End-Ischemic Reconditioning of Liver Allografts: Controlling the Rewarming

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1 ORIGINAL ARTICLE HOYER ET AL. End-Ischemic Reconditioning of Liver Allografts: Controlling the Rewarming Dieter Paul Hoyer, 1 Andreas Paul, 1 Sebastian Luer, 1 Henning Reis, 2 Patrik Efferz, 1 and Thomas Minor 1 1 General, Visceral and Transplantation Surgery and 2 Institute of Pathology, University Hospital Essen, University Duisburg-Essen, Essen, Germany Different nonhypothermic preservation modalities have shown beneficial effects in liver transplantation models. This study compares controlled oxygenated rewarming (COR) to normothermic machine perfusion (NMP) to resuscitate liver grafts following cold storage (CS). Porcine livers were preserved for 18 hours by CS. Before reperfusion, the grafts were put on a machine perfusion device (Liver Assist) for 3 hours and were randomly assigned to COR (n 5 6) or NMP (n 5 5) and compared to standard CS. COR was carried out with the new Custodiol-N solution, slowly increasing temperature from 8 8C to208c during the first 90 minutes. NMP was carried out with diluted autologous blood at 37 8C for 3 hours. In both cases, the perfusate was oxygenated to partial pressure of oxygen > 500 mm Hg. Then liver viability was tested for 180 minutes during in vitro isolated sanguineous reperfusion. Activity of the mitochondrial caspase 9 was lower after COR. Measurement of tissue adenosine triphosphate and total adenine nucleotides at the end of the reconditioning period showed better energetic recovery after COR. COR also resulted in significantly lower enzyme leakage and higher bile production (P < 0.05) during reperfusion. This first comparison of COR and NMP as end-ischemic reconditioning modalities demonstrates superior results in terms of mitochondrial integrity resulting in better energetic recovery, less hepatocellular injury, and ultimately superior function in favor of COR. Liver Transplantation AASLD. Received February 19, 2016; accepted June 28, The period of cold storage (CS) remains detrimental for all kinds of solid organ transplantation as the organs are exposed to hypoxic, hypoenergetic, and hyponutritional environments. Utilization of static hypothermia proved to sufficiently reduce the metabolism of isolated Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; ATP, adenosine triphosphate; AU, arbitrary units; CN, Custodiol-N; COR, controlled oxygenated rewarming; CPD, citrate phosphate dextrose; CS, cold storage; H & E, hematoxylineosin; IRI, ischemia/reperfusion injury; LPO, lipid peroxidation; MP, machine perfusion; NMP, normothermic machine perfusion; PO 2, partial pressure of oxygen; TAN, total adenine nucleotides. Address reprint requests to Dieter P. Hoyer, General, Visceral and Transplantation Surgery, University Hospital Essen, University Duisburg-Essen, Hufelandstrasse 55, Essen, Germany. Telephone: ; FAX: ; dieter.hoyer@uk-essen.de Copyright VC 2016 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI /lt Potential conflict of interest: Nothing to report. organs and has been used as a primary preservation modality for decades. Besides achieving the primary goal of preservation, it has the advantage of being an easy procedure and is associated with simple logistics. However, with increasing knowledge about CS of liver allografts, it has been shown that at the end of cold preservation, the cells of the allografts are energy depleted; hence, they are in suboptimal conditions to cope with the challenges of warm blood reperfusion in the recipient. This reperfusion is associated with the generation of oxygen radicals (1,2) from the suddenly accelerated metabolism. Accordingly, advancements of preservation methods should aim at priming the livers for this demanding period and should therefore recondition the allografts before reperfusion. Reconditioning of organs at the end of the cold preservation period has the advantage of using existing logistical resources and affecting preservation procedures only in the accepting transplantation center. Recent developments in this field include hypothermic machine perfusion (HMP) (3-8) and normothermic machine perfusion (NMP). (9-11) Both methods demonstrated favorable results in comparison to CS in preclinical and first emerging clinical studies. HMP acts ORIGINAL ARTICLE 1223

2 HOYER ET AL. LIVER TRANSPLANTATION, September 2016 at low metabolic activity and was suggested to protect the allografts from mitochondrial and nuclear injury and to reduce endothelial damage, (3) as well as the alloimmune response in the recipient. (12) NMP in turn targets the metabolically more active organ and is suggested to improve metabolism and function, (13,14) as well as offering optimized viability assessment of the isolated graft. A first comparative study has been published recently, suggesting favorable effects of HMP over NMP. (15) Our group has added a new principle to liver preservation represented by a period of slow and controlled oxygenated rewarming (COR) up to subnormothermic temperatures after initial HMP. With this slow reawakening of the metabolism of the allograft, superior restitution of hepatocellular function was achieved compared to solely hypothermic or subnormothermic MP. (16) COR was recently successfully applied in clinical transplantation (17) and is suggested to prepare a transplant for the posttransplant challenges. (18) The aim of this study was to establish the first direct comparison of COR and NMP as reconditioning tools after CS. Materials and Methods All experiments were performed in accordance with the federal law regarding the protection of animals and received humane care according to the criteria outlined in Guide for the Care and Use of Laboratory Animals (NIH). Female German Landrace pigs weighing between 25 kg and 30 kg were premedicated with ketamine (90 mg/kg), xylazine (10 mg/kg), and atropine (10 mg/kg). This was administered intramuscularly 10 minutes before induction of anesthesia. General anesthesia was induced by midazolam (0.5 mg/kg) and fentanyl (12.5 mg/kg), administered intravenously and maintained after intubation by mechanical ventilation with isoflurane in air/oxygen. Under general anesthesia, the liver was dissected free and then perfused by gravity (60 cm H 2 O) via the portal vein with 2 L of new histidine tryptophan ketoglutarate preservation solution (Custodiol-N [CN], Koehler Chemie, Germany) at 4 8C (Table 1). Livers were then excised; the hepatic artery was flushed on the back table with an additional 100 ml of CN; and the graft was stored overnight in CN for 18 hours. After static CS at 4 8C, the grafts were randomly assigned to 1 of the following groups: 1. NMP (n 5 5): Three hours prior to reperfusion, grafts were put on a machine perfusion (MP) device (Liver Assist) and were perfused at 37 8C with autologous TABLE 1. Composition of CN Solution blood, diluted with gelofundin to a hematocrit of approximately 20 without the addition of vasodilators. As recommended in earlier reports, (19,20) portal perfusion pressure was maintained constant at 8 mm Hg, and pulsatile arterial perfusion pressure was at 60/ 40 mm Hg by means of pressure-controlled electronic regulation of 2 individual rotary pumps. Because of the low hematocrit of the perfusate, partial pressure of oxygen (PO 2 ) was targeted at supranormal values to approximately 500 mm Hg. (19) 2. COR (n 5 6): COR was achieved by putting the graft on the MP circuit for a gentle warming up from the cold by a gradual increase of the perfusate temperature, basically as described previously. (21) COR was carried out with portal and arterial perfusion for 3 hours using CN solution as the perfusate, slowly increasing the temperature from 8 8C to 208C during the first 60 minutes followed by continuous perfusion at 20 8C for the remaining 2 hours. 3. CS (n 5 5): For comparison, a control group was also investigated composed of livers that were cold stored in the standard fashion for 18 hours at 4 8C in CN solution. ISOLATED LIVER PERFUSION MODEL Amount Sodium 16 Potassium 10 Magnesium 8 Calcium 0.02 Chloride 29 Histidine 124 N-Acetylhistidine 57 Mannitol Sucrose 33 a-ketoglutarate 2 Aspartate 5 Glycine 10 Alanine 5 Tryptophan 2 Arginine 3 Deferoxamine LK ph 7.0 Osmolality, mosmol/kg 304 NOTE: Values are given in mm (mmol/l) unless stated otherwise. All livers were rinsed with 500 ml of cold saline solution and kept at room temperature for 30 minutes to 1224 ORIGINAL ARTICLE

3 LIVER TRANSPLANTATION, Vol. 22, No. 9, 2016 HOYER ET AL. simulate the time of engrafting. (22) Then liver viability was tested for 180 minutes during in vitro isolated sanguineous reperfusion. (23) The perfusion medium consisted of autologous blood that was retrieved from the donor during the organ recovery operation and preserved overnight in citrate phosphate dextrose (CPD) blood bags. Prior to the experiment, 1 L of blood was diluted with 500 ml of balanced salt solution (Jonosteril, Fresenius Kabi, Bad Homburg, Germany) to which 4% dextran 40 was added. The final perfusate was supplemented with sodium bicarbonate and Ca 2 Cl as required to bring the ph and calcium concentration within the normal physiological range. Total hemoglobin in the final perfusate ranged between 5 and 6 g/dl. Livers were placed in a moist temperature chamber and perfused at 38 8C. Circulating blood was oxygenated in a temperature-controlled hollow fiber oxygenator (Hilite LT 2400, Medos, Stolberg, Germany). Gas flow to the oxygenator (air, oxygen, and CO 2 ) was differentially regulated so as to achieve physiological blood gas values (PO 2, mm Hg; partial pressure of carbon dioxide mm Hg). Temperature was regulated by a circulating thermostat, connected to a perfusion chamber and oxygenator. Hepatic artery perfusion pressure was set at 80 mm Hg and automatically maintained by a servo-controlled roller pump, connected to a pressure sensor placed in the inflow line immediately prior to the arterial cannula. Perfusion of the portal vein was performed in a flow-constant manner (0.8 ml/g/minute) driven by a centrifugal blood pump (Bio-Pump BP-50, Medtronic Inc., MN), whereas portal venous pressure was recorded using a water column connected to the inflow tract. Erythrocyte count and hemoglobin values remained unchanged during liver perfusion over the whole 240 minutes of investigation. During liver perfusion, heparin was infused at 1000 U/hour. A mixtureof80mlofglucose5%and20mlofamino acid solution (RPMI x, Sigma-Aldrich, Munich, Germany) was supplemented with 10 IU of insulin and 1 mg of taurocholic acid and infused to the reservoir at 15 ml/minute throughout the experiment to provide nutritional support and substrate for hepatic bile production. Hepatic oxygen consumption (ml/100 g/minute) was calculated from arterial, portal, and hepatic venous oxygen saturations, total hemoglobin, and oxygen partial pressures as detailed by Koetting et al., (23) taking into account the respective flow rates and liver mass. Serum enzyme activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were assessed photometrically using commercialized standard kits (Fa. Roche, Mannheim, Germany). Hepatic arterial and portal vascular perfusion resistance was calculated from independently measured flow and pressure values normalized to liver mass and expressed in mm Hg/L/ minute/100 g. BILE PRODUCTION The common bile duct of the livers was cannulated with polyethylene tubing. Bile was collected during the whole reperfusion period; hepatic bile production was calculated as ml/kg/hour. TISSUE EXTRACTION AND ASSAY OF ENERGETIC STATUS Tissue specimens for assessment of high-energy phosphates were taken with precooled steel tongs, immersed in liquid nitrogen, and stored at 80 8C for later analysis. High-energy phosphates were determined enzymatically in the neutralized supernatant after protein extraction with perchloric acid of freeze-dried tissue samples as described elsewhere. (6) The results were corrected for the respective dry weight to wet weight ratio of the tissue samples and expressed as mmol/g dry weight. Total adenine nucleotides (TAN) were calculated as tissue contents of adenosine triphosphate (ATP) 1 adenosine diphosphate 1 adenosine monophosphate. MITOCHONDRIAL INDUCTION OF APOPTOSIS Functional activity of caspase 9 was analyzed from homogenized tissue lysates in 96 well plates using a fluorimetric assay kit (Calbiochem, Darmstadt, Germany) based on the detection of the cleavage product 7-amino-4-trifluoromethyl coumarin, which emits yellow-green fluorescence peaking at 505 nm upon excitation at 400 nm. Measurements were done on a fluorescence microplate reader (Tecan, Grailsheim, Germany). Enzymatic activities of caspase 9 in the experimental groups are presented as the percentage increase with respect to baseline values obtained from nonischemic control tissue. LIPID PEROXIDATION Oxygen free radical induced tissue injury was approximated by the degree of lipid peroxidation (LPO) in the tissue at the end of reperfusion. Liver samples were ORIGINAL ARTICLE 1225

4 HOYER ET AL. LIVER TRANSPLANTATION, September 2016 taken after completion of the experiment, and LPO was evaluated by fluorimetry in the deproteinized tissue using the adduct formation with thiobarbituric acid as detailed elsewhere. (24) HISTOLOGY Immediately after completion of the experiments the liver tissue was collected, cut into small blocks, and fixed by immersion in 4% buffered formalin. These blocks were embedded in paraffin, and 2-4 mm tissue slides were prepared using a microtome (SM 2000R, Leica Instruments, Nußloch, Germany). Hematoxylineosin (H & E) staining was conducted adherent to inhouse standards and used to assess morphological integrity of the parenchyma. Sections were examined at 200-fold magnification (Nikon Eclipse E800, Nikon, Tokyo, Japan), and the extent of necrotic injury was semiquantitatively graded in a 4-stage system ranging from 0 (no necrosis) to 3 (severe necrosis with disintegration of hepatic cords) as described elsewhere, (25) by a blinded independent investigator (H.R.). STATISTICS All values were expressed as means 6 standard deviation. After proving the assumption of normality, differences between the groups were tested by analysis of variance followed by multiple comparisons of the means with the Student-Newman-Keuls test (Sigma- Plot 13.0; Systat Software Inc, San Jose, CA), unless otherwise indicated. Statistical significance was set at P < Results MACHINE PERFUSION Cumulative hepatocellular enzyme leakage during reconditioning demonstrated less enzyme release after COR (AST, U/L) compared to NMP (AST, U/L). Lactate concentrations were in the normal range at the end of reconditioning in both groups (COR versus NMP, mmol/l versus mmol/l, respectively). Analysis of graft weight before and after reconditioning demonstrated significantly less weight gain after COR (before/after weight ratio, ) compared to NMP (before/after weight ratio, ; P < 0.001). FIG. 1. Hepatocellular enzyme leakage during blood reperfusion (data as mean 6 SD). *: P vs CS; #: P vs NMP. BLOOD REPERFUSION Application of different reconditioning methods resulted in different patterns of hepatic damage and function during the reperfusion period. Measurement of AST in serum samples obtained at several time points during ex vivo blood reperfusion was used as a surrogate of general liver injury (Fig. 1). In all groups, a constant increase of AST release from hepatocytes was noted during the reperfusion period. COR-treated organs demonstrated the lowest values of all groups with statistical significance when compared to NMP (P ) and CS (P ). Leakage of ALT into the serum was lower after NMP compared to CS but without statistical significance. MITOCHONDRIAL INTEGRITY Caspase 9 activity was measured as a representative marker for mitochondrial impairment and induction of apoptosis. After CS, a more than 3-fold increase was observed in respect to baseline values. A 1.9-fold increase was observed in COR allografts after reperfusion with blood, which proved to be significantly lower than in CS (P ). In NMP grafts, a 2.7-fold increase was documented without significance to the other groups (Fig. 2). The energetic recovery of the allografts, and therefore, mitochondrial function was judged by measurement of tissue ATP and TAN at the end of the reconditioning 1226 ORIGINAL ARTICLE

5 LIVER TRANSPLANTATION, Vol. 22, No. 9, 2016 HOYER ET AL. period. Results of these investigations are depicted in Fig. 3A,B. These data demonstrate significantly higher ATP concentration after reconditioning with COR compared to CS (P ). Tissue TANs demonstrated similarly higher values after COR, proving statistical significance when compared to CS (P ) and NMP (P ). No differences were observed between NMP and CS for ATP or TANs. For further assessment of the metabolic activity of the organs, the oxygen uptake during the blood reperfusion period was calculated. In grafts preserved by COR, mean oxygen utilization amounted to ml/ 100 g/minute compared to ml/100 g/minute in NMP-preserved grafts (P ), and ml/100 mg/minute in CS-preserved grafts (P ). However, recent works demonstrated low sensitivity of this parameter. (11,16,26) PARENCHYMAL FUNCTION The complex parenchymal function of the allografts demanding energetically potent cells was assessed by production of bile. Results are depicted in Fig. 4. A significant difference in favor of COR was found between treatment groups (COR versus NMP, P ). NMP resulted in slightly better values than after simple CS, but without reaching statistical significance. This pattern of cell function is in line with the hepatocellular damage described above (enzyme leakage) and demonstrates the influence of the reconditioning modality on the allograft. FIG. 2. Caspase 9 activity after blood reperfusion. *: P vs CS; BL 5 baseline. LIPID PERIOXIDATION Degree of LPO at the end of reperfusion is depicted in Fig. 5. Reconditioning by COR led to significantly less LPO compared to NMP (P ). Furthermore, COR showed less LPO compared to CS without statistical significance. FIG. 3. Measurement of tissue ATP (*: P vs CS) and TAN (*: P vs CS; reconditioning period (data as mean 6 SD). #: P vs NMP) at the end of the ORIGINAL ARTICLE 1227

6 HOYER ET AL. LIVER TRANSPLANTATION, September 2016 FIG. 4. Bile production during reperfusion (data as mean 6 SD). *: P vs CS; #: P vs NMP. FIG. 5. Degree of lipid peroxidation at the end of reperfusion (data as mean 6 SD). #: P vs NMP. VASCULAR INTEGRITY In addition to hepatocellular damage and function, the vascular integrity was rated by hepatic arterial and portal flows during the ex vivo reperfusion period. COR-preserved grafts demonstrated mean flow rates of ml/minute during the observation time, whereas NMP-preserved grafts showed mean flow rates of ml/minute and grafts preserved by CS demonstrated mean flow rates of ml/ minute, without statistical significance between groups. For portal flows, no differences were observed between groups. HISTOLOGY Semiquantitative analysis of the H & E stained sections revealed signs of morphological alterations of highest degree after CS ( arbitrary units [AU]). Reconditioning by NMP showed slightly less morphological alterations ( AU). After COR, the histological morphology demonstrated only minor changes ( AU). Discussion The aim of this study was to compare COR and NMP as liver reconditioning tools after CS. Both methods are outstanding insofar as they target the metabolically more active organ. They are supposed to provide resumption of mitochondrial work for replenishment of the energy-depleted organ seen at the end of For theoretical reasons, the ultrastructural integrity and intact function of the mitochondria, which facilitate energy equivalents, are elementary prerequisites for a successful reconditioning. In this context, a clear delineation of mitochondrial integrity and function as a function of the applied reconditioning method was documented. Activity of caspase 9 as a marker for mitochondrial infraction and induction of apoptosis was far less pronounced after reconditioning by COR compared to CS. In contrast, reconditioning by NMP showed no difference compared to CS. The energetic recovery profiles of the different methods again in favor of COR further underscore these findings on a functional level. This has direct consequences on subcellular structures: superoxide anion radicals result from decoupling of the respiratory chain, and the suddenly accelerated metabolism and therefore throughput of the xanthine-oxidase (1,2) or reduced nicotinamide adenine dinucleotide reductase reaction. Such injurious byproducts cannot be cleared in energy-depleted cells. These oxygen radicals react harmfully with other macromolecules as 1 key component of the ischemia/reperfusion injury (IRI). (29,30) Accordingly, hepatocellular injury as an endpoint of IRI was significantly reduced by COR with a nearly halved aminotransferase peak after reperfusion compared to NMP. Additionally, the integrity of energy-dependent liver function represented by the amount of bile produced during reperfusion was far superior after COR compared to NMP. ischemia. (16,27,28) 1228 ORIGINAL ARTICLE

7 LIVER TRANSPLANTATION, Vol. 22, No. 9, 2016 HOYER ET AL. This study strongly suggests that a gentle rewarming process accounts for beneficial effects and the decrease of IRI, not the blunt activation of the metabolism for reconditioning. Abrupt temperature shift induced organ injuries are attributed to the opening of a mitochondrial transition pore (31) and subsequent mitochondrial dysfunction. The gentle rewarming process seems to alleviate this phenomenon. Interestingly, some earlier studies have demonstrated that even a short period of CS is deleterious for ischemically damaged livers. NMP did not recover such grafts. (32) This stands in contrast to the positive effects of NMP that have recently been reported: improved metabolic, functional, and morphological parameters (13,14) ; graft viability testing (19,33) ; as well as successful transplantation of normal, steatotic, and ischemically damaged livers (34,35) were documented. Parameters of liver injury stabilized after initiation of NMP. Although these studies used NMP for continuous preservation, the study at hand investigates the potential of NMP as an end-ischemic preservation modality. On the basis of the present results, immediate rewarming after CS might increase the reoxygenation injury in the energy-depleted altered tissue without beneficial conditioning effects. Therefore, the suitability of NMP for reconditioning purposes is questioned in its present form. It might be counterintuitive that COR, which uses lower temperatures and therefore lower metabolic activity than NMP, results in better energetic recovery after reperfusion. Interestingly, work by Rauen et al. (36) indicated that cold-induced/rewarming injury occurs at temperatures between 4 8C and 168C and not in the subnormothermic range above 20 8C. COR conducts a gentle transition of this critical temperature range and exceeds it clearly once the rewarming process is completed. Therefore, temperature shift-induced injury might be avoided and will not occur during reperfusion with warm recipient blood. Moreover, recent studies demonstrated that livers perfused up to 20 8C showednosigns of liver injury or anaerobiosis in contrast to livers perfused at higher temperatures. (37) Accordingly, rewarming up to 20 8C is sufficient for liver reconditioning purposes and results in the described superior results. For this first comparative investigation between the 2 preservation modalities, an established ex vivo reperfusion model (26,38,39) waschosentoreducebiologicalvariability. Therefore, emphasis was placed on the distinct effects of preservation. On the other hand, some limitations of such a model must be kept in mind for the correct interpretation of the presented data. The ex vivo reperfusion is restricted to a short time period, therefore providing information for the early reperfusion period only. Extrapolation of the data to later periods of reperfusion should be avoided. Of course, some aspects of liver injury and function, like posttransplant ischemic cholangiopathy, cannot be assessed based on the present data. Additionally, lack of transplantation into recipients means that an important aspect of reperfusion, namely, the interaction with the recipient, is not simulated by the present model. Therefore, future investigations should include in vivo transplantation models as the next step to confirm the present observations. In this context, comparisons to other recent emerging preservation modalities, especially HMP, which demonstrated interesting successful results, (7,40) should be carried out. The comparison of all the different perfusion modalities in future studies will yield knowledge of great interest and are awaited with great anticipation. It is of note that the present study represents a proofof-concept study. Both preservation modalities were carried out as previously described in the literature. (16,19,20) Obvious differences between methods, like preservation solutions, the use of oxygen carriers, temperature, etc., should not be considered as independent factors because these are prerequisites, respectively. In conclusion, for the first time, this study reports on the direct comparison of COR and NMP as endischemic reconditioning modalities. Superior results after application of COR were documented in terms of mitochondrial integrity resulting in better energetic recovery, less hepatocellular injury, and ultimately superior function in an ex vivo reperfusion model. On the basis of these results, COR should be preferred over NMP for reconditioning after CS, which should be validated by future investigations in transplantation models. Acknowledgments: The authors thank J. Weiler and Y. Yilmaz for editing this manuscript. REFERENCES 1) Khandoga A, Enders G, Luchting B, Axmann S, Minor T, Nilsson U, et al. Impact of intraischemic temperature on oxidative stress during hepatic reperfusion. Free Radic Biol Med 2003; 35: ) Toledo-Pereyra LH. Organ Preservation for Transplantation. 2nd ed. Austin, TX: Landes Biosci. 3) Schlegel A, Rougemont Od, Graf R, Clavien PA, Dutkowski P. Protective mechanisms of end-ischemic cold machine perfusion in DCD liver grafts. J Hepatol 2013;58: ) Dutkowski P, Schlegel A, de Oliveira M, M ullhaupt B, Neff F, Clavien PA. HOPE for human liver grafts obtained from donors after cardiac death. J Hepatol 2014;60: ORIGINAL ARTICLE 1229

8 HOYER ET AL. LIVER TRANSPLANTATION, September ) Schlegel A, Kron P, Dutkowski P. Hypothermic oxygenated liver perfusion: basic mechanisms and clinical application. Curr Tansplant Rep 2015;2: ) Stegemann J, Minor T. Energy charge restoration, mitochondrial protection and reversal of preservation induced liver injury by hypothermic oxygenation prior to reperfusion. Cryobiology 2009;58: ) Guarrera JV, Henry SD, Samstein B, Odeh-Ramadan R, Kinkhabwala M, Goldstein MJ, et al. Hypothermic machine preservation in human liver transplantation: the first clinical series. Am J Transplant 2010;10: ) Guarrera JV, Henry SD, Samstein B, Reznik E, Musat C, Lukose TI, et al. Hypothermic machine preservation facilitates successful transplantation of orphan extended criteria donor livers. Am J Transplant 2015;15: ) Nicholson ML, Hosgood SA. Renal transplantation after ex vivo normothermic perfusion: the first clinical study. Am J Transplant 2013;13: ) BagulA,HosgoodSA,KaushikM,KayMD,WallerHL,Nicholson ML. Experimental renal preservation by normothermic resuscitation perfusion with autologous blood. Br J Surg 2008;95: ) Tolboom H, Izamis ML, Sharma N, Milwid JM, Uygun B, Berthiaume F, et al. Subnormothermic machine perfusion at both 20 8C and 30 8C recovers ischemic rat livers for successful transplantation. J Surg Res 2012;175: ) Schlegel A, Kron P, Graf R, Clavien PA, Dutkowski P. Hypothermic Oxygenated Perfusion (HOPE) downregulates the immune response in a rat model of liver transplantation. Ann Surg 2014;260: ) Banan B, Chung H, Xiao Z, Tarabishy Y, Jia J, Manning P, et al. Normothermic extracorporeal liver perfusion for donation after cardiac death (DCD) livers. Surgery 2015;158: ) Xu H, Berendsen T, Kim K, Soto-Gutierrez A, Bertheium F, Yarmush ML, Hertl M. Excorporeal normothermic machine perfusion resuscitates pig DCD livers with extended warm ischemia. J Surg Res 2012;173:e ) Schlegel A, Kron P, Graf R, Dutkowski P, Clavien PA. Warm vs. cold perfusion techniques to rescue rodent liver grafts. J Hepatol 2014;61: ) Minor T, Efferz P, Fox M, Wohlschlaeger J, L uer B. Controlled oxygenated rewarming of cold stored liver grafts by thermally graduated machine perfusion prior to reperfusion. Am J Transplant 2013;13: ) Hoyer DP, Mathe Z, Gallinat A, Canbay AC, Treckmann JW, Rauen U, et al. Controlled oxygenated rewarming of cold stored livers prior transplantation: first clinical application of a new concept. Transplantation 2016;100: ) Leuvenink HG. Warming up--not only essential for athletes? Transplantation 2016;100: ) op den Dries S, Karimian N, Sutton ME, Westerkamp AC, Nijsten MW, Gouw AS, et al. Ex vivo normothermic machine perfusion and viability testing of discarded human donor livers. Am J Transplant 2013;13: ) Fondevila C, Hessheimer AJ, Maathuis MH, Mu~noz J, Taura P, Calatayud D, et al. Superior preservation of DCD livers with continuous normothermic perfusion. Ann Surg 2011;254: ) Moore DE, Feurer ID, Speroff T, Gorden DL, Wright JK, Chari RS, Pinson CW. Impact of donor, technical, and recipient risk factors on survival and quality of life after liver transplantation. Arch Surg 2005;140: ) Minor T, Yamaguchi T, Isselhard W. Effects of taurine on liver preservation in UW solution with consecutive ischemic rewarming in the isolated perfused rat liver. Transpl Int 1995;8: ) Koetting M, L uer B, Efferz P, Paul A, Minor T. Optimal time for hypothermic reconditioning of liver grafts by venous systemic oxygen persufflation in a large animal model. Transplantation 2011;91: ) Minor T, K otting M. Gaseous oxygen for hypothermic preservation of predamaged liver grafts: fuel to cellular homeostasis or radical tissue alteration? Cryobiology 2000;40: ) Camargo CA Jr, Madden JF, Gao W, Selvan RS, Clavien PA. Interleukin-6 protects liver against warm ischemia/reperfusion injury and promotes hepatocyte proliferation in the rodent. Hepatology 1997;26: ) Bell R, Shiel AG, Dolan P, Mears DC, Woodman K. The evaluation of the isolated perfused liver as a model for the assessment of liver preservation. Aust N Z J Surg 1993;63: ) Sch on MR, Kollmar O, Wolf S, Schrem H, Matthes M, Akkoc N, et al. Liver transplantation after organ preservation with normothermic extracorporeal perfusion. Ann Surg 2001;233: ) Ravikumar R, Leuvenink H, Friend PJ. Normothermic liver preservation: a new paradigm? Transpl Int 2015;28: ) Rauen U, Polzar B, Stephan H, Mannherz HG, de Groot H. Cold-induced apoptosis in cultured hepatocytes and liver endothelial cells: mediation by reactive oxygen species. FASEB J 1999;13: ) Rauen U, Klempt S, de Groot H. Histidine-induced injury to cultured liver cells, effects of histidine derivatives and of iron chelators. Cell Mol Life Sci 2007;64: ) Leducq N, Delmas-Beauvieux MC, Bourdel-Marchasson I, Dufour S, Gallis JL, Canioni P, Diolez P. Mitochondrial permeability transition during hypothermic to normothermic reperfusion in rat liver demonstrated by the protective effect of cyclosporin A. Biochem J 1998;336(pt 2): ) Reddy SP, Bhattacharjya S, Maniakin N, Greenwood J, Guerreiro D, Hughes D, et al. Preservation of porcine nonheart-beating donor livers by sequential cold storage and warm perfusion. Transplantation 2004;77: ) Bellomo R, Marino B, Starkey G, Fink M, Wang BZ, Eastwood GM, et al. Extended normothermic extracorporeal perfusion of isolated human liver after warm ischaemia: a preliminary report. Crit Care Resusc 2014;16: ) Brockmann J, Reddy S, Coussios C, Pigott D, Guirriero D, Hughes D, et al. Normothermic perfusion: a new paradigm for organ preservation. Ann Surg 2009;250: ) Jamieson RW, Zilvetti M, Roy D, Hughes D, Morovat A, Coussios CC, Friend PJ. Hepatic steatosis and normothermic perfusion-preliminary experiments in a porcine model. Transplantation 2011;92: ) Rauen U, Kerkweg U, de Groot H. Iron-dependent vs. ironindependent cold-induced injury to cultured rat hepatocytes: a comparative study in physiological media and organ preservation solutions. Cryobiology 2007;54: ) Ferrigno A, Di Pasqua LG, Bianchi A, Richelmi P, Vairetti M. Metabolic shift in liver: correlation between perfusion temperature and hypoxia inducible factor-1a. World J Gastroenterol 2015;21: ) Mets B, Rose-Innes C, Lotz Z, Hickman R, Chalton D. Comparison of in vivo and ex vivo porcine liver function using the same liver. J Hepatol 1993;17: ) Imber CJ, St Peter SD, Lopez de Cenarruzabeitia I, Pigott D, James T, Taylor R, et al. Advantages of normothermic perfusion over cold storage in liver preservation. Transplantation 2002;73: ) Dutkowski P, Polak WG, Muiesan P, Schlegel A, Verhoeven CJ, Scalera I, et al. First comparison of hypothermic oxygenated perfusion versus static cold storage of human donation after cardiac death liver transplants: an international-matched case analysis. Ann Surg 2015;262: ORIGINAL ARTICLE