Cold flush after dynamic liver preservation protects against ischemic changes upon reperfusion - an experimental study
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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.Keywords:
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Ex vivo
Warm ischemia
Adenosine triphosphate
Abstract Background Non-heart-beating donor (NHBD) livers represent an important organ pool, but are seldom utilized clinically and require rapid retrieval and implantation. Experimental work with oxygenated perfusion during preservation has shown promising results by recovering function in these livers. This study compared sanguinous perfusion with cold storage for extended preservation of the NHBD liver in a porcine model. Methods Porcine livers were subjected to 60 min of in vivo total warm ischaemia before flushing, after which they were preserved by one of two methods: group 1 (n = 4), University of Wisconsin (UW) solution by standard cold storage for 24 h; group 2 (n = 4), oxygenated autologous blood perfusion on an extracorporeal circuit for 24 h. All livers were subsequently tested on the circuit during a 24-h reperfusion phase. Results Livers in group 1 showed no evidence of viability during the reperfusion phase with no bile production or glucose utilization; they also displayed massive necrosis. Livers in group 2 demonstrated recovery of function by synthetic function, substrate utilization and perfusion haemodynamics; these livers displayed less cellular injury by hepatocellular enzymes. All differences in parameters between the two groups were statistically significant (P < 0·05). These findings were supported by histological examination. Conclusion Warm ischaemia for 1 h and simple cold storage (UW solution) for 24 h renders the liver non-viable. Oxygenated, sanguinous perfusion as a method of preservation recovers liver function to a viable level after 24 h of preservation.
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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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Waiting lists for transplantation have stimulated interest in the use of non-heart-beating donor (NHBD) organs. Recent studies on organ preservation have shown advantages of machine perfusion (MP) over cold storage (CS). To supply the liver with specific nutrients during MP, the preservation solution Polysol was developed. The aim of our study was to compare CS in University of Wisconsin solution (UW) with MP using UW-gluconate (UW-G) or Polysol in an NHBD model. After 30 minutes of warm ischemia, livers were harvested from rats for preservation by either CS, MP-UW-G, or MP-Polysol. After 24 hours of preservation, livers were reperfused with Krebs-Henseleit buffer (KHB). Perfusate samples were analyzed for liver damage and function. Biopsies were examined by hematoxylin and eosin staining and transmission electron microscopy. Liver damage was highest after CS compared with the MP groups. MP using Polysol compared with UW-G resulted in less aspartate aminotransferase (AST) and alanine aminotransferase (ALT) release. Perfusate flow, bile production, and ammonia clearance were highest after MP-Polysol compared with CS and MP-UW-G. Tissue edema was least after MP-Polysol compared with CS and MP-UW-G. In conclusion, preservation of the NHBD rat liver by hypothermic MP is superior to CS. Furthermore, MP using Polysol results in better-quality liver preservation compared with using UW-G. (Liver Transpl 2005;11:1379–1388.)
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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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Warm ischemia
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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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