End-ischemic pharmacological cocktail treatment to mitigate rewarming/reperfusion injury.

Increasing shortage of donor organs leads to the acceptance of less than optimal grafts for transplantation, up to and including organs donated after circulatory standstill of the donor. Therefore, protective strategies and pharmacological interventions destined to reduce ischemia induced tissue injury are considered a worthwhile focus of research. The present study evaluates the potential of a multidrug pharmacological approach as single flush at the end of static preservation to protect the liver from reperfusion injury. Livers were retrieved from male Wistar rats 20 min after cardiac standstill. The organs were cold stored for 18 h, flushed with 20 ml of saline, kept at room temperature for 20 min, and reperfused at 37 °C with oxygenated Williams E solution. In half of the cases, the flush solution was supplemented with a cocktail containing metformin, bucladesine and cyclosporin A. Upon reperfusion, treated livers disclosed a massive mitigation of hepatic release of alanine aminotransferase and aspartate aminotransferase, along with a significant approximately 50 % reduction of radical mediated lipid peroxidation, caspase activation and release of TNF-alpha. Even after preceding cold preservation, a pharmacological cocktail given as single flush is capable to mitigate manifestations of reperfusion injury in the present model.

Role of perfusion medium in rewarming machine perfusion from hypo- to normothermia.

BACKGROUND: Gradual warming up of cold stored organ grafts using a controlled machine perfusion protocol facilitates restitution of cellular homeostasis and mitigates rewarming injury by adapted increase of temperature and metabolism. The aim of the present study was to compare intra- and extracellular type perfusion media for the use in machine perfusion-assisted rewarming from hypo- to normothermia. METHODS: Rat livers were retrieved 20 min after cardiac arrest. After 18 h of cold storage (CS) with or without additional 2 h of rewarming machine perfusion from 8°C up to 35°C with either diluted Steen solution or with Belzer MPS, liver functional parameters were evaluated by an established ex vivo reperfusion system. RESULTS: Rewarming machine perfusion with either solution significantly improved graft performance upon reperfusion in terms of increased bile production, less enzyme release, and reduced lipid peroxidation compared to CS alone. Cellular apoptosis (release of caspase-cleaved keratin 18) and release of tumor necrosis factor were only reduced significantly after machine perfusion with Belzer MPS. Histological evaluation did not disclose any major morphological damage in any of the groups. CONCLUSION: Within the limitation of our model, the use of Belzer MPS seems to be an at least adequate alternative to a normothermic medium like Steen solution for rewarming machine perfusion of cold liver grafts.

Noninvasive measurement of 13Carbon turnover for evaluation of porcine renal grafts during ex vivo machine perfusion.

BACKGROUND: Kidney transplantation suffers from a shortage of donor organs. Despite this, a lot of grafts are discarded due to inadequate quality. As many kidneys are afflicted by transient filtration failure early after preservation, classical renal function tests are not applicable to differentiate between prospective recovery or continuing deficit of renal function. METHODS: Using normothermic machine perfusion as a platform for pre-implantation evaluation of the graft, we present a novel evaluative approach based on the metabolic turnover of 13C-acetate during isolated perfusion. After injection of the tracer, 13CO2 as a metabolic end-product can be quantified by high-precision laser-based spectroscopy in the gas outflow of the oxygenator. Three groups of porcine kidneys with graduated ischemic injury were investigated. RESULTS: This quantitative approach is able to discriminate acceptable quality kidneys, most likely to recover within days from poor kidney grafts that are unlikely to regain notable glomerular function with high discriminatory power (area under the ROC curve 0.91; P < 0.001 By contrast, conventional renal function tests are rather ineffective under these circumstances. CONCLUSIONS: This assessment method offers the potential to quantitatively assess donor kidney quality using a measurable output, salvaging donors that would otherwise have been discarded.

Kidney transplant surgery continues to face donor shortages. One consideration to tackle the shortage is to improve the way in which donor organ quality is assessed, so that fewer organs may be discarded. Here, we develop and test a non-invasive quantitative method that can measure the biochemical reaction that correlates to the viability of isolated kidneys using pig kidneys as a model system. We find the method could discriminate donor kidneys of good, intermediate, or poor quality with a discriminatory power superior to all other conventional parameters. Although the application needs to be tested in human kidney donors, it offers the potential to improve evaluation criteria for kidney grafts for transplantation.

Successful temporary machine perfusion of marginal liver grafts early after retrieval or shortly before implantation.

OBJECTIVES: The benefit of machine perfusion during storage of liver grafts retrieved after cardiac death should be investigated as applied either at the beginning or near the end of the preservation period. METHODS: Rat livers were explanted 20 min after cardiac arrest of the donor and cold-stored (CS) for 18 h. Other grafts were additionally subjected to 2 h of normothermic machine perfusion (MP) either 3 h after retrieval (early MP) or 3 h before reperfusion (late MP), thus extending total ischemic time to 20 h. The 3 h period should represent a short transport period between a resident regional pumping center and the explant or implant hospital, respectively. Viability of all livers was assessed thereafter by warm reperfusion in vitro. RESULTS: In comparison to the controls, both regimens significantly improved hepatic recovery upon post-preservation reperfusion as evaluated by enzyme release, bile production, and energetic recovery. Molecular upregulation of pro-inflammatory signals was also significantly mitigated. No functional differences between early and late machine perfusion could be disclosed. CONCLUSION: Our data suggest that it might not be necessary to hurry with the attempt to connect the graft to a machine early after retrieval.

Comparison Between Terminal or Preterminal Conditioning of Donor Livers by Ex Situ Machine Perfusion.

BACKGROUND: The successful implementation of end-ischemic normothermic machine perfusion (NMP) into clinical practice comes along with unusual demands for trained personnel and technical facilities in the implantation clinic. This creates an interest to bundle expertise and professional equipment for execution of MP at regional pump centers at the disadvantage of adding a second short period of cold preservation while sending the reconditioned grafts to the actual implant clinic. Differences of liver recovery upon reperfusion either immediately after NMP or after 3 h of cold storage subsequent to NMP should therefore be evaluated. METHODS: Rat livers were cold stored for 18 h, subjected to 2 h of NMP, and then either directly evaluated by ex vivo reperfusion or exposed to a second cold storage period of 3 h to simulate transport from the hub center to the implant clinic. Livers stored for 18 h by cold storage only served as controls. RESULTS: Both MP regimens significantly reduced hepatic enzyme release and improved bile production, clearance of lactate, and energetic recovery compared with the controls. However, no differences were seen between the 2 MP groups. CONCLUSIONS: The study provides first evidence that machine perfusion at regional perfusion centers may be a safe and economical alternative to the widespread individual efforts in the respective implantation clinics.

The impact of oxygen supply and erythrocytes during normothermic kidney perfusion.

The influence of erythrocytes and oxygen concentration on kidneys during long-term normothermic kidney perfusion is under debate. This study compares acellular and erythrocyte-based NMP with focus on oxygen delivery to the tissue as well as the effects of high oxygenation on tissue integrity. Pig kidneys were connected to NMP for six hours. The first group (n = 6; AC500) was perfused without addition of oxygen carriers, arterial perfusate pO2 was maintained at 500 mmHg. In the second group (n = 6; RBC500) washed erythrocytes were added to the perfusate at pO2 of 500 mmHg. Third group (n = 6; RBC200) was perfused with erythrocyte containing perfusate at more physiological pO2 of 200 mmHg. Addition of RBC did not relevantly increase oxygen consumption of the kidneys during perfusion. Likewise, there were no differences in kidney functional and injury parameters between AC500 and RBC500 group. Expression of erythropoietin as indicator of tissue hypoxia was comparable in all three groups. Cell free NMP at supraphysiological oxygen partial pressure seems to be a safe alternative to erythrocyte based perfusion without adverse effect on kidney integrity and provides a less cumbersome application of NMP in clinical practice.

Controlled oxygenated rewarming as novel end-ischemic therapy for cold stored liver grafts. A randomized controlled trial.

Abrupt return to normothermia has been shown a genuine factor contributing to graft dysfunction after transplantation. This study tested the concept to mitigate reperfusion injury of liver grafts by gentle warming-up using ex vivo machine perfusion prior to reperfusion. In a single center randomized controlled study, livers were assigned to conventional static cold storage (SCS) alone or to SCS followed by 90 min of ex vivo machine perfusion including controlled oxygenated rewarming (COR) by gentle and protracted elevation of the perfusate temperature from 10°C to 20°C. Primary outcome mean peak aspartate aminotransferase (AST) was 1371 U/L (SD 2871) after SCS versus 767 U/L (SD 1157) after COR (p = 0.273). Liver function test (LiMAx) on postoperative day 1 yielded 187 µg/kg/h (SD 121) after SCS, but rose to 294 µg/kg/h (SD 106) after COR (p = 0.006). Likewise, hepatic synthesis of coagulation factor V was significantly accelerated in the COR group immediately after transplantation (103% [SD 34] vs. 66% [SD 26]; p = 0.001). Fewer severe complications (Clavien-Dindo grade ≥3b) were reported in the COR group (8) than in the SCS group (15). Rewarming/reperfusion injury of liver grafts can be safely and effectively mitigated by controlling of the rewarming kinetics prior to blood reperfusion using end-ischemic ex vivo machine perfusion after cold storage.

Clinical use of controlled oxygenated rewarming of kidney grafts prior to transplantation by ex vivo machine perfusion. A pilot study.

BACKGROUND: Sudden restoration of normothermic conditions upon reperfusion of cold-stored grafts has been suggested to entail a massive energy demand not yet met by the cells that still suffer from hypothermic torpor. An adapted and gentle rise of graft temperature by ex-vivo machine perfusion has, therefore, been proposed. This should now be tested in the clinical setting. METHODS: In a first clinical series, six ECD-kidneys were subjected to controlled oxygenated rewarming (COR) during short term pre-implantation machine perfusion. Matched kidneys that were conventionally kept on ice served as controls. RESULTS: Early allograft function after transplantation was significantly improved by COR. On post-operative day 7, clearance of creatinine was more than twofold higher after COR and fractional excretion of sodium in the normal range, while significantly elevated in control kidneys. Good correlations were seen between ulterior graft function and real-time parameters obtained during pre-transplant machine perfusion (Lactate: r2  = .9; TIMP2: r2  = .74). Conventional denominators of graft viability like kidney donor risk index KDRI were far less predictive (r2  = .26). CONCLUSION: It is concluded that COR can be safely applied to renal grafts and appears to be a valuable tool to predict and improve early renal function after transplantation.

Comparison of thermal variations in post-retrieval graft conditioning on rat livers.

BACKGROUND: Machine perfusion was found an effective tool to recover organ grafts from ischemic insults during preservation. It could be observed that organ integrity is significantly affected by abrupt temperature shifts during hypothermic storage and implantation periods. Studies showed that a gentle and controlled rise of the temperature during oxygenated machine perfusion prior to implantation can protect the tissue from reperfusion injury. Now, the possible role of temperature kinetics upon retrieval of the graft and prior to later cold storage should be investigated. METHODS: Rat livers were retrieved after cardiac arrest and subjected to a brief ex situ machine perfusion with either hypothermic resuscitation (HR) at 8°C, near-normothermic resuscitation (NR) at 30°C or progressive resuscitation with lowering the temperature in a controlled fashion from 30°C to 8°C (PR). After cold storage (CS), liver functional parameters were evaluated by an established ex vivo reperfusion system. RESULTS: NR and PR resulted in significantly lower release of hepatic enzymes and less production of tumor necrosis factor upon reperfusion compared to CS while HR had a far less protective effect. An increase in bile production was only observed in the PR group, which also significantly increased the recovery of tissue adenosine triphosphate, the amount of which was, however, nearly paralleled by the NR protocol. CONCLUSION: Within the limitations of this model, it seems that normothermic recirculation appears to be a superior approach for the restitution of warm-ischemically injured liver grafts than immediate hypothermic machine perfusion.

Controlled Oxygenated Rewarming Compensates for Cold Storage-induced Dysfunction in Kidney Grafts.

BACKGROUND: Normothermic machine perfusion (NMP) provides a promising strategy for preservation and conditioning of marginal organ grafts. However, at present, high logistic effort limits normothermic renal perfusion to a short, postponed machine perfusion at site of the recipient transplant center. Thus, organ preservation during transportation still takes place under hypothermic conditions, leading to significantly reduced efficacy of NMP. Recently, it was shown that gentle and controlled warming up of cold stored kidneys compensates for hypothermic induced damage in comparison to end ischemic NMP. This study aims to compare controlled oxygenated rewarming (COR) with continuous upfront normothermic perfusion in a porcine model of transplantation. METHODS: Following exposure to 30 min of warm ischemia, kidneys (n = 6/group) were removed and either cold stored for 8 h (cold storage [CS]), cold stored for 6 h with subsequent controlled rewarming up to 35 °C for 2 h (COR), or directly subjected to 8 h of continuous NMP. Kidney function was evaluated using a preclinical autotransplant model with follow-up for 7 d. RESULTS: NMP and COR both improved renal function in comparison to CS and displayed similar serum creatinine and urea levels during follow-up. COR resulted in less tenascin C expression in the tissue compared with CS, indicating reduced proinflammatory upregulation in the graft by gentle rewarming. CONCLUSIONS: COR seems to be a potential alternative in clinical application of NMP, thereby providing logistic ease and usability.

Preservation of Mitochondrial Coupling and Renal Function by Controlled Oxygenated Rewarming of Porcine Kidney Grafts.

BACKGROUND: Warm reperfusion after previous cold storage has been shown to have a negative impact on mitochondrial function of organ grafts. Here, we wanted to investigate whether a more controlled warming up of the cold graft by ex vivo machine perfusion with gradually elevated temperature from cold to normothermia (including comparison of two warming up protocols) prior to implantation would be effective in preventing mitochondrial dysfunction upon reperfusion. METHODS: All experiments were conducted on porcine kidneys retrieved 15 min after cardiac arrest. After 18 h of cold storage in HTK solution (CS, n = 6), kidneys (n = 6) were subjected to 2 h of reconditioning machine perfusion starting with a hypothermic period followed by a gradual increase in perfusion temperature up to 35 °C (controlled oxygenated rewarming-COR). For a second group (n = 6), the slow warming up was begun instantly after connecting the graft onto the machine (iCOR). Functional recovery of all grafts was then observed upon normothermic reperfusion in vitro. At the conclusion of the experiments, tissue specimens were taken for immediate isolation and analysis of renal mitochondria. RESULTS: COR resulted in a significantly and more than 3-fold increased glomerular filtration rate upon reperfusion, along with a significant higher tubular sodium reabsorption and lesser loss of glucose in comparison to the controls. Enzyme release (AST) was also massively reduced during the reperfusion period. Specific analysis at the mitochondrial level revealed significantly better coupling efficiency and spare respiratory capacity in the COR group compared to the cold storage group. Interestingly, additional experiments revealed that the omission of a hypothermic perfusion period did not deteriorate any of the results after COR, provided that the instant temperature increase from 10 to 35 °C was effectuated in the same controlled manner. CONCLUSION: Controlled rewarming after extended cold preservation effectively improves mitochondrial recovery upon reperfusion and early functional outcome of kidney grafts.

Use of the new preservation solution Custodiol-MP for ex vivo reconditioning of kidney grafts.

Organ shortage and the increasing use of extended criteria donor grafts for transplantation drives efforts for more efficient organ preservation strategies from simple cold storage toward dynamic organ reconditioning. The choice of a suitable preservation solution is of high relevance in different organ preservation or reconditioning situations. Custodiol-MP is a new machine perfusion solution giving the opportunity to add colloids according to organ requirements. The present study aimed to compare new Custodiol-MP with clinically established Belzer MPS solution. Porcine kidneys were ischemically predamaged and cold stored for 20 hours. Ex vivo machine reconditioning was performed either with Custodiol-MP (n = 6) or with Belzer MPS solution (n = 6) for 90 minutes with controlled oxygenated rewarming up to 20°C. Kidney function was evaluated using an established ex vivo reperfusion model. In this experimental setting, differences between both types of perfusion solutions could not be observed. Machine perfusion with Custodiol-MP resulted in higher creatinine clearance (7.4 ± 8.6 mL/min vs. 2.8 ± 2.5 mL/min) and less TNC perfusate levels (0.22 ± 0.25 ng/mL vs. 0.09 ± 0.08 ng/mL), although differences did not reach significance. For short-term kidney perfusion Custodiol-MP is safe and applicable. Particularly, the unique feature of flexible colloid supplementation makes the solution attractive in specific experimental and clinical settings.

Reduction of Renal Preservation/Reperfusion Injury by Controlled Hyperthermia During Ex Vivo Machine Perfusion.

The possible reno-protective effect of a controlled brief heat-shock treatment during isolated ex vivo machine perfusion of donor grafts prior to reperfusion should be investigated in a primary in vitro study. Porcine kidneys (n = 14) were retrieved after 20 minutes of cardiac standstill of the donor and subjected to 20 hours of static cold storage in University of Wisconsin solution. Prior to reperfusion, kidneys were subjected to 2 hours of reconditioning machine perfusion with gradual increase in perfusion temperature up to 35°C. In half of the kidneys (n = 7), a brief hyperthermic impulse (10 minutes perfusion at 42°C) was implemented in the machine perfusion period. Functional recovery of the grafts was observed upon normothermic reperfusion in vitro. Hyperthermic treatment resulted in a 50% increase of heat shock protein (HSP) 70 and HSP 27 mRNA and was accompanied by ~ 50% improvement of tubular re-absorption of sodium and glucose upon reperfusion, compared with the controls. Furthermore, renal loss of aspartate aminotransferase was significantly reduced to one-third of the controls as was urinary protein loss, evaluated by the albumin to creatinine ratio. It is concluded that ex vivo heat-shock treatment seems to be an easily implementable and promising option to enhance renal self-defense machinery against reperfusion injury after preservation that merits further investigation in preclinical models.

Long-term Outcomes After Controlled Oxygenated Rewarming of Human Livers Before Transplantation.

Controlled oxygenated rewarming (COR) has been shown to be a feasible and safe method in clinical practice and to reduce peak serum transaminases after liver transplantation. This study aimed to demonstrate further clinical experience of this method of now 18 clinical liver transplantations utilizing COR and demonstrate the long-term results. METHODS: In this extended series of 18 patients, cold-stored livers were subjected to machine-assisted slow COR for ≈120 minutes before transplantation. A cohort of 178 patients transplanted during the same period with similar clinical characteristics were used for comparison of key outcomes. RESULTS: All livers were perfused in accordance to the COR protocol without incidences and transplanted successfully. Early allograft dysfunction was observed in 2 (11.1%) cases after COR. Liver elasticity measurements indicated normal healthy liver parenchyma at the last follow-up. Graft survival demonstrated excellent outcomes after COR. The 1-, 3-, and 5-year patient survival rates were 100%, 100%, and 93.8% compared with 84.5%, 82.0%, and 75.8% in the control group (P = 0.12). CONCLUSIONS: The present study demonstrates excellent clinical outcomes after COR before liver transplantation. Comparison with a control cohort shows superiority of graft survival. Further evidence is needed to assess this promising method to improve organ preservation, finally.

Effect of oxygen concentration in anterograde liver persufflation on high energy phosphates and graft function after ischemic preservation.

Here we evaluate the potential of anterograde gaseous oxygen persufflation for graft reconditioning after extended storage times. Pig livers were retrieved and cold-stored in HTK solution for 16 h. Some grafts were subsequently subjected to anterograde gaseous oxygen persufflation via the portal vein for 2 h. Oxygen concentrations for persufflation were either 100% or 40%. The gas was insufflated at a pressure adjusted to 18 mmHg, a pressure required to see gas bubbles leaving at the hepatic vein. Gas flow required for adequate maintenance of persufflation pressure amounted to approx. 300 ml/min in both groups. Only the use of 100% oxygen resulted in a significant increase of end-ischemic tissue ATP and improved bile flow upon reperfusion. Brief anterograde oxygen persufflation can improve energetic status of ischemic livers prior to transplantation, but the use of pure oxygen and adequate gas flow seems necessary to improve ulterior graft function.

First-in-man controlled rewarming and normothermic perfusion with cell-free solution of a kidney prior to transplantation.

Cold preservation sensitizes organ grafts to exacerbation of tissue injury upon reperfusion. This reperfusion injury is not fully explained by the mere re-introduction of oxygen but rather is pertinent to the immediate rise in metabolic turnover associated with the abrupt restoration of normothermia. Here we report the first clinical case of gradual resumption of graft temperature upon ex vivo machine perfusion from hypothermia up to normothermic conditions using cell-free buffer as a perfusate. A kidney graft from an extended criteria donor was put on the machine after 12.5 hours of cold storage. During ex vivo perfusion, perfusion pressure and temperature were gradually elevated from 30 mm Hg and 8°C to 75 mm Hg and 35°C, respectively. Perfusate consisted of diluted Steen solution, oxygenated with 100% oxygen. Final flow rates at 35°C were 850 mL/min. The kidney was transplanted without complications and showed good immediate function. Serum creatinine fell from preoperative 720 µmol/L to 506 µmol/L during the first 24 hours after transplantation. Clearance after 1 week was 43.1 mL/min. Controlled oxygenated rewarming prior to transplantation can be performed up to normothermia without blood components or artificial oxygen carriers and may represent a promising tool to mitigate cold-induced reperfusion injury or to evaluate graft performance.

Transient hyperthermia during oxygenated rewarming of isolated rat livers.

Pretransplant machine perfusion of donor grafts has gained clinical appreciation to improve graft function and survival after transplantation. This study was aimed as pilot investigation to evaluate the additive potential of a transient ex vivo heat shock treatment of the isolated organ during machine perfusion to further protect the graft from subsequent reperfusion injury. Rat livers were retrieved after 20 min of cardiac arrest and preserved for 18 h by cold storage in HTK solution. Prior to reperfusion, livers were subjected to 2 h of reconditioning machine perfusion with gradual increase in perfusion temperature up to 35 °C. In half of the livers (n = 7), a brief hyperthermic impulse (10 min perfusion at 42 °C) was implemented in the machine perfusion period. Functional recovery of the grafts was observed upon normothermic reperfusion in vitro. Induction of heat shock protein 70 was followed on the mRNA and protein level. Chaperone induction by transient hyperthermia was associated with a significant improvement of bile production upon reperfusion and significantly reduced enzyme loss of mitochondrial GLDH. Heat shock treatment further affected pro-inflammatory upregulation in the graft in significantly reducing gene expression as wells as protein release of TNF-alpha. It is concluded, that graft conditioning by controlled hyperthermia ex vivo may represent a feasible and useful tool to improve liver recovery after preservation.

Rewarming Injury after Cold Preservation.

Organ dysfunction pertinent to tissue injury related to ischemic ex vivo preservation during transport from donor to recipient still represents a pivotal impediment in transplantation medicine. Cold storage under anoxic conditions minimizes metabolic activity, but eventually cannot prevent energetic depletion and impairment of cellular signal homeostasis. Reoxygenation of anoxically injured tissue may trigger additional damage to the graft, e.g., by abundant production of oxygen free radicals upon abrupt reactivation of a not yet equilibrated cellular metabolism. Paradoxically, this process is driven by the sudden restoration of normothermic conditions upon reperfusion and substantially less pronounced during re-oxygenation in the cold. The massive energy demand associated with normothermia is not met by the cellular systems that still suffer from hypothermic torpor and dys-equilibrated metabolites and eventually leads to mitochondrial damage, induction of apoptosis and inflammatory responses. This rewarming injury is partly alleviated by preceding supply of oxygen already in the cold but more effectively counteracted by an ensuing controlled and slow oxygenated warming up of the organ prior to implantation. A gentle restitution of metabolic turnover rates in line with the resumption of enzyme kinetics and molecular homeostasis improves post transplantation graft function and survival.

Cold flush after dynamic liver preservation protects against ischemic changes upon reperfusion - an experimental study.

Ex vivo machine perfusion of the liver after cold storage has found to be most effective if combined with controlled oxygenated rewarming up to (sub)-normothermia. On disconnection of the warm graft from the machine, most surgeons usually perform a cold flush of the organ as protection against the second warm ischemia incurred upon implantation. Experimental evidence, however, is lacking and protective effect of deep hypothermia has been challenged for limited periods of liver ischemia in other models. A first systematic test was carried out on porcine livers, excised 30 min after cardiac arrest, subjected to 18 h of cold storage in UW and then machine perfused for 90 min with Aqix-RSI solution. During machine perfusion, livers were gradually rewarmed up to 20 °C. One group (n = 6) was then reflushed with 4 °C cold Belzer UW solution whereas the second group (n = 6) remained without cold flush. All livers were exposed to 45 min warm ischemia at room temperature to simulate the surgical implantation period. Organ function was evaluated in an established reperfusion model using diluted autologous blood. Cold reflush after disconnection from the machine resulted in a significant increase in bile production upon blood reperfusion, along with a significant reduction in transaminases release alanine aminotransferase and of the intramitochondrial enzyme glutamate dehydrogenase. Interestingly, free radical-mediated lipid peroxidation was also found significantly lower after cold reflush. No differences between the groups could be evidenced concerning histological injury and recovery of hepatic energy metabolism (tissue content of adenosine triphosphate). Post-machine preservation cold reflush seems to be beneficial in this particular setting, even if the organs are warmed up only to 20 °C, without notion of adverse effects, and should therefore be implemented in the protocol.