Donation after circulatory determination-of-death (DCD) donors have significant potential to expand the donor pool. The ischemic injury they suffer during cardiac arrest (CA) increases the risk for immediate and delayed graft complications (1–3), however, and their livers are infrequently recovered. A primary factor limiting the broader clinical application of DCD liver transplantation is that cold storage (CS) inadequately maintains graft viability. Using CS merely slows organ degradation during ischemia. Machine perfusion, in contrast, delivers metabolic substrates, eliminates toxins, and may help revert preextraction injury and prevent further damage from developing. The conditions under which machine perfusion should be performed, however, are debated.
The logic behind using oxygenated hypothermic machine perfusion (HMP) is that mitochondrial electron transport and oxidative energy production continue at low temperatures (4–6), and HMP provides a continuous oxygen supply for adenosine triphosphate production during preservation (7). From a logistical standpoint, HMP is relatively easier to perform. Unlike normothermic machine perfusion (NMP), it does not require a heat exchanger or an energy source. Moreover, the demand for oxygen under hypothermic conditions is significantly reduced, and the perfusion solution does not require an oxygen carrier or the full range of physiological substrates.
Hypothermic machine perfusion may lead to better kidney preservation (8, 9), in particular among grafts arising from DCD donors (10), and early studies using HMP to preserve livers from dogs and pigs were promising (11, 12). More recently, Guarrera et al. described three porcine transplants (13) and the first human series of liver transplants performed in which extracorporeal preservation included HMP (14). In the human study, normal livers were perfused for up to 7 hr before transplantation. Compared with an historical control group of grafts preserved with CS, HMP led to improvements in viability markers and hospital stay, but because it was a pilot study, there were no significant differences in outcome. Also, the Zurich group studied the use of HMP as an end-ischemic treatment to improve DCD livers in a porcine transplant model. Although HMP improved hepatocellular preservation and energy status among DCD livers, posttransplant survival was low: all recipients died or required sacrifice during the first day (15). Finally, the Leuven group recently compared HMP and CS during 4 hr of extracorporeal preservation using normal pig livers. On transplantation, only 13% of livers preserved with HMP survived 3 days versus 83% with CS (16).
The limited number of clinically applicable studies on hepatic HMP that have been performed have achieved varied results and have largely failed to examine the effects of HMP in DCD livers, where its use is most relevant. We hypothesized that using HMP to preserve DCD livers—although it might lead to some improvements—would ultimately lead to significant injury and failure among these grafts. To test this hypothesis, we compared oxygenated HMP with static CS by mimicking DCD in a clinically relevant liver transplant model.
Five-day survival was 0 and 20% in CS and HMP, respectively (Fig. 1). Although survival was improved in HMP, the difference was not significant (log-rank test statistic, 0.073). No deaths were due to procedure-related causes.
Parameters During HMP
Portal vein flow (PVF), hepatic artery flow (HAF), aspartate aminotransferase (AST), pH, partial pressure of oxygen (PO2), sodium, and potassium remained stable during HMP; no significant differences were seen when starting values were compared with those measured at the end of HMP (Table 1). Furthermore, starting graft weight was similar to the weight at the end of HMP: 715 g (25–75% interquartile range, 678–771 g) versus 771 g (688–772 g), respectively (P=0.675).
Transplantation and Postoperative Function
Cold ischemia times were 272 min (242–291 min) and 40 min (39–44 min) in CS and HMP, respectively; in the latter, cold ischemia was the time it took to extract, prepare, and place the organ on the machine. Reperfusion warm ischemia times were 26 min (25–29 min) and 25 min (24–25 min), respectively.
After reperfusion, HAF was significantly higher in HMP versus CS: 105 mL/min (range, 95–120 mL/min) versus 30 mL/min (range, 25–30 mL/min), respectively (P=0.021). Postreperfusion PVF was also higher in HMP than in CS, although the difference was not significant: 900 mL/min (830–1020 mL/min) versus 700 mL/min (540–830 mL/min), respectively. Mean arterial pressure worsened from baseline during the anhepatic phase, remained unchanged after portal reperfusion, and improved somewhat by the time the hepatic artery was reperfused. No recipient developed severe hemodynamic instability on reperfusion, and no significant differences were found when values in CS and HMP were compared.
In CS, serum AST levels rose dramatically after reperfusion until death in all animals. In HMP, the rise in AST was slower than that observed in CS, with significant differences existing between the groups at 1, 3, and 6 hr (P=0.006, P=0.018, and P=0.021, respectively). Among surviving animals in HMP, AST levels rose slowly from 12 to 48 hr and then increased considerably between 48 and 72 hr (Fig. 2A).
Serum bilirubin rose progressively after reperfusion until death in CS. In HMP, bilirubin remained stable during the first 48 hr and rose progressively thereafter among surviving animals (Fig. 2B). Similarly, bile salts rose continuously after reperfusion until death in CS, whereas they were initially stable in HMP but began to rise after 48 hr among surviving animals (Fig. 2C).
On reperfusion, the quick prothrombin time (QPT) declined to a significantly greater extent in CS versus HMP (P=0.014, P=0.006, and P=0.027 at 1, 3, and 6 hr, respectively). In HMP, QPT improved between 3 and 24 hr but fell again after 24 hr (Fig. 2D).
Tissue sampled at various time points during the procedure was evaluated. In the donor, the sinusoidal spaces were narrow and the hepatocyte cords well preserved (Fig. 3A). After CA, there was significant vascular congestion, affecting both the sinusoids and the peribiliary arterial plexus, and diffuse hepatocellular hydropic changes (Fig. 3B). After HMP, the sinusoids were somewhat dilated, and hydropic changes persisted. There was also sinusoidal endothelial cell (SEC) shrinking and detachment (Fig. 3C). After reperfusion in CS, sinusoidal dilatation was significant, and there were areas of intraparenchymal hemorrhage, diffuse hydropic changes, and numerous foci of hepatocellular necrosis (Figs. 3D, G). Among grafts preserved with HMP, the hepatic microarchitecture was better preserved; hydropic changes and areas of necrosis were fewer (Figs. 3E, H). On the fifth day in the surviving animal in HMP, there was ongoing evidence of necrosis, most notable in zone 3 (Fig. 3F). The most striking finding, however, was that of intrahepatic bile ducts lined by swollen cholangiocytes with peripherally displaced nuclei (Fig. 3I).
Cytokines and Proinflammatory Response
Interleukin (IL)-6 messenger RNA (mRNA) expression was similar at baseline and after CA, although it was significantly higher in CS after reperfusion: 1.20-fold change (0.88- to 1.23-fold change) versus 0.60-fold change (0.26- to 0.64-fold change), respectively (P=0.009). On day 5, IL-6 mRNA had returned to baseline in the remaining animal in HMP (Fig. 4A). These results were confirmed in the serum, in which IL-6 peaked between 3 and 6 hr and was significantly higher in CS at the later time point (P=0.020; Fig. 4B).
Conversely, the expression of tumor necrosis factor (TNF) mRNA was similar at baseline and after CA but was significantly higher in HMP after reperfusion: 1.26-fold change (1.02- to 1.49-fold change) versus 2.64-fold change (2.32- to 2.75-fold change), respectively (P=0.010). By day 5, TNF mRNA had returned to baseline in the remaining animal (Fig. 4C). In serum, TNF peaked at 3 hr and was significantly higher in HMP (P=0.016; Fig. 4D).
Sinusoidal Endothelial Cell Activation and Injury
Intragraft expression of E-selectin mRNA did not vary significantly between the groups after reperfusion, with 0.59-fold change from baseline (0.36- to 0.63-fold change) in CS versus 0.24-fold change (0.13- to 0.59-fold change) in HMP. After 5 days, E-selectin expression remained increased in the remaining animal in HMP, with a 0.58-fold change from baseline (Fig. 4E).
After reperfusion, serum levels of von Willebrand factor (vWF) rose progressively among recipients in CS but remained lower than those measured in HMP. In HMP, levels peaked at 12 hr and were significantly higher than those measured in CS at the same point (P=0.032). Serum levels of vWF remained stable between 24 and 48 hr but rose progressively thereafter in the remaining animals in HMP (Fig. 4F).
It seems that hypothermic oxygenated machine perfusion does offer some benefits over CS in preserving DCD livers. In this study, no graft preserved with CS survived beyond 36 hr secondary to the development of primary nonfunction, whereas three grafts preserved with HMP survived beyond 48 hr. Markers of hepatocellular injury and stress (AST, bilirubin, bile salts, and IL-6) were initially lower, and postreperfusion biopsies were improved in grafts treated with HMP. Quick prothrombin time, an indicator of hepatic synthetic function, was better in HMP versus CS immediately after reperfusion. Nonetheless, after approximately 24 to 48 hr, all the aforementioned markers that had been stable began to deteriorate among remaining HMP grafts.
We initially performed experiments with shorter CA (40 or 60 min) and achieved excellent postoperative survival among control animals. One has to keep in mind that these are young, healthy pigs, and their livers have considerably more functional reserve than those arising from human DCD donors. Although it may seem extreme, we finally chose 90 min because it allowed us to achieve death—the ultimate measure of organ viability—among recipients in the control group. Furthermore, as we demonstrated in experiments with normothermic extracorporeal membrane oxygenation (NECMO) and NMP, the damage in pig livers subjected to 90 min CA is not irreversible (17). Rather, organ viability in this setting is a function of the preservation methods used after warm ischemia.
Although the progressive injury that ultimately led to graft failure appeared later, there were some early indicators that DCD livers preserved with HMP suffered a unique lesion. Although most injury markers were improved in HMP, levels of TNF measured immediately after reperfusion were considerably elevated among these grafts. Tumor necrosis factor is produced primarily by macrophages, which in the liver include Kupffer cells. Kupffer cells have been shown to be activated during cold preservation (18–20), in particular among DCD livers (21).
Limited studies have looked at the effects of HMP on Kupffer cells. Southard et al. (22) did not find increased Kupffer cell activation in normal rat livers preserved with 24 hr of HMP versus 24 hr of CS, whereas Guarrera et al. (14) indicated that HMP actually decreased TNF production versus CS in normal human livers. On the other hand, Monbaliu et al. (16) demonstrated significantly higher postreperfusion TNF levels in normal pig livers preserved with 4 hr of HMP versus 4 hr of CS. Also, in a porcine nontransplant model of HMP, healthy livers pumped 24 hr with University of Wisconsin machine perfusion solution (UW-MP) at 25% physiological pressures demonstrated endothelial fenestration and sinusoidal dilatation, with damaged SECs and Kupffer cells in the perihilar regions (23). It has been shown that cooling cells to near-freezing temperatures leads to dysfunctional regulation of ions and irreversible disruption of the plasma membrane, independent of cellular ischemia (24–28), and abnormal shear stresses arising from altered flow dynamics in hypothermia merely serve to intensify these effects. Hence, the finding that there was considerable Kupffer cell activation among DCD livers perfused continuously in the cold is not entirely unexpected.
Postreperfusion levels of vWF, a marker of SEC injury, were also elevated in HMP. Like Kupffer cells, SECs are susceptible to hypothermic injury (29). Serum levels of vWF rose steadily after reperfusion, reaching an initial peak at 12 hr. E-selectin gene expression, another marker of SEC activation, was measured 1 hr after reperfusion. It was not significantly different in HMP at that point, possibly indicating that the sample was taken too early to reflect a lesion still in progress. The observation that HMP leads to SEC activation and injury is not unique to this study (30–33).
Performing HMP at very low pressures may help reduce SEC and Kupffer cell injury in DCD liver transplant. However, using UW-MP at 4°C, pressures should be at least 25% physiological values to achieve complete perfusion of the graft (34). Another option that has been proposed is exposing the graft to intermittent positive and negative pressures during HMP, resulting in an oscillating intrasinusoidal flow profile (35). This method was tested by the Zurich group as part of an end-ischemic treatment to improve the viability of DCD livers (15). Although it did seem to lead to better SEC preservation in a porcine model, posttransplant survival after the use of this method was just as poor as that described in the present study, with no recipient animal surviving beyond 25 hr after surgery. Furthermore, it is unclear whether better preservation of the sinusoidal endothelium was due to oscillating intrasinusoidal flow or to the fact that HMP was used only briefly.
Although it is more complex to perform, NMP avoids the adverse effects of hypothermia altogether. The use of NMP has led to sinusoidal preservation and improved postoperative survival in porcine DCD liver transplantation (36, 37). At our institution, we use NECMO to maintain our human DCD donors in situ (38, 39), and we recently described the use of continuous normothermic perfusion (NECMO+NMP) in a DCD liver transplant model (17). Livers preserved continuously under normothermic conditions demonstrated no typical reperfusion injury when transplanted, and no grafts failed during follow-up. Other recent studies on the perfusion of livers at subnormothermic temperatures indicate that results improve as temperatures approach 37°C, provided that adequate oxygen is supplied (40–42). Overall, NMP, which provides metabolic substrates under physiological conditions, seems to be a better option to repair injury and improve the quality of damaged livers for transplantation (43).
In summary, although HMP seems to offer some advantages over simple CS in the preservation of DCD livers, it simultaneously to lead to higher rates of sinusoidal endothelial and Kupffer cell injury, which may ultimately contribute to graft failure in a more delayed fashion.
MATERIALS AND METHODS
Twenty-two outbred male weanling pigs (30–35 kg) were used (11 donor-recipient pairs). Animals were divided between two groups:
- – CS group: 90 min of CA+4 hr of CS+implant (n=6)
- – HMP group: 90 min of CA+4 hr of HMP+implant (n=5)
Donor Procedure and Machine Perfusion
The liver, hilum, and abdominal aorta were dissected; cholescystectomy was performed. Hepatic artery flow and PVF were measured using ultrasonic flow probes and a flowmeter (HT107; Transonic Systems, Ithaca, NY).
Heparin (3 mg/kg intravenously) was administered as the aorta was cross-clamped, and the heart was stopped with intravenous potassium chloride. The abdomen was closed, and a heating pad was placed. Approximately 90 min later, the abdomen was opened, and the abdominal aorta and portal vein were cannulated. The liver was perfused with 1 L of University of Wisconsin cold storage solution (UW-CS) both portally and arterially; UW-CS was used because it is the gold standard solution for static preservation of abdominal organs. The liver was removed, prepared, and placed in CS at 4°C.
After the liver was removed, the portal vein, celiac trunk, and bile duct were cannulated. The liver was connected to a prototype of the Groningen hypothermic liver perfusion pump (Organ Assist, BV, Groningen, the Netherlands) (23). In brief, the device consisted of a receptacle for the liver; two centrifugal pumps (Deltastream DPII; MEDOS Medizintechnik AG, Stolberg, Germany) delivering continuous and pulsatile flow, respectively; a HILITE 800LT membrane oxygenator (MEDOS); an oxygen canister; and a measurement and control device connected to an interface. The pulsatile pump delivered perfusate from the receptacle, through the oxygenator, and into the hepatic artery; the continuous pump perfused the portal vein without oxygenation. Previous experiments demonstrated that an oxygen flow of 100 mL/min resulted in a constant PaO2 of 95±3 kPa (713 mm Hg) (23). Before placing the liver, the machine was primed with 2 L of UW-MP, which is the only clinically registered solution for HMP. Portal and arterial perfusion pressures were maintained constant at 4 mm Hg and 30/20 mm Hg (25% physiological values) (34), and the temperature of the UW-MP solution was 4°C. Flows were recorded and the perfusate sampled throughout HMP. Samples of the perfusate were analyzed for hepatic transaminases, electrolytes, oxygenation, and acid-base balance using an automatic analyzer.
Recipient Procedure and Postoperative Management
Four hours after the start of CS or HMP, the liver graft was flushed with warm Ringer’s lactate solution and transplanted. The anhepatic phase was less than 20 min. Neither venovenous bypass nor vasoactive substances were used. The administration of intravenous fluids was standard (20–25 mL/kg isotonic crystalloid solution per hour during the hepatectomy, and after reperfusion, 500 mL of colloid during the anhepatic phase) (44). On completion of the procedure, anesthesia was weaned, and the recipient was followed intensively for up to 5 days, as described previously (17, 44).
Blood and Serum Analysis
Blood was sampled in the recipients at baseline and 1, 3, 6, 12, and 24 hr after reperfusion and daily thereafter. Serum levels of AST, bilirubin, bile salts, IL-6, TNF, and vWF and QPT were measured, as previously described (17).
Hepatic tissue was sampled at baseline in the donor, at the end of CA and HMP, and 1 hr and 5 days after portal reperfusion. Biopsies were divided into two sections: one preserved in 10% formaldehyde for inclusion in paraffin and one diced finely and snap-frozen in liquid nitrogen for RNA extraction.
Real-time quantitative TaqMan reverse-transcriptase polymerase chain reaction analyses of IL-6, TNF, and E-selectin gene expression were performed to detect inflammation, Kupffer cell activation, and SEC injury, as described previously (17).
Data and Statistical Analysis
Values are expressed as median (25%–75% interquartile range), unless otherwise specified. Continuous variables were compared using the Mann-Whitney U test; P<0.05 was considered significant. Survival was analyzed according to the method of Kaplan-Meier; differences between groups were evaluated using the log-rank test.
Ethical and Humane Considerations
The experimental protocol was approved by the Hospital Clinic Institutional Review Board and the University of Barcelona Committee on Ethics in Animal Experimentation.
The authors thank Gerhard Rakhorst and Arjan van der Plaats of Organ Assist, BV, for providing the prototype of the Groningen hypothermic liver perfusion pump and the disposable machine components.
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