Historically, continuous oxygenation was considered a crucial component of organ preservation by maintaining cellular homeostasis and preventing cell damage.1 However, with the introduction of the more sophisticated preservation solutions (eg, University of Wisconsin [UW] solution), organ preservation without additional oxygenation was possible and became the standard of organ preservation. Although the evidence comparing oxygenated with nonoxygenated preservation is limited, it seems that oxygenation may be particularly beneficial in extended criteria donation, such as donation after circulatory death (DCD). These organs have a higher likelihood to develop primary nonfunction and are predisposed to ischemic cholangiopathy.1-4 It has been proposed that supplementation of oxygen during organ preservation may support oxidative phosphorylation thus driving adenosine triphosphate (ATP) production. Cells then use ATP to uphold metabolic processes that protect the cell from ischemic damage.5,6 Novel preservation strategies may allow utilization of a larger pool of potential donors.
Perfluorocarbons (PFC) are biologically inert substances with a high solubility for oxygen approximately a hundred times higher than plasma. The amount of oxygen dissolved in plasma is less than 1% of the total amount of oxygen in the blood, and the addition of PFC increases the content of dissolved oxygen to 10% to 15%.7 A negligible O2-binding constant of PFC also allows the release of oxygen into the surrounding tissue more effectively than hemoglobin.8 PFC-dissolved oxygen is characterized by a high extraction ratio, making it immediately available to tissues. Importantly, O2 dissociation and tissue release are maintained at low temperatures.9 Another advantage of PFC is its small particle size. Varying from 1/10th to 1/100th the size of an erythrocyte, PFC has a larger relative surface-to-volume ratio, which contributes to better oxygenation of the microvasculature.10 This is of particular importance to DCD grafts because the microvasculature can be compromised by microthrombi in the setting of circulatory death.11 As a result of this unique combination of properties, PFC-based solutions have been examined as an oxygen carrier for blood substitutes, myocardial protection, ventilatory support, cell culture, and organ preservation before transplantation.12
PFC-based preservation solutions have been used in many models of organ preservation, including ischemic livers.13-18 In addition, it has been investigated in an animal liver transplant model.19 The aim of this study was to optimize oxygen delivery during the cold storage period by adding PFC to standard UW preservation solution and confirm this using a rat liver transplant model. It is known that the period of warm ischemia associated with DCD exhausts ATP stores. We hypothesize that the addition of PFC may allow increased oxidative phosphorylation during cold storage, resulting in a more effective ATP recovery upon transplantation. This can alleviate ischemia reperfusion injury to the liver parenchyma and biliary tree and result in better outcomes posttransplantation.
MATERIALS AND METHODS
Perfluorocarbon (perfluorodecadolin; FluoroMed L.P., Round Rock, TX) was used in the concentration of 20% v/v and was preloaded with oxygen (1 L/min) through direct bubbling for 30 minutes before preservation. Because the PFC has high density and does not mix with UW solution, the graft was placed at the interface between PFC and UW solution (double layer method).9
Animals received humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Massachusetts General Hospital. After circulatory arrest livers from C57/B6 mice were flushed with UW solution and stored in either preoxygenated UW solution or UW + PFC solution in 3 different temperature groups (4°C for 24 hours, 37°C for 60 minutes, and 37°C for 45 minutes followed by 36 hours at 4°C; Figure 1). The preoxygenation time was 30 minutes with an O2 rate of 1 L/min. We used PFC at 20% v/v (3 mL PFC + 12 mL UW solution) versus 15 mL of UW in the control group. Storage of grafts at 37°C for 45 minutes followed by 36 hours at 4°C was used to simulate the warm ischemia experienced by DCD donors.
Liver biopsies were taken for analysis of tissue ATP content at different time points as described before (every 15 minutes in the warm ischemia group and every 2 hours in the first 12 hours, then every 6 hours to 36 hours in the cold ischemia group).5 Immediately after biopsy, the samples were snap frozen in liquid nitrogen and stored at −80°C. Before homogenization of the tissue, biopsies were mechanically pulverized in liquid nitrogen. Pulverized tissue was analyzed for ATP content using a luminescence-based cell viability assay (BioVision Inc.). Luminescence was measured using a single-tube detector (Molecular Devices, Sunnyvale, CA). ATP content was normalized to protein content by the Bradford method, and measured spectrophotometrically after reaction with Coomassie dye.
Rat Orthotopic Liver Transplantation
All animals were transplanted by an experienced microsurgeon (T.A.B.) according to the established protocol by Kamada et al and optimized by Delriviére et al,20,21 and were divided in 2 groups differing only by the presence of PFC in the UW solution (n = 6/group; Figure 2). Male Lewis rats weighing 250 to 300 g (Charles River Laboratories, Boston, MA) were used as donors and recipients. The animals were maintained in accordance with National Research Council guidelines, and the experimental protocols were approved by the Subcommittee on Research Animal Care, Committee on Research, Massachusetts General Hospital. All surgical procedures were performed under aseptic conditions. The animal was anesthetized with 5% isoflurane induction, followed by 1% to 2% maintenance (Baxter, Deerfield, IL). Donor animals received heparin (1000 units intravenously [IV]) before opening of the chest and exsanguination of the animal until cardiac arrest occurred. We left the liver in situ for 30 minutes (period of warm ischemia) and then flushed it either with oxygenated UW or with oxygenated UW + PFC. After that, the liver was removed and placed in a container containing UW solution or UW + PFC at 4°C for an additional 4 hours (period of cold ischemia). Both solutions were preoxygenated for 30 minutes before storage of the graft. Grafts were flushed with normal saline to remove the preservation solutions before transplantation. Livers were transplanted using the cuff technique and without arterial anastomosis because an arterialized model was not available in our laboratory.20
Liver parenchyma biopsies were fixed in 10% formalin. After paraffin embedding, samples were sectioned and stained with hematoxylin and eosin. We used cytokeratin 19 antibody (sc-33119; Santa Cruz Biotechnology). Samples were stained for caspase-3 as markers of apoptosis. Bile duct damage was analyzed by light (hematoxylin and eosin, and cytokine-19 immunohistochemical staining) and by electron microscopy.
Common bile duct specimens were fixed in a mixture of 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer. After dehydration in a graded series with ethanol, samples were embedded in Epon 812 resin. Excised transplants were fixed in 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3). Electron microscopy was performed with a transmission electron microscope (EM902; Carl Zeiss, Oberkochen, Germany) at 80 kV. Images were recorded with a digital camera.
Continuous data are presented as mean ± standard deviation. We used either analysis of variance or Student t test to compare continuous variables in different groups. P values less than 0.05 were considered statistically significant.
Oxygenated PFC + UW Solution Improves ATP Stores After Warm and Cold Ischemia
First, the oxygen content (PaO2) of the solutions was measured in the absence of a liver graft. After 30 minutes of preoxygenation, the PaO2 of the UW + PFC solution, as expected, was increased when compared with preoxygenated UW solution (Figure 3). In the first hours, the oxygen content of both groups dropped with a similar trend, whereas the PFC + UW group retained higher dissolved oxygen content after longer preservation.
The solutions were then tested in an extreme warm ischemia model of the mouse liver. After a period of 45 minutes of warm ischemia at 37°C, the livers preserved in PFC + UW solution showed over 30% higher levels of ATP in the liver parenchyma when compared with UW solution (Figure 4A). In comparison, livers that underwent cold ischemia only manifested a markedly slower decrease of ATP over many hours (Figure 4B). Livers preserved in PFC + UW solution had ATP levels multifold higher than UW preservation alone up to 24 hours (P < 0.05), after which ATP was depleted in both groups. To simulate the clinical situation with both warm and cold ischemia, we included a group that underwent both warm (45 minutes) and cold (36 hours) ischemia, and followed ATP. In this setting, livers stored in PFC + UW solution also retained significantly higher ATP levels after 6 hours (P = 0.021), 12 hours (P = 0.007), and 18 hours (P = 0.02) (Figure 4C).
Liver Grafts Preserved in PFC + UW Solution Show Better Histological Morphology
As expected for a nontransplant model and with short-term preservation, the histological changes in livers after 45 minutes of warm ischemia observed with light microscopy were modest. However, storage in UW + PFC was associated with less ballooning of hepatocytes (Figure 5A). Using light microscopy, we found marked changes in the liver parenchyma between the 2 groups only after 72 hours of cold ischemia. The PFC + UW solution grafts show less cellular edema and necrosis (Figure 5E). On the other hand, the ischemic damage suffered by the biliary epithelium was more intense after warm ischemia than the damage found in the liver parenchyma. Oxygenated PFC + UW solution was associated with improved preservation of the biliary epithelium. The epithelium of the gallbladder and bile duct (nontransplanted) preserved in oxygenated UW solution after 45 minutes of warm ischemia was flat and started sloughing-off (Figure 6). When examined using EM, biliary epithelium preserved in UW solution without PFC showed loss of apical microvilli and increased apoptosis (Figure 7D–F) and increased expression of caspase 3 (Figure 8). These characteristics were improved in oxygenated PFC + UW solution (Figures 7C and 8B).
PFC + UW Solution Improves Survival of DCD Liver Grafts
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).
Importance of Oxygenation in Liver Preservation
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.
PFC Use in Graft Preservation and Clinical Transplantation
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
Limitations of PFC Therapy and Study Limitations
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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