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.

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.

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.

Controlled Rewarming after Hypothermia: Adding a New Principle to Renal Preservation.

Early graft dysfunction due to preservation/reperfusion injury still represents a notable issue after kidney transplantation, affecting long term prognosis of graft viability. One trigger of postischemic cell dysfunction could be recognized in the abrupt temperature shift from hypo- to normothermia, leading to mitochondrial dysfunction and proapoptotic signal transduction. Here we propose a technique to cope with this "rewarming injury" by interposing a period of gentle warming up by hypo- to subnormothermic machine perfusion of the isolated graft prior to warm reperfusion. Porcine kidneys were subjected either to 18 hours of hypothermic machine preservation (HMP) or 18 hours static cold storage + 3 hours of gentle, machine controlled oxygenated rewarming (COR). Functional integrity was evaluated in both groups by subsequent normothermic reperfusion in vitro. The functional benefit of COR was documented by an approximately twofold increase in renal clearances of creatinine as well as urea upon warm reperfusion, compared to controls. This was accompanied with a notable mitigation of postischemic mitochondrial dys-homeostasis. COR significantly improved renal oxygen consumption and maintained total NAD tissue content upon reperfusion. Mitochondrial initiation of cellular apoptosis, as evidenced by activation of caspase 9 was also largely prevented after COR but not in controls. The concept of gentle regenerative graft rewarming could become a valuable adjunct in renal transplantation.

Ex vivo use of a Rho-kinase inhibitor during renal preservation improves graft function upon reperfusion.

BACKGROUND: Activation of the Rho-Rho-kinase pathway has been shown to cause vasoconstriction in renal afferent arterioles. Vascular dysfunction plays a pivotal role in triggering reperfusion injury after kidney transplantation. Therefore, the effect of a Rho-kinase inhibitor, added to the preservation solution, on renal function after 18 h of storage at 4 °C was evaluated. METHODS: Porcine kidneys were preserved with cold HTK-solution. During preservation, in the study group, HTK was supplemented with the Rho-kinase inhibitor HA1077, whereas the control group received no further treatment (n=6, respectively). Kidney function after 18 h of storage at 4 °C was evaluated by 90 min of isolated reperfusion in vitro. RESULTS: Rho-kinase inhibition (RKI) was associated with significantly higher renal perfusate flow compared to the control group. Endothelial function, as measured by perfusate levels of nitric oxide and gene expression of eNOS, was significantly increased in the study group. In our model, RKI also significantly improved glomerular function (clearance of creatinine) as well as tubular cell integrity as reflected by reduced fractional sodium excretion and release of fatty acid binding protein, a specific tubular cell marker. CONCLUSION: Our results indicate that blocking the Rho-kinase pathway during cold preservation may lead to a better graft function upon reperfusion.

Adding pulsatile vascular stimulation to venous systemic oxygen persufflation of liver grafts.

The effect of adding pulsatility to gaseous oxygen persufflation during liver preservation was studied in an isolated rat liver model. Livers from male Wistar rats were retrieved 30 min after cardiac arrest of the donor and subjected to 18 h of cold storage. Some grafts were subjected to nonpulsatile or pulsatile gaseous oxygen persufflation. Graft viability was assessed thereafter upon warm reperfusion in vitro (n = 5 per group). Pulsatile persufflation significantly improved parenchymal integrity (enzyme release, bile flow) upon reperfusion, with respect to nonpulsatile persufflation or cold storage (CS) (e.g., max. release of alanine aminotransferase: 44 ± 10 vs. 178 ± 29 vs. 345 ± 100 U/L; pulsatile vs. nonpulsatile persufflation vs. CS).The effect was associated with the prevention of the ischemic decline in gene and protein expression of the vasoprotective Krüppel-like factor 2, increased perfusate levels of the endogenous vasodilator nitric oxide, and reduced portal vascular resistance upon reperfusion, while nonpulsatile persufflation was less effective (e.g., vascular resistance: 1235 ± 108 vs. 1607 ± 155 vs. 2215 ± 208 Pa s/mL; pulsatile vs. nonpulsatile persufflation vs. CS). In conclusion, pulsatile mechanostimulation of the hepatovasculature seems a genuine protective mechanism, affecting early graft recovery upon reperfusion.

Role of pulsatility in hypothermic reconditioning of porcine kidney grafts by machine perfusion after cold storage.

BACKGROUND: Brief in-house machine perfusion after cold storage (CS) (hypothermic reconditioning) has been proposed as a convenient tool to improve kidney graft function. The present study aimed to investigate the mechanistic role of vascular pulsatility in this context. METHODS: Kidney function after cold preservation (4°C, 18 hr) and subsequent reconditioning by 90 min of pulsatile machine perfusion (PP) (30/20 mm Hg) or nonpulsatile machine perfusion (NPP) (30 mm Hg) was studied in an isolated kidney perfusion model in pigs (n=6 for both) and compared with simply CS grafts. RESULTS: Compared with CS, PP but not NPP significantly improved renal perfusate flow and urine production and significantly increased the reduction of perfusate levels of creatinine and urea during reperfusion. Perfusate levels of fatty acid binding protein, a marker of tubular cell injury, were dramatically reduced by PP but not NPP. PP and NPP lowered fractional excretion of sodium, but significance was only reached for PP. Molecular effects of PP comprised a significant (vs. CS) mRNA elevation of the endothelial anti-inflammatory transcription factor Krüppel-like factor 2 as well as endothelial nitric oxide synthase, along with significantly higher perfusate levels of the endogenous vasodilator nitric oxide. Functional efficiency of PP over CS was confirmed in additional porcine transplant experiments (n=5 for both) by, for example, up to threefold improved clearance of creatinine during the first days after transplantation. CONCLUSION: PP of 90 min shortly before transplantation seems to be an efficient mechanism to reduce proinflammatory endothelial phenotype and improve functional outcome of kidney grafts even after preceding static storage.

Use of the new preservation solution Custodiol-N supplemented with dextran for hypothermic machine perfusion of the kidney.

Custodiol-N is a new preservation solution specifically designed to prevent free radical-induced tissue alterations and to protect vascular integrity of the graft. Thus, Custodiol-N appears particularly suitable as base solution for oxygenated machine preservation and its putative benefit for renal preservation by hypothermic machine perfusion (HMP) was investigated using a porcine in vitro model. Kidneys were retrieved from German Landrace pigs and preserved for 20 h by pulsatile oxygenated HMP on a Lifeport kidney transporter (syst. pressure 30 mmHg, 30cycles/min). Each graft was randomly assigned to the use of one of the following preservation solutions: Custodiol-N solution supplemented with 50 g/l dextran 40 (CND) or kidney perfusion solution 1 (KPS-1). Renal viability was evaluated upon reperfusion in vitro with diluted autologous blood from the donor for 120 min at 37°C. After 2h of postischemic reperfusion CND-preserved kidneys exhibited significantly higher renal blood flow and urine production. Oxygen consumption was also higher in the CND group than in KPS-1 kidneys. Clearance of creatinine increased during reperfusion of CND kidneys but declined in KPS-1 grafts ending in significantly higher values in CND kidneys. No differences between the groups were seen for enzyme release or fractional excretion of sodium. In conclusion the data presented provide first experimental evidence for adequate organ protective potential of CND in HMP as compared to the gold standard KPS-KPS-11.

Role of oxygenation in hypothermic machine perfusion of kidneys from heart beating donors.

UNLABELLED: Dynamic preservation of organ grafts by hypothermic machine perfusion (HMP) has regained broader interest to provide better outcome after transplantation. One pivotal aspect still under debate is the role of oxygenation during HMP. The present study investigates functional and molecular aspects of active oxygenation during HMP of kidneys from heart beating donors. METHODS: Kidneys were retrieved from Landrace pigs (25-30 kg body weight) and preserved by pulsatile HMP for 21 hr. All kidneys were randomly assigned to either anoxic perfusion (MPanox) or active oxygenation of the perfusate (MPox). All grafts were then autotransplanted, and the remaining native kidney was removed at the same time. Renal integrity and function was evaluated during perfusion and for 1 week after the transplantation and the removal of the remaining native kidney. RESULTS: Oxygenation during HMP resulted in lower endischemic vascular resistance and slightly elevated free radical-mediatedtissue injury during HMP. After reperfusion, radical mediated lipid peroxidation was twofold higher in the MPanox group. Renal clearance of creatinine was found significantly better during the first 2 days after transplantation after MPanox than after MPox. Molecular expression of erythropoietin was increased threefold to baseline levels after MPanox, indicating renal hypoxia during preservation, but was remained unchanged after MPox. Gene expression of sodium-glucose transporter reflected similar functional outcome in both groups. Fractional excretion of Na(+), proteinuria, or serum levels of lactate dehydrogenase were similar in both groups. CONCLUSION: The present data do not support the use of active oxygenation during hypothermic perfusion of kidneys from donors with intact circulation.

Hypothermic reconditioning by gaseous oxygen persufflation after cold storage of porcine kidneys.

BACKGROUND: Delayed graft function still represents a major complication in clinical kidney transplantation. Here we tested the possibility to improve functional outcome of cold stored kidneys a posteriori by hypothermic reconditioning using retrograde oxygen persufflation (ROP) immediately prior to reperfusion. METHODS: Kidneys from female German Landrace pigs were flushed with Histidine-Tryptophan-Ketoglutarate (HTK) solution and cold-stored for 18 h (control). Some grafts were subsequently subjected to 90 min of retrograde oxygen persufflation (ROP) via the renal vein during cold preservation. Early graft function of all kidneys was assessed thereafter by warm reperfusion in vitro (n=6, resp.). RESULTS: Renal function upon reperfusion was significantly enhanced by ROP with an approximately twofold increase in renal clearances of creatinine and urea. ROP also led to higher renal vascular flow rates, enhanced urine output and mitigated histological alterations. CONCLUSION: It is concluded that initial graft function can be improved by 90 min of hypothermic gaseous oxygenation after arrival of the preserved organ in the transplantation clinic.

Hypothermic reconditioning of porcine kidney grafts by short-term preimplantation machine perfusion.

BACKGROUND: Clinical trial data suggest that continuous hypothermic machine perfusion (HMP) during the entire preservation period reduces the incidence of delayed graft function and improves graft survival. This study evaluates whether short-term MP after cold storage (CS) is also effective. METHODS: Kidney function after cold preservation (4°C, 21 hr) and transplantation was studied in an autotransplant model using Landrace pigs (25-30 kg; n=5 per group) with 1 week follow-up. Preservation was performed by conventional CS or HMP with a modified Lifeport Kidney Transporter either continuously during the entire preservation period or only for 2 hr of hypothermic reconditioning (HR) subsequent to conventional CS. RESULTS: HMP and HR similarly improved cortical microcirculation and significantly reduced maximal serum creatinine levels and recovery of creatinine clearance to normal values compared with CS. Fractional excretion of Na+ was unaltered after HMP and HR but significantly increased until postoperative day 5 on CS. On a molecular level, HR reduced innate immunoreactivity (toll-like receptor 4 expression and high mobility group protein B1 [HMGB-1] release) and normalized antiinflammatory tissue expression of von Kruppel-like Factor-2. CONCLUSION: Short-term reconditioning after CS proves to be as effective as continuous MP during the whole storage time. Because of its logistical convenience, the concept of an a posteriori treatment recommends itself to be evaluated in clinical trials.

Energetic recovery in porcine grafts by minimally invasive liver oxygenation.

BACKGROUND: Gaseous insufflation of oxygen via the venous vascular system has proven to be an effective tool for preventing anoxic tissue injury after extended time periods of ischemic liver preservation. Most experimental studies so far have been undertaken in rat models and include a series of pinpricks into postsinusoidal venules as an outlet for the insufflated gas. Here, we describe a simplified technique for minimally invasive liver oxygenation in porcine grafts, representing a hassle-free access to organ oxygenation without vascular lesions. METHODS: We retrieved livers from Landrace pigs and cold-stored them in histidine-tryptophan-ketoglutarate solution. Subsequent to 18 h preservation, we treated some livers for an additional 2 h with gaseous oxygen, insufflated via silicone tubing inserted into the suprahepatic caval vein. Gas pressure was limited to 18 mm Hg. We occluded the infrahepatic caval vein with a bulldog clamp. Gas bubbles left the graft via the portal vein. We assessed liver integrity by energetic tissue status and by controlled in vitro reperfusion with autologous blood. RESULTS: Magnetic resonance imaging demonstrated homogeneous gas distribution in the persufflated tissue without major shunting. Biochemical analyses revealed effective and homogeneous restoration of energetic homeostasis in the ischemic graft before reperfusion. Sinusoidal endothelial clearance of hyaluronic acid was significantly improved upon reperfusion, as was hepatic arterial flow. Parenchymal enzyme loss was concordantly mitigated after minimally invasive liver oxygenation. CONCLUSIONS: Our results indicate that gaseous oxygen persufflation of the porcine liver is possible without tissue trauma, and significantly enhances post-preservation recovery of the graft.

Dopamine improves hypothermic machine preservation of the liver.

BACKGROUND: Hypothermic machine preservation (HMP) is currently reconsidered as alternative to standard cold storage of organs from non-heart-beating donors. The present study was aimed at investigating the possible synergistic effect of HMP and the addition of dopamine to the circulating perfusate during preservation. METHODS: Cardiac arrest was induced in male Wistar rats (250-300 g) by phrenotomy. Thirty minutes later livers were flushed via the portal vein and subjected to 20 h of HMP at 5ml/min at 4°C. During HMP the preservation solution was equilibrated with 100% oxygen and dopamine was added at 0, 10, 50 or 100 µM (D0, D10, D50, D100; n=6 resp.). Graft viability was assessed thereafter upon warm reperfusion in vitro for 2h. RESULTS: During HMP, D50 and D100 significantly reduced hepatic release of ALT to about 50%. No influence of dopamine was found on vascular resistance, oxygen uptake or lactate production at any concentration. D50 significantly reduced enzyme release during reperfusion (∼50%), enhanced bile flow and oxygen consumption. D10 was less effective while D100 even rose enzyme release compared with D0. Enhanced oxygen free radical mediated lipid peroxidation (LPO), found in the tissue of D0 livers was significantly reduced by D50; D50 significantly abrogated molecular upregulation of vWillebrand factor upon reperfusion suggesting vascular protection of the endothelial cell. CONCLUSION: Efficiency of HMP might be increased by stimulating livers with dopamine during ex vivo preservation, limiting vascular side effects and improving functional recovery upon early reperfusion.

Optimal time for hypothermic reconditioning of liver grafts by venous systemic oxygen persufflation in a large animal model.

BACKGROUND: Quality of cold-stored livers declines beyond 12 hr of ischemia, increasing the risk of primary dysfunction. Here we evaluate the potential and optimal treatment interval of gaseous oxygen persufflation for grafts reconditioning after long storage times in an experimental pig liver model. METHOD: Porcine livers (n=6/group) were cold stored at 4°C for 18 hr in histidine-tryptophan-ketoglutarate solution. Hypothermic reconditioning (HR) was performed in some livers, by insufflation of gaseous oxygen through the caval vein for 1, 2, or 3 hr subsequent to cold storage. Liver integrity was assessed by controlled in vitro reperfusion with autologous blood. RESULTS: HR resulted in a 40% to 50% reduction of serum levels of aspartate aminotransferase, lactate dehydrogenase, and tumor necrosis factor-α with a maximal effect after 2 hr of HR (P<0.05). Functional parameters (bile production, cholinesterase and energetic recovery) were likewise enhanced (P<0.05). Two hours of HR also improved hepatic arterial flow and abrogated the postischemic increase in portal venous perfusion resistance compared with untreated (P<0.05). Gene expression of Toll-like receptor-4 was reduced by 2 hr of HR as was platelet adherence in the reperfused graft (P<0.05), in line with a trend toward lower expression of von Willebrand factor. CONCLUSION: HR effectively ameliorated graft dysfunction after extended preservation of porcine livers. Two hours of "a posteriori" treatment provide the maximal effect and are recommended for further application.

Role of oxygen during hypothermic machine perfusion preservation of the liver.

Grafts from non-heart-beating donors are thought to be best preserved by hypothermic machine perfusion (HMP). Controversy exists concerning the role of oxygenation during HMP. In this study, we wanted to evaluate the relative role of oxygenation for graft integrity during and after HMP. Cardiac arrest was induced in male Wistar rats (250-300 g) by phrenotomy. Thirty minutes later, livers were flushed via the portal vein and subjected to 18 h of HMP at 5 ml/min at 4 degrees C. During HMP, the preservation solution was equilibrated with 100% oxygen (HMP100), with air (HMP20) or not oxygenated at all (HMP0). Graft integrity was assessed thereafter upon warm reperfusion in vitro. During preservation, oxygenation of the perfusate reduced alanine aminotransferase release by 50% compared with HMP0. HMP100 resulted in reduced oxygen free radical-mediated lipid peroxidation upon warm reperfusion compared with both HMP20 and HMP0. One hundred per cent oxygenation during HMP also significantly enhanced the activation of AMPK salvage pathway, and upstream activation of protein kinase A when compared with HMP0. Enzyme release during reperfusion was reduced by approximately 40% (HMP20) or approximately 70% (HMP100) after oxygenation compared with HMP0. Functional recovery (bile production) was only enhanced by HMP100 (approximately twofold increase vs. HMP20 and HMP0, P < 0.05). Efficiency of HMP might be markedly increased by additional aeration of the perfusate, most successfully by equilibration with 100% oxygen.