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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.

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. CA Minor

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.

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 up- front normothermic perfusion in a porcine model of transplantation. Methods Following exposure to 30 minutes of warm ischemia kidneys (n=6/group) were removed and either cold stored for 8 hours (CS), cold stored for 6 hours with subsequent controlled rewarming up to 35 °C for 2 hours (COR) or directly subjected to 8 hours of continuous normothermic machine perfusion (NMP). Kidney function was evaluated using a preclinical autotransplant model with follow up for 7 days. 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 to CS, indicating reduced proinflammatory upregulation in the graft by gentle rewarming. Conclusion COR seems to be a potential alternative in clinical application of NMP, thereby providing logistic ease and usability.

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.

Importance Continuous hypothermic machine perfusion during organ preservation has a beneficial effect on graft function and survival in kidney transplant when compared with static cold storage (SCS). Objective To compare the effect of short-term oxygenated hypothermic machine perfusion preservation (end-HMPo2) after SCS vs SCS alone on 1-year graft survival in expanded criteria donor kidneys from donors who are brain dead. Design, Setting, and Participants In a prospective, randomized, multicenter trial, kidneys from expanded criteria donors were randomized to either SCS alone or SCS followed by end-HMPo2 prior to implantation with a minimum machine perfusion time of 120 minutes. Kidneys were randomized between January 2015 and May 2018, and analysis began May 2019. Analysis was intention to treat. Interventions On randomization and before implantation, deceased donor kidneys were either kept on SCS or placed on HMPo2. Main Outcome and Measures Primary end point was 1-year graft survival, with delayed graft function, primary nonfunction, acute rejection, estimated glomerular filtration rate, and patient survival as secondary end points. Results Centers in 5 European countries randomized 305 kidneys (median [range] donor age, 64 [50-84] years), of which 262 kidneys (127 [48.5%] in the end-HMPo2 group vs 135 [51.5%] in the SCS group) were successfully transplanted. Median (range) cold ischemia time was 13.2 (5.1-28.7) hours in the end-HMPo2 group and 12.9 (4-29.2) hours in the SCS group; median (range) duration in the end-HMPo2 group was 4.7 (0.8-17.1) hours. One-year graft survival was 92.1% (n = 117) in the end-HMPo2 group vs 93.3% (n = 126) in the SCS group (95% CI, −7.5 to 5.1; P = .71). The secondary end point analysis showed no significant between-group differences for delayed graft function, primary nonfunction, estimated glomerular filtration rate, and acute rejection. Conclusions and Relevance Reconditioning of expanded criteria donor kidneys from donors who are brain dead using end-HMPo2 after SCS does not improve graft survival or function compared with SCS alone. This study is underpowered owing to the high overall graft survival rate, limiting interpretation. Trial Registration isrctn.org Identifier: ISRCTN63852508

Orthotopic liver transplantation (OLT) using allografts from donation after circulatory death (DCD) is potentially associated with compromised clinical outcomes due to ischemia-reperfusion injury (IRI)-induced organ damage and graft-related complications. The aim of this study was to provide in vivo data on the effects of adenosine A2a receptor stimulation in a clinically relevant large animal model of DCD liver transplantation. Cardiac arrest was induced in German Landrace pigs (n = 10, 20&ndash, 25 kg). After 30 min of warm ischemia, the donor liver was retrieved following a cold flush with 3 L of histidine-tryptophan-ketoglutarate-HTK solution. Animals of the treatment group (n = 5/group) received a standard dose of the selective adenosine receptor agonist CGS 21680 added to the cold flush. All grafts were stored for 4.5 h at 4 &deg, C in HTK-solution before OLT. Hepatocellular injury, apoptosis, protein kinase A-PKA activity, graft microcirculation, liver function, and animal survival were assessed. Compared to untreated livers, adenosine A2a receptor stimulation resulted in improved tissue microcirculation (103% &plusmn, 5% vs. 38% &plusmn, 4% compared to baseline, p &lt, 0.05), accelerated functional recovery of the graft (indocyanine green-plasma disappearance rate (ICG-PDR) of 75% &plusmn, 18% vs. 40% &plusmn, 30% after 3 h), increased PKA activity ratio (56% &plusmn, 3% vs. 32% &plusmn, 3%, 0.001 after 1 h), and consequently reduced tissue necrosis and apoptosis. The potent protective effects were clinically manifested in significantly improved survival in the treatment group after 72 h (100% vs. 40%, p = 0.04). The ex vivo administration of adenosine A2a receptor agonist during the back-table flush mitigates IRI-mediated tissue damage and improves functional graft recovery and survival in a large animal model of DCD liver 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.

Contains fulltext : 226016.pdf (Publisher’s version ) (Closed access) In several deceased donor kidney allocation systems, organs from elderly donors are allocated primarily to elderly recipients. The Eurotransplant Senior Program (ESP) was implemented in 1999, and since then, especially in Europe, the use of organs from elderly donors has steadily increased. The proportion of ≥60-year-old donors reported to the Collaborative Transplant Study (CTS) by European centers has doubled, from 21% in 2000-2001 to 42% in 2016-2017. Therefore, in the era of organ shortage it is a matter of debate whether kidney organs from elderly donors should only be allocated to elderly recipients or whether

Background. 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. CA - Hoyer

The concept of "controlled oxygenated rewarming" (COR) using ex vivo machine perfusion after cold storage was evaluated as tool to improve renal graft function after transplantation. Renal function after 20 min warm ischemia and 21 h cold storage was studied in an auto-transplant model in pigs (25-30 kg, n = 6 per group). In the study group, preimplant ex vivo machine perfusion for 90 min was added after cold storage, including gentle warming up of the graft to 20°C (COR). Kidneys that were only cold stored for 21 h served as controls. In vivo follow up was one week; the remaining native kidney was removed during transplantation. COR significantly improved cortical microcirculation upon early reperfusion and reduced free radical mediated injury and cellular apoptosis. Post-transplant kidney function (peak levels in serum) was also largely and significantly improved in comparison to the control group. A weak inverse correlation was found between renal flow during COR and later peak creatinine after transplantation (r2 = 0.5), better values were seen for oxygen consumption, measured during machine perfusion at 20°C (r2 = 0.81). Gentle graft rewarming prior to transplantation by COR improves post-transplant graft outcome and may also be a valuable adjunct in pretransplant graft assessment.

Background Recently, the postoperative Model for End stage Liver Disease score (POPMELD) was suggested as a definition of postoperative graft dysfunction and a predictor of outcome after liver transplantation (LT). Aim The aim of the present study was to validate this concept in the context of extended criteria donor (ECD) organs. Patients and methods Single-center prospectively collected data (OPAL study/01/11-12/13) of 116 ECD LTs were utilized. For each recipient, the Model for End stage Liver Disease (MELD) score was calculated for 7 postoperative days (PODs). The ability of international normalized ratio, bilirubin, aspartate aminotransferase, Donor Risk Index, a recent definition of early allograft dysfunction, and the POPMELD was compared to predict 90-day graft loss. Predictive abilities were compared by receiver operating characteristic curves, sensitivity and specificity, and positive and negative predictive values. Results The median Donor Risk Index was 1.8. In all, 60.3% of recipients were men [median age of 54 (23-68) years]. The median POD1-7 peak-aspartate aminotransferase value was 1052 (194-17 577) U/l. The rate of early allograft dysfunction was 22.4%. The 90-day graft survival was 89.7%. Out of possible predictors of the 90-day graft loss MELD on POD5 was the best predictor of outcome (area under the curve=0.84). A MELD score of 16 or more on POD5 predicted the 90-day graft loss with a specificity of 80.8%, a sensitivity of 81.8%, and a positive and negative predictive value of 31 and 97.7%. Conclusion A MELD score of 16 or more on POD5 is an excellent predictor of outcome in ECD donor LT. Routine evaluation of POPMELD scores might support clinical decision-making and should be reported routinely in clinical trials.

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.

Der steigende Organmangel fuhrte zu einem Wandel in der experimentellen Erforschung von Organkonservierungsstrategien in der Transplantationsmedizin. Die einfache kalte Lagerung der Transplantate als Standardkonservierungsverfahren bietet fur Spenderorgane mit erweiterten Kriterien nicht immer optimale Bedingungen. Als klassische dynamische Konservierungsmethode gilt die hypotherme oxygenierte Maschinenperfusion (HMP). Durch die HMP wird das Gewebe mit Sauerstoff und Nahrstoffen versorgt und eine metabolische Erholung des Transplantats vor Implantation ermoglicht. Ein moderneres Konzept ist die normotherme Maschinenperfusion (NMP), die durch Simulation physiologischer Konditionen eine Evaluation und Behandlung des Organs vor Transplantation ermoglicht. Studien zur NMP zeigten allerdings, dass eine vorgeschaltete Periode kalter Lagerung den funktionellen Vorteil der NMP abschwacht. Das kontrolliert oxygenierte Wiedererwarmen (COR) ist eine Strategie, diesen Nachteil ausgleichen, indem das kalt gelagerte Transplantat langsam und schrittweise auf subnormotherme oder normotherme Temperaturen erwarmt wird. So kann eine schonende Adaptation des Energiestoffwechsels stattfinden und dem Wiedererwarmungsschaden vorgebeugt werden.

Background: Adequate microcirculatory perfusion is essential for the provision of oxygen to the liver following transplantation. Data from the Oxygen Persufflation in Liver Transplantation (OPAL) study (ISRCTN00167887) were analyzed from liver transplants performed at a single center to determine the role of factors affecting the hepatic microcirculation and early allograft dysfunction (EAD). Material/Methods: Retrospective data from 116 patients from the Oxygen Persufflation as Adjunction in Liver Transplantation (OPAL) study who underwent liver transplantation at a single center were analyzed. Oxygen saturation of hemoglobin (SO₂), relative capillary hemoglobin concentration (rHb), relative tissue blood flow (rBF) using laser Doppler flow measurements, and the Oxygen-to-See (O2C) spectrometry were measured and with post-transplant allograft function were analyzed using univariate and multivariate logistic regression statistics. Results: Livers donors had a median donor risk index of 1.8. Most liver transplant recipients were men (60.3%), with a median age of 54 years (IQR, 23–68 years). Mean post-transplant 3-month survival was 90.5%. The EAD rate was 22.4%, the median SO₂ was 78% (IQR, 29.5–95.8%), the median rHb was 55.6 AU (IQR, 16.8–74.8 AU), and the median rBF was 110.1 AU (IQR, 35.8–406.8 AU). Multivariate logistic regression analysis showed that tissue SO₂ (p=0.01), body mass index (BMI) of the transplant recipient (p=0.002), serum alanine transaminase (ALT) of the donor (p=0.02), and portal blood flow (p=0.01) were predictive factors for EAD. Conclusions: Non-invasive investigations of the liver microcirculation and hemoglobin oxygenation were shown to be predictive factors for EAD following liver transplantation. CA - Benkö

Short term normothermic reconditioning by machine perfusion after cold storage has shown beneficial effects in renal transplantation models. Systematic investigations concerning the inclusion of washed erythrocytes as oxygen carriers are lacking in this context. Porcine kidneys were subjected to 20 h of static cold storage. Prior to reperfusion, grafts were put on a machine for 2 h of oxygenated (95% O2 ; 5% CO2 ) rewarming perfusion. In one group (n = 6) washed erythrocytes were added to the perfusate after temperature has reached 20°C; the other group (n = 6) was run without additives. Control kidneys (n = 6) were immediately reperfused without treatment. Upon reperfusion in vitro, a more than twofold improvement of renal clearance of creatinine, urinary protein loss, fractional excretion of sodium, efficiency of oxygen utilization (TNa/VO2 ) and a significant reduction of innate immune activation (HMGB1, tenascin C, expression of TLR4) was seen after machine perfusion, compared with the controls. However, no advantage could be obtained by the addition of erythrocytes and inner cortical tissue pO2 always remained above normal values during cell-free machine perfusion. Our data strongly argue in favor of a rewarming perfusion of cold stored donor kidneys but do not substantiate an indication for adding oxygen carriers in this particular setting.

Background: Although organ shortage is a rising problem, organs from hepatitis C virus (HCV) ribonucleic acid (RNA)-positive donors are not routinely transplanted in HCV-negative individuals. Because HCV only infects hepatocytes, other organs such as kidneys are merely contaminated with HCV via the blood. In this study, we established a protocol to reduce HCV virions during the cold ischemic time. Methods: Standard virological assays were used to investigate the effect of antivirals, including methylene blue (MB), in different preservation solutions. Kidneys from mini pigs were contaminated with Jc1 or HCV RNA-positive human serum. Afterwards, organs were flushed with MB. Hypothermic machine perfusion was used to optimize reduction of HCV. Results: Three different antivirals were investigated for their ability to inactivate HCV in vitro. Only MB completely inactivated HCV in the presence of all perfusion solutions. Hepatitis C virus-contaminated kidneys from mini pigs were treated with MB and hypothermic machine perfusion without any negative effect on the graft. Human liver-uPA-SCID mice did not establish HCV infection after inoculation with flow through from these kidneys. Conclusions: This proof-of-concept study is a first step to reduce transmission of infectious HCV particles in the transplant setting and might serve as a model for other relevant pathogens.

Background The long-term outcome of intestinal transplantations is still not favorable, which is partly due to the intestinal susceptibility to ischemia. There are several indications that the inflammatory response to ischemia-reperfusion injury is mediated by cyclooxygenases and that their inhibition may be associated with improved organ function. The aim of this study was to analyze if cyclooxygenase (COX) inhibitors could improve the early posttransplant outcome after orthotopic small bowel transplantation. Methods Small bowel transplantation was performed between rats to test the impact of nonselective (Piroxicam), preferential (Meloxicam), and selective COX-2 inhibitors (Parecoxib). The donor intestines were either perfused and stored with inhibitor or had inhibitor administered intravenously after transplantation. Results Using COX inhibitors, a sequential increase of posttransplantation intestinal integrity could be shown, with Parecoxib the least effective and Meloxicam the most effective treatment. These differences were in line with the downregulation of COX-2 activity by the inhibitors. Functionally, the same tendency could be seen in diminished expression of proinflammatory molecules, decreased leucocyte inflammation, and significantly improved graft microcirculation. In most cases, the intravenous administration was more effective. However, the COX inhibitors used were shown to cause relevant hepatotoxicity under nearly all conditions, but particularly under intravenous administration. Only Meloxicam in histidine-tryptophan-ketoglutarate was demonstrated to be a safe drug without hepatotoxic side effects. Conclusions The activity of COX contributes to ischemia-reperfusion injury after intestinal transplantation. In this comparative study, the administration of the preferential COX-2 inhibitor Meloxicam via histidine-tryptophan-ketoglutarate showed the best graft-protective attributes and the lowest hepatotoxic side effects.

Background Post-transplant function of suboptimal kidney grafts can be improved but not accurately predicted by hypothermic machine perfusion. Therefore, a new concept of ex situ pre-implantation machine perfusion with controlled rewarming up to subnormothermic temperatures was developed and evaluated. Methods Porcine kidneys (n= 6/ group) were retrieved before or 30 min after cardiac arrest of the donor and subjected to 18 hours of static cold storage. In some cases 90 min of machine controlled oxygenated rewarming (COR) was added thereafter. Functional integrity was evaluated in all kidneys by subsequent normothermic reperfusion in vitro. After supplementation of the preservation solution (Custodiol-N solution + 5g/L dextran 40) with 10mg/dL creatinine, ex situ renal function was assessed by monitoring urine output, urinary creatinine and creatinine clearance at 20°C. Functional integrity was evaluated in all kidneys by normothermic reperfusion. Results COR resulted in a more than twofold improvement of post reperfusion creatinine clearance, oxygen consumption and enzyme release upon reperfusion, when compared with static cold storage. Predictive discrimination between kidneys with good or impaired function upon reperfusion based on parameters during perfusion at 4°C was only moderate. This improved significantly at 20°C. Correlation with renal clearance upon reperfusion was weak for vascular resistance at 8° (r2=0.2) and 20°C (r2=0.41). Best correlation was found for clearance measurements at 20° (r2=0.81). Conclusion Reconditioning by controlled oxygenated rewarming up to 20°C improves renal function after reperfusion and can be utilized to assess graft integrity of pre-damaged donor kidneys. This article is protected by copyright. All rights reserved.

Within the scope of transplantation research, ex vivo kidney perfusion has been proven an attractive model to study ischemia-reperfusion and preservation injury. Renal perfusion techniques also occupy scientists with the aim to optimize organ reconditioning and preparation prior to transplantation. This study investigated the influence of a pulsatile perfusion pattern that brings flow conditions closer to physiological situations, on renal perfusion characteristic and kidney function in the isolated perfused kidney. Kidneys were perfused via a roller pump at constant pressure set to 90 mmHg, or with addition of pulsatile pressure peaks (90/70 mmHg; 60/min) using an adjustable positive displacement pump. It was found that pulsatile pressure significantly enhanced renal flow rates as compared to non-pulsatile perfusion mode, especially after preceding preservation of the kidney by static cold storage. The improved vascular conductivity went along with a notable improvement of clearance of creatinine, sodium reabsorption and reduced tubular cell injury (Loss of fatty acid binding protein). The better vascular conductance upon pulsatile perfusion could be attributed to improved endothelial release of nitic oxide and reduced secretion of endothelin-1 into the perfusate. It is concluded, that pulsatile perfusion mode should be preferred in isolated kidney perfusion as resulting in better preservation/recovery of renal perfusion and function.

Summary Custodiol-N, a new preservation solution, has been shown particularly suitable for hypothermic machine perfusion preservation (HMP) in isolated porcine kidneys. These preliminary results should be confirmed in an actual transplant model in vivo. Kidney function after 21 h of HMP was studied in an autotransplant model using Landrace pigs (25–30 kg; n = 6 per group). Perfusion was performed with oxygenated perfusate, using either Custodiol-N solution including 50 g/l dextran 40 (CND) or kidney perfusion solution 1 (KPS-1) as gold standard. Viability of the grafts was followed for 1 week after bilateral nephrectomy in the recipient pigs. HMP with CND resulted in less acute tubular injury, evaluated by levels of fatty acid-binding protein and better clearance function during the first 24 h after Tx than with KPS-1 (P

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.

Organ transplantation is still affected by a notable degree of preservation-associated ischemia and reperfusion injury, which can seriously hamper early graft function. The increasing extension of the criteria for donor organ acceptance, especially for organs that have suffered from periods of warm ischemic injury prior to graft retrieval, results in even higher demands on preserving these ischemia-sensitive grafts. Growing attention is thus directed towards more dynamic preservation methods instead of simple static storage. Particularly in grafts that are retrieved after cardiac standstill of the donor, provision of oxygen to enable some kind of regenerative metabolism appears to be desirable, although the optimal temperature for oxygenated preservation/revitalization is still under debate. Hybrid solutions, comprising conventional cold storage for ease of graft procurement and transportation together with more sophisticated ‘in-house' reconditioning protocols after arrival at the implantation clinic, might help to minimize graft injury during the critical transition from preservation to reperfusion. © 2014 S. Karger AG, Basel

The concept of "controlled oxygenated rewarming" (COR) using ex vivo machine perfusion after cold storage was evaluated as tool to improve renal graft function after transplantation. Renal function after 20 min warm ischemia and 21 h cold storage was studied in an auto-transplant model in pigs (25-30 kg, n = 6 per group). In the study group, preimplant ex vivo machine perfusion for 90 min was added after cold storage, including gentle warming up of the graft to 20°C (COR). Kidneys that were only cold stored for 21 h served as controls. In vivo follow up was one week; the remaining native kidney was removed during transplantation. COR significantly improved cortical microcirculation upon early reperfusion and reduced free radical mediated injury and cellular apoptosis. Post-transplant kidney function (peak levels in serum) was also largely and significantly improved in comparison to the control group. A weak inverse correlation was found between renal flow during COR and later peak creatinine after transplantation (r

PURPOSE OF REVIEW Organ shortage in transplantation medicine forces surgical research toward the development of more efficient approaches in organ preservation to enable the application of 'less than optimal' grafts. This review summarizes current techniques aiming to recondition cold-stored organ grafts prior to transplantation to reduce reperfusion-induced tissue injury and improve postimplantation graft function. RECENT FINDINGS End-ischemic reconditioning has classically been attempted by cold oxygenated perfusion. By contrast, evaluation of graft performance prior to transplantation might be facilitated by perfusion at higher temperatures, ideally at normothermia. A drastic temperature shift from cold preservation to warm perfusion, however, has been incriminated to trigger a so-called rewarming injury associated with mitochondrial alterations. A controlled gradual warming up during machine perfusion could enhance the restitution of cellular homeostasis and improve functional outcome upon warm reperfusion. SUMMARY Machine perfusion after conventional cold storage is beneficial for ulterior function after transplantation. Cold grafts should be initially perfused at low temperatures allowing for restitution of cellular homeostasis under protective hypothermic limitation of metabolic turnover. Delayed slow rewarming of the organ might further mitigate rewarming injury upon reperfusion and also increases the predictive power of evaluative measures, taken during pretransplant perfusion.

Donor livers marginally acceptable or acceptable according to extended criteria are more frequently transplanted due to the growing discrepancy between demand and availability of donor organs. One type of marginally acceptable graft is a steatotic donor liver, because it is more sensitive to ischemia-reperfusion injury. Thus, quantitative assessment of steatosis is crucial prior to liver transplantation. Extent of steatosis of 49 pre-reperfusion liver biopsies from patients who received orthotopic liver transplantation was assessed by three techniques: semi-quantitative histological evaluation, computerized histomorphometry, and NMR-based estimation of fat content. The findings were correlated to clinical data and to histological examination of corresponding post-reperfusion biopsies for quantification of ischemia-reperfusion injury. We found that values obtained through all three assessment methods were positively correlated. None of the values obtained by the three applied methods correlated with clinical outcome or extent of ischemia-reperfusion injury. Quantitative evaluation of steatosis by NMR yields results comparable to histological and morphometrical assessment. This technique is rapid (

This clinical study evaluates end-ischemic hypothermic machine perfusion (eHMP) in expanded criteria donors (ECD) kidneys. eHMP was initiated upon arrival of the kidney in our center and continued until transplantation. Between 11/2011 and 8/2014 eHMP was performed in 66 ECD kidneys for 369 (98-912) minutes after 863 (364-1567) minutes of cold storage (CS). In 49 of 66 cases, the contralateral kidney from the same donor was preserved by static CS only and accepted by another Eurotransplant (ET) center. Five (10.2%) of these kidneys were ultimately judged as "not transplantable" by the accepting center and discarded. After exclusion of early unrelated graft losses, 43 kidney pairs from the same donor were eligible for direct comparison of eHMP vs CS only: primary non-function and delayed graft function (DGF) were 0% vs 9.3% (P=.04) and 11.6% vs 20.9% (P=.24). There was no statistically significant difference in 1-year graft survival (eHMP vs CS only: 97.7% vs 88.4%, P=.089). In a multivariate analysis, eHMP was an independent factor for prevention of DGF (OR: 0.28, P=.041). Development of DGF was the strongest risk factor for 1-year graft failure (Renal resistance: 38.2, P

Background Hypothermic machine perfusion (HMP) for preservation led to compelling success for outcomes of renal transplantation. Application of different concentrations of oxygen during renal HMP has not been systematically analyzed. This study investigates the aspects of renal function and morphology in dependence of oxygen concentrations during HMP in a porcine donation after circulatory death model. Methods After 30 min of warm ischemia, porcine kidneys were randomly assigned to preservation for 21 hr by HMP without oxygenation (HMPnoox), oxygenated HMP with air (HMPair), or 100% oxygen (HMPox100%). Afterward, kidneys were reperfused for 2 hr in an ex vivo model for assessment of function and integrity. Results Application of HMPox100% led to significantly increased blood flow during reperfusion compared to HMPnoox. Preservation by HMPox100% led to a doubling of creatinine clearance after 90 and 120 min of reperfusion (13.4 and 12.0 mL/min) compared to preservation by HMPnoox (7.3 and 7.7 mL/min; P=0.01). Oxygenated HMP with air led to results between the two other groups. Fractional excretion of sodium demonstrated a strong tendency of higher values after HMPnoox compared to HMPox100% (P=0.096) and HMPair (P=0.09). Analysis of structural integrity during reperfusion demonstrated significantly higher values of lactate dehydrogenase resembling cell damage (P=0.02), higher values of gamma-glutamyl-transferase (gGT) resembling tubulus injury (P=0.048), and more pronounced tubular dilatation (P=0.02) after HMPnoox compared to HMPox100%. Conclusion The present study demonstrates that application of HMPox100% for kidneys from donations after circulatory death results in better renal function during early reperfusion compared to HMPnoox.

Background: Ischemia/reperfusion injury (IRI) is one of the major clinical problems in liver and transplant surgery. Livers subjected to warm ischemia in vivo often show a severe dysfunction and the release of numerous inflammatory cytokines and arachidonic acid metabolites. Cyclooxygenase (COX)-2 is the inducible isoform of an intracellular enzyme that converts arachidonic acid into prostaglandins. The aim of the study was to evaluate the effect of COX-2 inhibition and the role of Kupffer cells in IRI of the liver. Methods: Male Wistar rats [250- 280 g body weight (BW)] were anesthetized and subjected to 30-min warm ischemia of the liver (Pringle's maneuver) and 60-min reperfusion after median laparotomy. The I/R group received no additional treatment. In the COX-2 inhibitor (COX-2I) group, the animals received 1 mg/kg BW meloxicam prior to operation. Gadolinium chloride (GdCl3) (10 mg/kg BW) was given 24 h prior to operation in the GdCl3 and GdCl3 + COX-2I groups for the selective depletion of Kupffer cells. The GdCl3 + COX-2I group received both GdCl3 and meloxicam treatment prior to operation. Blood and liver samples were obtained at the end of the experiments for further investigations. Results: After 30 min of warm ischemia in vivo, severe hepatocellular damage was observed in the I/R group. These impairments could be significantly prevented by the selective COX-2 inhibition and the depletion of Kupffer cells. Alanine aminotransferase was significantly reduced upon meloxicam and GdCl3 treatment compared to the I/R group: I/R, 3,240 ± 1,262 U/l versus COX-2I, 973 ± 649 U/l, p < 0.001; I/R versus GdCl3, 1,611 ± 600 U/l, p < 0.05, and I/R versus GdCl3 + COX-2I, 1,511 ± 575 U/l, p < 0.01. Plasma levels of tumor necrosis factor alpha (TNF-α) were significantly reduced in the COX-2I treatment group compared to I/R (3.5 ± 1.5 vs. 16.3 ± 11.7 pg/ml, respectively; p < 0.05). Similarly, the amount of TxB2, a marker for COX-2 metabolism, was significantly reduced in the meloxicam treatment groups compared to the I/R group: I/R, 22,500 ± 5,210 pg/ml versus COX-2I, 1,822 ± 938 pg/ml, p < 0.001, and I/R versus GdCl3 + COX-2I, 1,530 ± 907 pg/ml, p < 0.001. All values are given as mean ± SD (n = 6). Conclusion: These results suggest that the inhibition of COX-2 suppressed the initiation of an inflammatory cascade by attenuating the release of TNF-α, which is an initiator of the inflammatory reaction in hepatic IRI. Therefore, we conclude that preferential inhibition of COX-2 is a possible therapeutic approach against warm IRI of the liver.

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 Kruppel-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.

The quality of cold-stored livers declines with the extension of ischemic time, increasing the risk of primary dys or nonfunction. A new concept to rescue preserved marginal liver grafts by gentle oxygenated warming-up prior to blood reperfusion was investigated. Porcine livers were preserved by cold storage (CS) in modified HTK-solution for 18 h. Some grafts were subsequently subjected to 90 min of controlled oxygenated rewarming (COR) by machine perfusion with gradual increase of perfusate temperature up to 20°C or simple oxygenated machine perfusion in hypothermia (HMP) or subnormothermia (SNP). Graft viability was assessed thereafter by 4 h of normothermic blood reperfusion ex vivo. Endischemic tissue energetics were significantly improved by COR or SNP and to a notably lesser extent by HMP. COR significantly reduced cellular enzyme loss, gene expression and perfusate activities of TNF-alpha, radical mediated lipid peroxidation (LPO) and increase of portal vascular perfusion resistance upon reperfusion, while HMP or SNP were less protective. Only COR resulted in significantly more bile production than after CS. Histological injury score and caspase 3-activation were significantly lower after COR than after CS. Oxygenated rewarming prior to reperfusion seems to be a promising technique to improve subsequent organ recovery upon reperfusion of long preserved liver grafts.

Background: Owing to the shortage of donors, organs with an increased risk potential such as grafts recovered from expanded criteria donors are increasingly being used in transplants. Machine perfusion (MP) technology offers the possibility of determining the biomarkers in the perfusion solution so that conclusions might be drawn regarding the effectiveness of organ preservation and organ viability.Methods: All kidneys from the MP arm of our multicenter, randomized, controlled trial of MP whose donors were aged 55 years or older were included in the present study. The biomarkers, total glutathione-S-transferase (GST), alpha-GST, and pi-GST and markers for lipid peroxidation and cell decay were determined in the MP perfusate and correlated with the outcomes after kidney transplantation.Results: A total of 111 machine perfused kidneys were included in the present study. The mean donor age was 64.1 +/- 6.6 y. The average cold ischemic time was 13.8 +/- 5.3 h. Total GST, alpha-GST, and lipid peroxidation markers were significantly elevated at the end of MP. However, according to the multivariate analysis, only the lipid peroxidation markers were independent predictors of delayed graft function after transplantation.Conclusions: Our data suggest that routine determination of lipid peroxidation markers in the perfusate of expanded criteria donor kidneys can provide the opportunity to identify the kidneys that have sustained severe oxidative damage and should be excluded from transplantation. Additional analysis of a larger cohort with more primary nonfunction cases is needed to confirm these findings. (c) 2013 Elsevier Inc. All rights reserved.

OA gold

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 = 6) or NMP (n = 5) and compared to standard CS. COR was carried out with the new Custodiol-N solution, slowly increasing temperature from 8 °C to 20 °C during the first 90 minutes. NMP was carried out with diluted autologous blood at 37 °C 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

Abrupt temperature shift from hypothermia to normothermia incurred on reperfusion of organ grafts has been delineated as a genuine factor contributing to reperfusion injury and graft dysfunction after transplantation.In a first clinical series of 6 patients, cold-stored livers, all allocated by the rescue offer mechanism by Eurotransplant, were subjected to machine-assisted slow controlled oxygenated rewarming (COR) for 90 minutes before engrafting. A historical cohort of 106 patients basically similar in graft (all rescue offer organs) and recipient factors was used for comparison.The clinical benefit of COR was documented by a significant reduction by approximately 50% in peak serum transaminases after transplantation compared to untreated controls (AST 563.5 vs. 1204 U/L, P = 0.023). After 6 months graft survival was 100% in the COR group and 80.9% in the controls (P = 0.24). Respective patient survival was 100% and 84.7% (P = 0.28). Real-time assessment of glucose concentration in the perfusion solution correlated well with postoperative synthetic graft function (r = 0.78; P0.02). All treated recipients had normal liver function after a 6-month follow-up and are well and alive.This first clinical application suggests that controlled graft rewarming after cold storage is a feasible and safe method in clinical praxis and might become an adjunct in organ preservation.

Myocardial ischemia leads to energetic, morphologic, metabolic, and functional alterations. To evaluate differences in ischemia tolerance between neonatal and adult hearts, we investigated gap junction uncoupling (GJU) and its correlation to myocardial intracellular edema formation during normothermic ischemia.Hearts of landrace piglets (neonates, 7.4 ± 1.9 days of age, body weight 2.9 ± 0.5 kg, n = 5 and adults, 84 ± 9 days of age, body weight 30.5 ± 3.9 kg, n = 5) were investigated. After we harvested the hearts, the bioelectrical impedance spectra were measured continuously during normothermic global ischemia (35°C). Spectra of the dielectric permittivity, ε'(frequency), and conductivity, σ(frequency), were calculated from the impedance measurements, and GJU was identified in the sigmoidal time course of ε' (13 kHz). The extracellular volume was estimated by the ratio σ (100Hz)/σ (1MHz). Dielectric data were correlated with electron-microscopical images.Intraischemic GJU was observed in neonates after 54 ± 9 minutes of ischemia and thus significantly earlier than in adults (90 ± 7 minutes, P .05). A more than 20% increase of intercalated water was found in tissue samples of neonates after 20 ± 2 minutes, in contrast to adults after 137 ± 8 minutes (P .05).Intraischemic formation of edema and earlier GJU indicate faster intraischemic changes in neonates compared with adults. Intraischemic GJU and determination of intracellular water shifts are an experimental approach to establish the period of life-threatening damage. Because both parameters are linked and occur significantly earlier in neonates, they distinctly demonstrate the lower ischemia tolerance of neonatal hearts as both events interact.

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 5 ml/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 2 h. 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.