In a rat model of orthotopic liver transplantation, 4 (66%) of 6 animals that received DCD grafts (30 minutes of warm ischemia + 4 hours of cold ischemia) preserved in UW + PFC solution survived long term (>100 days), whereas all animals of the control group died within 24 hours after liver transplant (P = 0.0005). Two animals of the PFC + UW group survived for more than 6 months (Figure 9).
It has been suggested that oxygenation during organ storage is beneficial to extended criteria organs.1,45 During liver machine preservation, oxygenation of the perfusate reduces alanine aminotransferase compared with controls. This also resulted in reduced oxygen free radical-mediated lipid peroxidation upon reperfusion and activated the AMP salvage pathway. Enzyme release during reperfusion was reduced by 70% with additional oxygenation compared with controls. Functional recovery (bile production) was enhanced by approximately twofold with high oxygenation of the perfusate.22
Our group and others have demonstrated that liver metabolism can be optimized before transplantation to improve postoperative outcome.2,5,6,23-27,40,46 Energy repletion processes during preservation are correlated with resumption of normal metabolic function of the liver.5,20,23-26,28 We show that ATP stores drop dramatically after warm ischemia, and that subsequent addition of PFC mitigates this drop in ATP levels.
Oxygen carriers have been used in experimental models of preservation of various organs (eg, kidney, liver, pancreas, heart, lung, and intestine),13,15,17,18,29,42,43 and in clinical transplantation studies of kidney and pancreas.8,30 These studies show that PFC oxygenation increased graft tolerance to ischemia.44 When the pancreas is preserved in PFC-based preservation solution, the organ continuously generates ATP for up to 96 hours,18 which it uses to maintain cell integrity. Thus, PFC-based solution prevents pancreas swelling more effectively than UW solution alone.31,32,41 Furthermore, it improves the viability of vascular endothelium and microcirculation.33 In a rat small-bowel transplantation model, PFC-based solution successfully preserved grafts twice as long as UW storage only.15 Similar results were seen in a canine small bowel model and a rat heart transplant model. Seven of 8 recipients of small bowel preserved with UW solution died within 3 days, whereas grafts preserved in PFC + UW solution survived.16,34 Kuroda et al33 showed that after 90 minutes of warm ischemic injury, the canine pancreas grafts lost ATP and were no longer viable. However, when the damaged pancreas was resuscitated by PFC-based solution for 24 to 48 hours at 4°C, the grafts regained viability.33
PFC-based solutions are simpler to use and cheaper than oxygenated machine preservation. To our knowledge, there is no report of liver preservation with PFC-based solutions in the clinical setting. However, in a rat model, Bezinover et al13 reported that when livers were flushed with PFC added to the preservation solution and stored for 8 hours, they showed less damage based on aspartate aminotransferase levels, histology score, and caspase-3 expression by immunohistochemistry.13 In another study, Bezinover et al showed that preservation of ischemic rat liver grafts with oxygenated UW solution (with or without PFCs) produces superior preservation of the graft, based on the pattern of hepatic gene expression, intracellular fat score, degree of caspase-3 activation, as well as the adenosine diphosphate/ATP ratio when compared with standard storage in UW solution without oxygenation.14 Another study using a rat DCD model showed that liver grafts subjected to 30 minutes of warm ischemia after cardiac arrest followed by 18 hours of cold ischemia could be reconditioned by gaseous oxygenation (persufflation) in the first 2 hours of cold storage.35 However, the last 3 studies were not performed in a transplantation model. Our results are consistent with more recent rat liver transplant experiments performed by Okumura et al.19 With regard to the diffusion of O2 through our perfusion system, we hypothesize that O2 preferentially flows from the area of highest affinity to lowest in the graft. In PFC alone after O2 infusion, a PaO2 of approximately 600 mm Hg was measured, compared with nearly 900 mm Hg in PFC + UW. Because PFC and UW do not homogenize, the graft remains immersed in UW and is fed by O2 moving from the PFC phase, through the UW and finally into graft tissue across the capsule. However, due to the passive nature of the O2 diffusion, we recognize that deeper parenchymal tissue may be unequally exposed as those nearest the surface of the organ. DCD donor livers preserved in 20% preoxygenated UW solution for 3 hours before transplant showed a significant reduction in serum malondialdehyde levels (P = 0.03) compared with control livers perfused in UW alone, indicating reduced oxidative stress.19 Furthermore, histological examination of livers treated with preoxygenated UW demonstrated less focal necrosis and architecture loss plus significantly improved Suzuki ischemia reperfusion injury scores (P < 0.001) compared with controls.19 The authors argue that reduced oxidative stress likely attenuated hepatocyte mitochondrial swelling, thereby mitigating histological damage. Biliary damage assessed with transmission electron microscopy showed preserved microvilli in the canaliculi of their UW + PFC group with significant loss of microvilli in controls. We also observe blunting of canaliculi in mouse bile ducts with UW only perfusion in contrast to UW + PFC. However, as neither this study nor ours investigated biliary complications after transplant — a major problem in using DCD grafts in humans — we are limited in drawing conclusions regarding biliary function after using PFC. In terms of overall survival after transplant, Okumura et al19 show that DCD graft preservation in UW + PFC increased the 14-day survival from 28.6% to 85.7% (P = 0.02). Interestingly, as in our study, mortality of control animals with plain UW preservation grafts was striking.
Most of the experience with PFC-based solution was obtained with respect to the pancreas. Currently, PFC is used by a number of centers in pancreatic islet preservation.8 For whole-pancreas preservation, it was clinically used for the first time in 1999 at the University of Minnesota. In the first clinical trial of 10 pancreas transplants, no adverse effects on the recipients after transplantation were reported.8 In a clinical study with 58 DCD kidney grafts flushed with 20% PFC added to supplemented UW solution, Reznik showed that the incidence of delayed graft function was reduced by 30% and serum creatinine was half that of the control group 21 days after transplant.30
Perfluorocarbons are biologically inert and cannot bind to any protein or enzyme.10 Intravenous PFC forms emulsions with other agents and is cleared from the blood through phagocytosis by reticuloendothelial macrophages before ultimately being eliminated through the lungs approximately 4 to 12 hours after infusion. Although PFC use for organ or islet transplantation has not been associated with side effects,9 larger volumes of IV PFC can elicit flu-like symptoms and cutaneous flushing in rare cases. These effects are reversible.36 In the 1980 to 1990s, PFCs were pursued as “blood substitutes.” In 1989, the United States Food and Drug Administration approved the PFC emulsion Fluosol-DA-20% for IV use.37 Fluosol was approved as an “oxygen therapeutic” for treatment of myocardial ischemia at the time of balloon angioplasty.37 At least 15 000 patients received Fluosol-DA-20%.36 For organ transplantation, the risks of PFC therapy are even more negligible once the graft is flushed before transplantation to remove the potassium rich UW preservation solution.
One limitation of static PFC oxygenation is that oxygenation is done passively by diffusion from the oxygen-rich (PFC) compartment in the bottom of the solution to the lower oxygen compartment (UW solution + graft) on the top. This creates a gradient of oxygen delivery that is not equally divided over the organ.38 Another limitation of our study is that we could not assess long-term complications of ischemic cholangiopathy because the transplant model we used is not arterialized (technique not available in our laboratory because of technical challenges). This creates a challenge in assessing the contribution of this organ preservation method on biliary complications.39 In addition, no rat in the control group survived more than 1 day to allow meaningful comparison.
In conclusion, we show herein that PFC addition to UW preservation solution slows the decrease of ATP, thus reducing tissue damage/apoptosis and improving posttransplant survival in a rat liver transplant model. This suggests that a PFC based preservation solution may extend tolerance to ischemic damage and open a wider pool of extended criteria donation donors in human transplant.
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