis confirmed these findings and showed that PVB measurements were significantly lower for the ITBL group at time 1 versus the ITBL group at time 0 and the non-ITBL and CRC groups (they were largest in the CRC group). Thus, the PVB dimensions decreased in the ITBL group in comparison with the time 0 biopsy samples, and they were significantly smaller at time 1 in comparison with the dimensions for the non-ITBL and CRC groups. In conclusion, a smaller PVB lumen size in postreperfusion biopsy samples from liver grafts, suggesting a relatively decreased portal blood flow, is associated with a higher incidence of ITBLs. These findings support recent clinical studies suggesting a possible pathophysiological role of portal blood flow in the oxygenation of the biliary tree after LT.

Roles of dendritic cells in murine hepatic warm and liver transplantation-induced cold ischemia/reperfusion injury

Dendritic cells (DCs) induce and regulate both innate and adaptive immune responses; however, their in vivo functional importance in hepatic ischemia/reperfusion (IR) injury is perplexing. We hypothesized that liver-resident DC and locally recruited blood-borne DC might have distinctive roles in hepatic IR injury. We tested this hypothesis by using DC-deficient, fms-like tyrosine kinase 3 ligand (Flt3L) knockout (KO) mice in hepatic warm (70% partial clamping for 60 minutes) and cold IR injury (liver transplant [LTx] with 24-hour cold storage). Flt3L KO liver and lymphoid organs contained virtually no CD11c+ F4/80- DC. Hepatic warm IR injury was significantly lower in Flt3L KO than in wildtype (WT) mice with lower alanine aminotransferase (ALT) levels, reduced hepatic necrosis, and lower neutrophil infiltration. Hepatic messenger RNA (mRNA) and protein levels for inflammatory cytokines (tumor necrosis factor alpha [TNFα], interleukin [IL]-6) and chemokines (CCL2, CXCL2) were also significantly lower in Flt3L KO than in WT mice, indicating that lack of both liver-resident and blood-borne DC ameliorated hepatic warm IR injury. Adoptive transfer of splenic or hepatic WT DC into Flt3L KO or WT mice increased hepatic warm IR injury, suggesting injurious roles of DC infusion. When Flt3L KO liver was transplanted into WT mice, ALT levels were significantly higher than in WT to WT LTx, with enhanced hepatic necrosis and neutrophil infiltration, indicating a protective role of liver-resident DC. Using both warm and cold hepatic IR models, this study suggests differential roles of liver-resident versus blood-borne DC, and points to the importance of the local microenvironment in determining DC function during hepatic IR injury.

564 TLR4 INDUCTION BY TUDCA IN EXPERIMENTAL MODELS OF SYNGENEIC AND ALLOGENEIC STEATOTIC LIVER TRANSPLANTATION

3. Antioxidant capability of rMnSOD as a supplement of a commercially available preservation solution was evaluated in hepatic biopsies cold stored for transplantation. Results: 1. Cold storage induced a marked increase in O−2 levels and a decrease in NO bioavailability in SEC, those detrimental effects were abolished in cells preserved with rMnSOD. 2. In rats, administration of rMnSOD ameliorated hepatic injury and endothelial dysfunction derived from cold storage and warm reperfusion injuries. The beneficial effects of rMnSOD were associated with a reduction in hepatic O−2, ONOO − and inflammation together with an improved antioxidant activity and nitric oxide bioavailability. 3. rMnSOD added to a conventional preservation solution maintains its marked antioxidant activity avoiding oxidative stress formation in hepatic tissue preserved for transplantation. Conclusions: This study demonstrates that rMnSOD markedly improves liver viability and endothelial function after cold ischemia and warm reperfusion. rMnSOD represents a new therapeutic strategy to protect liver grafts undergoing transplantation.

Proteasome inhibitors protect the steatotic and non-steatotic liver graft against cold ischemia reperfusion injury

BackgroundThe dramatic shortage of organs leads to consider the steatotic livers for transplantation although their poor tolerance against ischemia reperfusion injury (IRI). Ubiquitin proteasome system (UPS) inhibition during hypothermia prolongs myocardial graft preservation. The role of UPS in the liver IRI is not fully understood. Bortezomib (BRZ) treatment at non-toxic doses of rats fed alcohol chronically has shown protective effects by increasing liver antioxidant enzymes. We evaluated and compared both proteasome inhibitors BRZ and MG132 in addition to University of Wisconsin preservation solution (UW) at low and non-toxic dose for fatty liver graft protection against cold IRI. ExperimentalSteatotic and non-steatotic livers have been stored in UW enriched with BRZ (100nM) or MG132 (25μM), for 24h at 4°C and then subjected to 2-h normothermic reperfusion (37°C). Liver injury (AST/ALT), hepatic function (bile output; vascular resistance), mitochondrial damage (GLDH), oxidative stress (MDA), nitric oxide (NO) (e-NOS activity; nitrates/nitrites), proteasome chymotrypsin-like activity (ChT), and UPS (19S and 20S5 beta) protein levels have been measured. ResultsChT was inhibited when BRZ and MG132 were added to UW. Both inhibitors prevented liver injury (AST/ALT), when compared to UW alone. BRZ increased bile production more efficiently than MG132. Only BRZ decreased vascular resistance in fatty livers, which correlated with an increase in NO generation (through e-NOS activation) and AMPK phosphorylation. GLDH and MDA were also prevented by BRZ. In addition, BRZ inhibited adiponectin, IL-1, and TNF alpha, only in steatotic livers. ConclusionMG132 and BRZ, administrated at low and non toxic doses, are very efficient to protect fatty liver grafts against cold IRI. The benefits of BRZ are more effective than those of MG132. This evidenced for the first time the potential use of UPS inhibitors for the preservation of marginal liver grafts and for future applications in the prevention of IRI.