Hypothermic continuous machine perfusion improves metabolic preservation and functional recovery in heart grafts
Olivier Van CaenegemChristophe BeauloyeJonathan VercruysseSandrine HormanLuc BertrandNoëlla BethuyneAlain PonceletPierre GianelloPeter DemuylderE. LegrandGwen BeaurinFrançoise BontempsLuc JacquetJean‐Louis Vanoverschelde
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Abstract In transplantation, livers are transported to recipients using static cold storage (SCS), whereby livers are exposed to cold ischemic injury that contribute to post-transplant risk factors. We hypothesized that flushing organs during procurement with cold preservation solutions could influence the number of donor blood cells retained in the allograft thereby exacerbating cold ischemic injury. We present the results of rat livers that underwent 24h SCS after being flushed with a cold University of Wisconsin (UW) solution versus room temperature (RT) lactated ringers (LR) solution. These results were compared to livers that were not flushed prior to SCS and thoroughly flushed livers without SCS. We used viability and injury metrics collected during normothermic machine perfusion (NMP) and the number of retained peripheral cells (RPCs) measured by histology to compare outcomes. Compared to the cold UW flush group, livers flushed with RT LR had lower resistance, lactate, AST, and ALT at 6 hours of NMP. The number of RPCs also had significant positive correlations with resistance, lactate, and potassium levels and a negative correlation with energy charge. In conclusion, livers exposed to cold UW flush prior to SCS appear to perform worse during NMP, compared to RT LR flush.
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Background. Hypothermic oxygenated perfusion (HOPE) improves outcomes of marginal liver grafts. However, to date, no preservation solution exists for both static cold storage (SCS) and HOPE. Methods. After 30 min of asystolic warm ischemia, porcine livers underwent 6 h of SCS followed by 2 h of HOPE. Liver grafts were either preserved with a single preservation solution (IGL2) designed for SCS and HOPE (IGL2-Machine Perfusion Solution [MPS] group, n = 6) or with the gold-standard University of Wisconsin designed for for SCS and Belzer MPS designed for HOPE (MPS group, n = 5). All liver grafts underwent warm reperfusion with whole autologous blood for 2 h, and surrogate markers of hepatic ischemia–reperfusion injury (IRI) were assessed in the hepatocyte, cholangiocyte, vascular, and immunological compartments. Results. After 2 h of warm reperfusion, livers in the IGL2-MPS group showed no significant differences in transaminase release (aspartate aminotransferase: 65.58 versus 104.9 UI/L/100 g liver; P = 0.178), lactate clearance, and histological IRI compared with livers in the MPS group. There were no significant differences in biliary acid composition, bile production, and histological biliary IRI. Mitochondrial and endothelial damage was also not significantly different and resulted in similar hepatic inflammasome activation. Conclusions. This preclinical study shows that a novel IGL2 allows for the safe preservation of marginal liver grafts with SCS and HOPE. Hepatic IRI was comparable with the current gold standard of combining 2 different preservation solutions (University of Wisconsin + Belzer MPS). These data pave the way for a phase I first-in-human study and it is a first step toward tailored preservation solutions for machine perfusion of liver grafts.
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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.
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Potential conflict of interest: Nothing to report. To the Editor: We read with great interest the paper by Jassem et al.,1 which provides detailed gene expression profiles of human livers during normothermic machine perfusion (NMP), compared with conventional cold storage. The authors should be congratulated on this extensive analysis, which shows for the first time transcriptional changes during any form of reperfusion under normothermic conditions.1 However, we would like to add a few critical comments. The authors analyzed liver samples at the end of cold storage or NMP, and after graft implantation. First, although changes in gene alteration during NMP are certainly relevant, we would like to mention that all livers in the NMP group underwent prolonged normothermic reperfusion on the device before the first biopsy, obtained after a considerable contact of liver cells with fully oxygenated blood. Another biopsy, for example, 1 hour after the start of NMP, would be important to confirm the hypothesis that NMP is in fact actively leading to down‐regulation of inflammatory pathways (Fig. 1). Importantly, for kidneys, lungs, hearts, and livers, current research has shown the opposite.2 Second, proper comparisons of NMP and cold storage require assessment of reperfusion injury after a similar duration of normothermic reperfusion, either on the NMP device or after reperfusion of cold stored livers in situ.1 (Fig. 1) Expectedly, gene expression profiles between NMP and cold storage appear very different, and parallel previous assessments after transplantation of subnormothermically perfused livers.4 Third, the results imply that NMP after cold ischemia triggers inferior outcome.1 Consistently, Watson et al. have demonstrated a clear inflammatory response when NMP was performed after significant cold storage in livers with a cumulative high risk.5 It may therefore be important to emphasize that especially high‐risk livers need either upfront NMP or other machine perfusion techniques targeting metabolic changes before implantation, such as hypothermic oxygenated perfusion or controlled oxygenated rewarming.2Figure 1: Overview of liver preservation and timepoints of gene expression profile analysis comparing upfront normothermic machine perfusion and cold storage.Based on this, we believe that the presented data show a minimization of inflammation and necrosis through replacement of cold ischemia by sophisticated NMP techniques. However, we do not think the data support the conclusion that NMP itself leads to down‐regulations of inflammatory pathways.
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End-stage liver diseases are nowadays effectively treated by transplantation of the affected liver. The transplantation procedure includes procurement of the liver from the donor and subsequently transport of the liver from donor to receiving patient (Chapter 1). To bridge the timespan of transport between donor operation and actual implantation of the organ in the receiving patient, the liver has to be optimally stored and preserved in order to maintain viability of the organ. To date, the conventional method of preservation is the Cold Storage (CS) preservation technique. The CS method implies a single flush of the liver in situ with an ice-cold preservation solution to wash-out remaining blood and immediately cool the organ. Subsequently, the liver is stored in a plastic bag containing cold preservation solution and transported in a cooling box filled with melting ice to maintain a lowered metabolism during hypothermia (0-4±C). The University of Wisconsin cold storage (UW-CS) solution is nowadays the golden standard in preservation solutions. Although CS preservation shows good results in preserving livers from brain-dead donors, who have an intact circulation, expansion of the donor pool with an important potential group of non-heart-beating donors (NHBDs), after cardiac arrest, requires improved preservation techniques. Hypothermic machine perfusion (HMP) is a dynamic preservation method that actively perfuses the liver. With HMP a continuous supply of oxygen and removal of waste products is obtained which improves preservation outcome. Especially marginal, older and NHB donor livers will benefit from this improved quality. The aim of this thesis was to develop a hypothermic machine perfusion system which is able to optimally preserve donor livers.
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Abstract In transplantation, livers are transported to recipients using static cold storage (SCS), whereby livers are exposed to cold ischemic injury that contribute to post-transplant risk factors. We hypothesized that flushing organs during procurement with cold preservation solutions could influence the number of donor blood cells retained in the allograft thereby exacerbating cold ischemic injury. We present the results of rat livers that underwent 24 h SCS after being flushed with a cold University of Wisconsin (UW) solution versus room temperature (RT) lactated ringers (LR) solution. These results were compared to livers that were not flushed prior to SCS and thoroughly flushed livers without SCS. We used viability and injury metrics collected during normothermic machine perfusion (NMP) and the number of retained peripheral cells (RPCs) measured by histology to compare outcomes. Compared to the cold UW flush group, livers flushed with RT LR had lower resistance, lactate, AST, and ALT at 6 h of NMP. The number of RPCs also had significant positive correlations with resistance, lactate, and potassium levels and a negative correlation with energy charge. In conclusion, livers exposed to cold UW flush prior to SCS appear to perform worse during NMP, compared to RT LR flush.
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P25 Aims: Hypothermic machine perfusion (HMP) provides better protection against cold-ischemic injury than cold-storage in marginal donor kidneys. Also, in liver transplantation a switch from static cold-storage to HMP could be beneficial as it would allow longer preservation times and the use of marginal donors. A critical question concerning application of HMP in liver preservation is the crucial balance between perfusion pressure and occurrence of endothelial injury. Methods: Rat livers were preserved using static cold-storage or continuous perfusion with the appropriate UW preservation solution. Cold-storage was compared to HMP preserved livers using an arterial perfusion pressure of 25 mmHg (mean) and a portal perfusion pressure of 4 mmHg (low pressure group), and to HMP at 50 mmHg and 8 mmHg perfusion, respectively (high pressure group). To stain for dead cells, the UW solution was enriched with 14.9 μM propidium iodide (PI), and with an additional 13.5 μM acridine orange, to stain for viable hepatocytes. After 24 h preservation the amount and anatomical localization of PI positive cells were assessed. Acridine orange (AO) used to stain viable hepatocytes, and the RECA-1 (rat endothelial cell antibody-1) and ED-1 (Kupffer cell marker) antibodies were used to identify which cells were PI positive. ATP levels were determined as a viability marker and to confirm that higher energy levels can be obtained with HMP than with cold-storage. Results: All livers in the cold-storage group and both HMP groups were completely perfused as shown with AO staining. Cold-storage preserved livers showed 75.1+/-6.2 dead PI positive cells per microscopic field. PI staining in the low-pressure group showed 64.4+/-7.8 and in the high-pressure group showed 93.4+/-5.9 PI positive cells per microscopic field. PI positive cells were non-parenchymal cells and correlated with the pattern found for RECA-1. The results for ED-1 staining did not correspond with the pattern found for PI positive cells. ATP levels were low after cold-storage (1.2 +/- 0.5) and significantly higher after HMP i.e. 44.5+/-5.9 (low-pressure) and 36.5+/-2.8 pmol/μg-protein (high-pressure), respectively. Conclusions: This study showed better ATP levels for HMP preserved livers in comparison to CS livers. The perfusion pressure showed a low PI-positive cell count in comparison to CS, when the HMP technique was used at a low perfusion pressure. A high HMP perfusion pressure resulted in more PI-positive cells compared to CS, indicating that the perfusion pressure is critical for HMP preservation of the liver. The PI-positive cells were found in the portal triad, sinusoids and central veins and were non-parenchymal in origin. The staining pattern with RECA-1 demonstrated that these non-parenchymal cells can be identified as endothelial cells. HMP preservation is effective for the liver with low-pressure perfusion and it is crucial to fine-tune the perfusion pressure to prevent endothelial injury.
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The UW solution effectively preserves the dog liver for up to 48 hr by simple cold storage. This solution contains lactobionate as the primary impermeant. Another solution developed for machine perfusion of the kidney is similar to the UW solution but contains gluconate in place of lactobionate. In this study the UW gluconate solution was used for the continuous hypothermic machine perfusion of dog livers for 72 hr. Dog livers were continuously perfused at 5°C through the portal vein at a pressure of 16–18 mm Hg and transplanted. Seven of 8 dogs survived for 7 or more days following orthotopic transplantation. The livers functioned as well as those preserved for 48 hr by cold storage in the UW solution as indicated by various liver-function tests. Successful machine perfusion was only achieved when the perfusate contained a high concentration of potassium (125 mM) but not with a high concentration of sodium (125 mM). This study demonstrates the feasibility of machine-perfusion preservation of the liver that yields longer preservation of equal quality compared to simple cold storage. For the development of truly long-term preservation (5 or more days) and better quality short-term preservation, machine perfusion may be the method of choice.
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