Advantages of normothermic perfusion over cold storage in liver preservation : Transplantation

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Experimental Transplantation

Advantages of normothermic perfusion over cold storage in liver preservation

Imber, Charles J.1; St. Peter, Shawn D.; Lopez de Cenarruzabeitia, Inigo; Pigott, Dave; James, Tim; Taylor, Richard; Mcguire, James; Hughes, David; Butler, Andrew; Rees, Michael; Friend, Peter J.

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Abstract

INTRODUCTION

The ideal method of preservation would prolong preservation time, reduce the rate of primary nonfunction, make use of marginal organs, and provide assessment of viability. This would reduce the pressure of urgent retransplantation on an already exhausted donor pool and loosen time constraints placed on transplant co-ordination worldwide. It may be possible in the future to immunomodulate the organ during the preservation period to enable tolerance induction or to transfect genes that may provide resistance to recurrent hepatitis infection. Normothermic, sanguineous machine perfusion is proposed as the preservation method that may achieve these goals, with the intuitive assumption that replicating the physiological environment is the least deleterious method of maintaining a liver after retrieval.

Standard clinical practice involves flushing the liver in situ with University of Wisconsin (UW) solution and storing it at 4°C for up to 15–18 hr before transplantation. The first critical comments on extended preservation time with UW solution entered the literature in 1990 (1), and subsequently the relationship between cold-ischemia time and postoperative primary graft function was clearly demonstrated. Primary nonfunction still occurs at an unacceptable rate ranging between 2% and 23% and is a major cause of death in transplantation (2). Additionally, there is clinical evidence that severe preservation injury is associated with an increase in liver graft rejection (1).

Preservation injury in liver allografts is the encompassing term used to describe the injury an organ sustains during the process of transplantation. It may be considered in four categories: prepreservation injury, cold-preservation injury, rewarming injury, and reperfusion injury. Warm perfusion during preservation can avoid cold-preservation injury and ameliorate prepreservation, rewarming, and reperfusion injury (3).

When blood flow is terminated during ischemic preservation, the supply of oxygen, cofactors, and nutrients is eliminated along with the vehicle for disposal of waste and metabolic by-products. During anoxia, ineffective anaerobic metabolism leads to depletion in energy stores (adenosine tri-phosphate [ATP]), with a concomitant build up of acidic by-products (lactic acid). This leads to loss of transcellular electrolyte gradients, cell swelling, influx of free calcium, and the subsequent activation of phospholipases. The breakdown of ATP during ischemia creates a setting for the production of reactive oxygen intermediates on reperfusion and a cascade of ischemic injury.

Preventing ATP loss and the injurious cascades that follow requires perfusion with an oxygenated solution (4). Studies have demonstrated the ability of oxygenated perfusion to prevent ATP loss during, and reset ATP levels after, periods of ischemia (5,6).

In this preclinical study, we directly compared standard cold storage in UW solution to normothermic, sanguineous perfusion over a 24-hr preservation period in a porcine liver model.

MATERIALS AND METHODS

Experimental Design

Preservation period.

Group C (n=5): Preserved in cold storage for 24 hr in UW solution.

Group W (n=5): Preserved on isolated perfusion circuit with whole blood at 38°C (Fig. 1).

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Figure 1:
Overview of experimental plan.

Cold-ischemia/rewarming period.

Both sets of livers were exposed to a realistic backbench/anastomosis period of 60 min between preservation and reperfusion. During this time group C livers were prepared and connected to the extracorporeal circuit ready for reperfusion. Group W livers, however, having been perfused during the preservation period, required a bypass to be incorporated in the circuit design (Fig. 2) so that the livers in this group could undergo 60 min of cold ischemia while the flow of perfusate through the pump remained uninterrupted.

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Figure 2:
(A) Bypass circuit. To keep the perfusion circuit running while the liver is undergoing a period of cold ischemia, a bypass circuit is included in the group W experiments. After 24 hr of warm preservation, the afferent limb to both hepatic artery (1) and portal vein (3) is clamped, as well as the caval outflow via (7), and the organ is cold flushed via (2) and (5). Flow of perfusate to the pump continues via the bypass circuit, which is opened at points (4) and (8). On reperfusion the liver is flushed with blood, and 500 mL of effluent admixed with the cold flush solution is lost via (6). The afferent limbs (1 and 2) are reopened and the bypass circuit closed (4). (B) Extracorporeal perfusion circuit.

Reperfusion period.

The livers in both groups were then reperfused for 24 hr on the isolated circuit as a surrogate for transplantation.

Sampling.

Perfusate was sampled at regular intervals during this phase (as described below) so that ischemic preservation injury could be compared and functional liver viability assessed. At the end of each perfusion, each liver was sectioned and assessed histologically for evaluation of reperfusion injury.

Animals

Twenty, large, white/landrace pigs were used in these experiments (mean weight 40 kg) in accordance with the Animal Protection Act 1986.

Blood Donor

Blood donor pigs were premedicated with 10 mg/kg of ketamine (Willows Francis Veterinary, Ketaset) and 1 mg/kg of midazolam (Roche, Hypnovel) intramuscularly (i.m.), followed by halothane (Zeneca, Fluothane) anesthesia. The common carotid artery was cannulated, 15,000 U of heparin was given, and blood was collected in citrated bags via gravity drainage until circulation ceased. Blood was used immediately or stored at 4°C and used within 1 day of collection.

Liver Donor

Each experiment in the study required an organ for extracorporeal perfusion. The same techniques that are standard practice for harvesting human donor organs were used. Animals were premedicated as above: an ear vein cannulated and anesthesia induced using 0.5 mg/kg of propofol i.v. (Zeneca, Diprivan). The animal was intubated using an extended endotracheal tube. Pulse oximetry via a tail probe was used to follow oxygenation, and end tidal CO2 was measured. After intubation, a 14-gauge cannula was sited to allow for fluid replacement during the hepatectomy, and an arterial line placed in the femoral artery. Anesthesia was maintained with i.v. propofol (1 mg/kg/hr) throughout the procedure. A midline abdominal incision was made. The bile duct was divided and the hepatic vessels identified and isolated in the standard fashion. The liver was dissected until connected to the donor only by its vascular attachments. Throughout this procedure, meticulous attention was paid to hemostasis to minimize subsequent blood loss on the heparinized extracorporeal circuit.

Heparin (20,000 U) was given intravenously and allowed to circulate. The infrarenal aorta was cannulated with a 10-gauge cannula (Medtronic) and connected to a closed system containing cold Eurocollins solution (Baxter, Soltran). The portal vein was cannulated with a 20-gauge cannula (Medtronic) in a similar manner and perfusion with cold Eurocollins solution via the portal vein and aorta was commenced via gravity as the suprahepatic inferior vena cava (IVC) was divided in the pericardium.

After 2 L of cold Eurocollins solution had perfused through the liver, it was removed by excising a cuff of diaphragm around the suprahepatic IVC, dividing the hepatic artery at the coeliac axis, the infrahepatic IVC at the level of the renal veins, and the portal vein at the level of the splenic vein. While continuing portal perfusion, the liver was removed from the animal and placed into a bowl at ice temperature. The diaphragmatic remnant was oversewn with running 3.0 Prolene to secure hemostasis. The suprahepatic IVC was cannulated with a 28-gauge cannula (Medtronic) with its orifice positioned at the level of the hepatic veins. Pressure-monitoring cannula (Portex) were placed in the IVC (directly 8 Fr) and portal vein (via a tributary 6 Fr). The hepatic artery was cannulated with a 10-Fr cannula (Medtronic). Arterial pressures were measured directly from the arterial limb of the circuit. Continuous pressure monitoring was achieved using a transducer (Viggo-Spectramed, Haemodynamic Monitoring Set) and a digital monitor (Datex, AS/3). The bile duct was cannulated with a 14-gauge silastic T-tube with the open end of the T-tube placed into a collection device to monitor hourly bile production.

Perfusion Circuit

While backbench preparation of the liver was performed, the perfusion apparatus (Fig. 2) was assembled and primed with 1500 mL of donor pig blood (2). The perfusion circuit consisted of an oxygenator (1500 ECMO Oxygenator Medtronic), a heat exchanger (Ecmotherm II HE Medtronic), a centrifugal pump (Medtronic BP50 Centrifugal Pump), a reservoir (Venous reservoir bag 800 mL Medtronic), tubing (Medtronic, PVC, 1/4 and 3/16 inch internal diameter [ID] with Medtronic Polycarbonate connectors), a gate clamp, pressure transducers (Baxter Triple Pressure Transducers), and flow probes (2 Medtronic DP 38P).

The inflow to the liver consisted of two circuits in parallel. The cannula within the hepatic artery was fed directly from the centrifugal pump, whereas the portal vein received blood that drained passively from a reservoir. In this way pressures within both systems can be maintained within physiological limits.

The oxygenator was attached to the heat exchanger to maintain the temperature of the blood at 39°C. During priming of the circuit, the pH, paCO2, and Ca2+ were adjusted to bring them within the normal physiologic range. In general the perfusion circuit was supplemented with 9.2 mmol of CaCl2, 20 mmol of sodium bicarbonate, and 7000 U of heparin. Once the blood within the circuit was optimized, a sample was obtained for full blood count, electrolytes, urea, creatinine, and liver function tests (LFT); and plasma was collected and stored in liquid nitrogen for further analysis. While the circuit was running, prostacyclin (Glaxowellcome, Flolan) (500 mcg in 50 ml, diluted in 250 mL of n/saline, 4 mL/min), 1% taurocholic acid (Sigma) at 7 mL/min, and total parenteral nutrition (TPN) (2 L of Clinifeed + 100 IU of Actrapid) at 17 mL/hr were all infused via an Imed pump (Imed, Gemini PC-4) into the perfusate.

Before connection of the liver to the perfusion circuit, the Eurocollins solution was flushed from the portal circulation using 1 L of colloidal infusion solution (Behring, Haemaccel). This also allowed complete exclusion of air from the liver and cannula. The liver was placed in an intestinal bag and then suspended in saline in a sterile perfusion chamber. Two soft plastic tubes were placed at the most dependent part of the intestinal bag. These tubes were then connected to the reservoir via an Imed pump (Imed, Gemini PC-4) to recirculate any free fluid that collected. The liver was connected to the primed perfusion apparatus avoiding any entrainment of air.

Normothermic Protocol

Once harvested the liver was attached to the circuit after backbench work, including cannula, and pressure line insertion was completed. This took a mean of 15 min (±2.8), during which time it was kept at 4°C with cold Soltran (Eurocollins) solution being dripped into the portal vein.

The first 24 hr on the circuit was the preservation period of the experiment. During this time, and during the reperfusion phase in both groups, total flow was maintained at approximately 1.5 L/min, by adjusting the rpm of the centrifugal pump. Arterial and venous blood gases were also checked, and the pO2 and pCO2 were kept within normal limits by adjusting the air/oxygen mixture supplying the oxygenator. The pH, however, was not corrected by external manipulation, because its regulation has been found to be a useful marker of liver viability. An intravenous bolus dose of heparin (3000 IU) was administered every 4 hr.

After the initial 24 hr, the bypass circuit was opened (Fig. 2), which allowed continued flow around the circuit while excluding the liver. This was then flushed with 2 L of cold Soltran (Eurocollins) and left cold for 60 min. After this period the bypass circuit was closed, Soltran was flushed from the liver with 1 L of colloid (Behring, Haemaccel), and blood from the preservation period was discarded. Reperfusion of the liver then commenced with fresh blood for a further 24 hr.

Hypothermic Protocol

This group of livers was harvested in an identical manner to the normothermic group and stored for 24 hr in cold 4°C UW solution (Du Pont). After this, the organs were flushed with 2 L of colloid solution (Behring, Haemaccel) and then attached to an identical circuit as for the warm group, except for the absence of the bypass tubing. They then underwent 24 hr of reperfusion on the circuit, with identical sampling of the perfusate as described above.

Data Collection

The reperfusion phase of the experiment acted as a surrogate for transplantation. During this time regular sampling of the perfusate was performed as follows:

  1. A full blood count (1 mL) was taken every 2 hr for 8 hr, and every 4 hr subsequently.
  2. 200 μL every hr, from both the afferent and efferent side of the oxygenator, were analyzed on a CIBA-CORNING 288 reflecting arterial and venous blood gases.
  3. Galactose maximum elimination capacity (Vmax) estimation at 12 and 24 hr of the reperfusion.
  4. 1 mL of bile and ascites every 4 hr of the perfusion.
  5. Whole blood (perfusate) was drawn off the circuit and centrifuged at 12,000 rpm for 8 min and the supernatant immersed in liquid nitrogen as follows:
  6. 900 μL of perfusate supernatant mixed with 100 μL of sodium citrate for Factor V analysis every 2 hr for the first 8 hr, every 4 hr subsequently.
  7. 500 μL of perfusate supernatant for urea/electrolytes and liver function tests every 1 hr for 8 hr, every 4 hr subsequently.
  8. 500 μL of perfusate supernatant for β-galactosidase estimation.
  9. 500 μL of perfusate supernatant for free hemoglobin estimation.

Electrolyte analysis.

Urea and electrolytes including liver function tests were measured on 500 μL of plasma using an automated analyser (Aeroset analyser, Abbott Diagnostics).

Factor V assay (MDA 180 method).

Dilutions of standard and test plasma were mixed with a substrate plasma that is completely deficient in Factor V (FV). A modified prothrombin time (PT) was then performed utilizing a multichannel discreet analyzer (MDA) coagulometer, and the ability of the test and standard plasmas to correct the prolonged PT of the substrate plasma compared.

Galactose elimination kinetics.

Galactose elimination kinetics were measured according to the protocol suggested by Uesugi et al. (7). The galactose concentration was measured on a Cobas Fara centrifugal analyser using a spectrophotometric assay kit (Boehringer Mannheim).

Hemolysis (cyanmethemoglobin colorimetric method).

Free hemoglobin was converted to cyanmethemoglobin by utilizing Drabkin solution. A direct spectrophotometric assay measuring cyanmethemoglobin was then utilized (8) (diluent volume 4.0 mL, perfusate sample volume 20 μL, dilution 1/201).

Liver weight.

Each liver was weighed on an electronic balance after retrieval and after 24 hr of reperfusion to compare changes during the experiment between the two groups.

Histology.

At the end of the 24-hr reperfusion phase, the liver was sectioned through its mid-portion. From this slice, five random blocks (5×10 mm) were immersed in formal saline for paraffin sectioning and hematoxylin and eosin staining.

Two independent blinded assessors using the system outlined in Figure 6 examined each slide. The total area of necrotic liver was determined for each specimen and expressed as a total percentage of the surface area of each slide. This was scored as 0%, <30%, 30–60%, and >60% scoring 0, 5, 10, and 20 points, respectively. The viable tissue was then assessed under the following headings: sinusoidal dilatation, congestion, hepatocellular vacuolization, mitosis, and apoptosis. This was again scored receiving points for being mild, moderate, or severe. The total score for each specimen was the sum of these two indices, which if less than 15 was determined to be healthy and if greater than 15 nonviable.

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Figure 6:
(A) Histology proforma. Semiquantitative scoring system used by two blinded assessors evaluating each slide. (B) Biopsy of porcine liver (cold-preserved). Hematoxylin and eosin-stained cold-preserved porcine liver having undergone 24 hr of reperfusion. Substantial areas of parenchymal necrosis and red cell infiltration are evident, with associated sinusoidal dilatation (magnification ×20). (C) Biopsy of porcine liver (warm-preserved). Hematoxylin and eosin-stained porcine liver having undergone 72 hr of normothermic machine perfusion. Viable hepatocellular structure is maintained (magnification ×20).

Statistical Analysis

Statistical analysis was performed using a Welch’s t test (assumption of unequal variances), with each time point reflecting the mean of five perfusions. P <0.05 was considered significant. The histology was compared using a Fisher’s exact test.

RESULTS

Liver Injury

Perfusate was sampled from the circuit during the 24 hr of reperfusion, plasma eluted after centrifugation and frozen and stored at −60°C. Blood levels of transaminases were measured on an automated analyser.

A significant difference (P <0.05) in alanine aminotransferase (ALT) between the two groups can be seen (Fig. 3), with a continual rise in the cold-preserved livers up to a level of 200 IU/L. The corresponding value in the warm-preserved group never exceeded 40 IU/L. A similar picture is seen in aspartate aminotransferase (AST) release, with correspondingly higher levels in cold-preserved livers, up to 6000 IU/L.

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Figure 3:
(A) ALT comparison between warm- and cold-preserved livers. Progressive increase in alanine aminotransferase was observed over the 24-hr reperfusion phase in the cold-preserved livers, with minimal changes in the corresponding normothermic group. Each time point represents the mean±SEM for five animals. x=(P <0.05) group W vs. group C. (B) Comparison of γ-glutamyltransferase (GT) between cold- and warm-preserved livers. Serial analysis of perfusate during the two groups of perfusions reveals a greater level of liver regeneration, as demonstrated by the γ-GT level, in hypothermically preserved livers. This trend is significant for the first 16 hr of the reperfusion phase when one would expect liver repair to be maximal. Each time point represents the mean±SEM for five animals. x=(P <0.05) group W vs. group C.

γ-Glutamyltransferase (GT) levels started higher in group C livers and remained greater than group W for the entire reperfusion phase, reaching a maximum of 70 IU/L within 2 hr. Levels in group W never rose above 20 IU/L (Fig. 3).

Biochemical Markers of Metabolic Liver Function

The cold-preserved livers displayed a persistent hyperglycemic state on reperfusion with blood glucose readings between 30 and 35 mmol/L (Fig. 4). In contrast glucose levels fell rapidly in group W perfusions to physiological levels, rising slightly to between 13 and 15 mmol/L from 20 hr onward. Significant differences were evident throughout the entire perfusion (P <0.05).

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Figure 4:
(A) Glucose comparison between cold- and warm-preserved livers. Glucose concentrations of the perfusate were analyzed using a standard glucometer. A constant supraphysiological level was seen in all cold-preserved livers, in contrast to the normothermic group in which the level fell for 10 hr and then stabilized. This difference was significant at all time points. Each time point represents the mean±SEM for five animals. x=(P <0.05) group W vs. group C. (B) Comparison of urate production between cold and warm livers. Progressive increases in urate concentration (end point of high energy phosphate metabolism) in the perfusate were noted over the 24-hr period in the cold group of livers compared to baseline levels noted in the warm group. Each time point represents the mean±SEM for five animals. x=(P <0.05) group W vs. group C.

A pharmacokinetic analysis was also used as a means of comparing different liver’s metabolic capabilities while on the perfusion circuit. The galactose elimination capacity (Vmax) was measured at two time points during each perfusion (12 and 23 hr) by analyzing the decrease in concentration of galactose within the perfusate over 1 hr after a bolus injection. This test was chosen because galactose elimination follows Michaelis-Menten kinetics and represents a zero order reaction at the dose of galactose utilized, which directly reflects the hepatic functional mass.

In the group C livers, the galactose Vmax at the end of 24 hr of reperfusion was 65 mg/min/kg, in contrast to the group W livers where the mean value was 76 mg/min/kg. These values are comparable to other group’s results in alloperfused livers (9) and 70% of values obtained in the pig in vivo.

The two groups display significantly differing urate levels during the reperfusion phase of the experiments (Fig. 4), with levels reaching 850 μmol/L in the cold-preserved group and remaining at low levels in group W.

Comparison of Synthetic Capability

The synthetic capabilities of the cold-preserved group of livers is shown to be inferior to the normothermic group, because the level of factor V diminishes throughout the reperfusion period with no evidence of de novo synthesis (Fig. 5). In contrast, the warm-preserved livers show an initial reduction in factor V levels, which plateaus and from 15 hr onward shows a significant increase compared with group C, toward its initial value.

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Figure 5:
(A) Comparison of Factor V production. Divergence of Factor V production in the two groups can be seen, with levels rising in Group W from 4 hr onward. Each time point represents the mean of five experiments±SEM. x=(P <0.05) group W vs. group C. (B) Comparison of bile production. Normothermically preserved livers are shown to produce larger quantities of bile for the entire 24 hr of the perfusion, a finding which is significant at the majority of time points. This is collected by cannulation of the common bile duct, and production stimulated in both groups by an infusion of taurocholate (2% taurocholic acid at 7 mL/min). Each time point represents the mean±SEM for five animals. x=(P <0.05) group W vs. group C.

Bile production commences soon after reperfusion in all livers and continues for the 24 hr of the experiment in both groups. However, the warm-preserved livers demonstrated significantly greater production throughout as illustrated in Figure 5 (P <0.05), maintaining levels of approximately 6 ml/hr. Both sets of livers received the same choleretic stimulus in the form of a taurocholate infusion.

Oxygen Consumption

Both groups of liver seem to consume oxygen at the same rate up to 15 hr of reperfusion (Table 1). At this point, however, a sustained increase in the hypothermic group occurs in contrast to a fall in the normothermic group toward baseline values. Levels are significantly greater in group C from 18 hr onward.

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Table 1:
Comparison of hemolysis (free hemoglobin levels) and oxygen consumption during the reperfusion period for warm- and cold-preservation groups

Red Cell Hemolysis

Free hemoglobin levels were significantly greater from 6 hr of reperfusion onward in group C livers (Table 1), reflecting greater red cell hemolysis in this group.

Hemodynamic Parameters

The total flow through the liver in the normothermic group starts at 2 L/min at the initial reperfusion phase and remains stable throughout. In contrast the cold group had a lower total flow around 1.5 L/min during reperfusion, which dropped significantly from 20 hr onward. This trend held true for portal flow, although there was no significant difference between the groups in respect of hepatic arterial flow. Portal pressures were raised in response to cold preservation. No significant differences were seen in arterial pressures or resistance between the two groups.

Liver Weight

The mean weight in the group C livers increased by approximately 40% of its starting value, whereas in group W livers the average weight dropped by 5% during the course of an experiment.

Histology

A significantly greater amount of reperfusion injury (Fishers exact test=0.024) was manifest by scoring each of the five sections stained per liver as described. The cold-preserved group (Fig. 6) displayed substantially greater proportions of necrotic parenchyma, as well as sinusoidal dilatation secondary to the generation of higher inflow pressures in this group.

DISCUSSION

During the first half of the twentieth century, Carrel (10) perfused organs with normothermic, oxygenated serum at supraphysiological volumes and demonstrated gross viability for several days. By 1967, advances in hypothermic storage solutions in addition to hypothermic machine perfusion allowed organ preservation to achieve new goals. Brettschneider et al. (11) successfully applied this technology to seven human cases. Organs underwent continual machine perfusion with a perfusate made up of autologous blood mixed with a preservation solution under refrigerated hyperbaric conditions for periods of 4–7 hr. In this work, they established the need to perfuse the liver through the portal vein alone or in combination with the hepatic artery.

With the development of Collins solution, however, machine perfusion was seen to present too many logistical constraints and was largely abandoned as a mechanism of preservation in the liver. Research into extracorporeal machine perfusions largely centered on liver support (12), although Belzer continued to work on hypothermic machine preservation of the kidney, a technique still favored in several units, particularly in the United States.

This body of work presents a large animal preclinical model comparing cold storage with UW solution and dual vessel normothermic sanguineous perfusion utilizing oxygenated blood as the perfusate. The circuit acts not only as a mechanism for performing machine perfusion but as a surrogate for transplantation, allowing quantification of the cumulative injury that a liver has received during the process of retrieval and preservation. The outcome after large animal transplantation studies can be influenced by many factors unrelated to the preservation of the grafted organ. By removing these, any conclusions drawn from this study can be attributed directly to the retrieval/preservation process. Several previous studies have used similar extracorporeal perfusion circuits to test different preservation models (13,14).

The pig has been favored as a research transplantation model because of its anatomical and physiological similarity to the human (15,16). It has the benefit of resembling the human situation in terms of preservation characteristics, although survival after liver transplantation has never been achieved with hypothermic preservation periods greater than 20 hr.

The circuit described has successfully maintained liver viability for periods as long as 72 hr. This far exceeds the current maximum period of successful hypothermic preservation and even surpasses what other groups have achieved with isolated liver perfusion. This may be explained by several factors unique to this experimental system.

All the components of the circuit, including the centrifugal pump as well as the heat exchanger and oxygenator, have been designed for the purpose of sustaining prolonged clinical cardiopulmonary bypass. Hence a sanguineous perfusion is achieved with minimal mechanical disruption to erythrocytes. Flow is controlled by adjusting pump head speed, and the liver can thus adjust resistance to maintain physiological pressures within afferent vessels and prevent barotrauma to sinusoidal lining cells. Prostacyclin is infused to the liver while on the circuit, and this has been shown within clinical trials to decrease ischemia reperfusion injury after transplantation (17). As well as this, the continual provision of oxygen and a supply of substrate in the form of parenteral nutrition may benefit the liver, as well as the continual stimulus to bile production with a constant infusion of cholic acid.

Various transaminases are present in hepatocytes and leak into blood with liver cell and mitochondrial membrane damage, representing nonspecific markers of global liver injury. Hepatocellular enzymes have long been the basis of viability testing. AST, ALT, and lactate dehydrogenase (LDH) were shown to correlate well with ischemic time in canine perfusion studies (18).

Of the enzymes assayed, ALT is the most sensitive indicator of liver cell damage, because it is found solely in the hepatocyte as opposed to aspartate transaminase, which is also found in erythrocytes. Measurement of the latter yields valuable information; several groups have shown that red cell hemolysis in an extracorporeal model reflects reperfusion injury.

γ-Glutamyltransferase is a liver microsomal enzyme derived from the endoplasmic reticulum of the cells of the hepatobiliary tract. It may be induced after acute liver injury and regeneration, such as in alcoholic liver disease. This also clearly demonstrated the fact that the hypothermic group of livers sustained a more severe injury (levels reaching approximately 60 IU/L) during the reperfusion phase of the experiments, displaying significantly higher levels of γ-GT than the normothermic group.

Glucose level within the perfusate represents the balance between supply (in the form of a constant infusion of total parenteral nutrition) and uptake by the hepatocyte. When faced with a glucose load in the portal vein, glycogen is synthesized and stored and glucose is converted into fatty acids, which are normally transported to the adipose tissue. In view of the fact that the level of glucose/insulin is consistent in all perfusions, the glucose level is an indirect measure of liver metabolism.

Hems et al. (19) have suggested that the functional state of an isolated liver can be gauged from its ability to perform gluconeogenesis. Both glucose concentration and galactose estimated elimination capacity (Vmax) seem to reflect trends in metabolic capabilities during the reperfusion period. The former remains “high” in cold-preserved livers, suggesting an inability of the liver to take up or metabolize glucose, as the galactose Vmax decreases over the 24-hr period to levels well below those expected in an extracorporeal model (9).

This is in contrast to the normothermic group of livers in which the glucose concentration fell for the first 10 hr of the reperfusion and then seemed to recover to a level around 10 mmol/L. This pattern of glucose uptake mimics that described by Abouna et al. (20), who demonstrated the initial fall to be due to glycogen formation and the subsequent rise to glycogen breakdown. The reasons for this are not clear, but Craig (21) showed that the isolated rat liver seems to maintain the blood glucose at approximately 15 mmol/L. He suggested that approximately 35% of the glucose taken by the liver during perfusion is used for glycogen synthesis.

Greater oxygen consumption was seen in the group C livers during the reperfusion period. This may be explained by the respiratory burst and subsequent oxygen debt that follows prolonged periods of cold ischemia and subsequent reperfusion.

Bell et al. (22) used an isolated perfusion model to compare different cold-preservation solutions and found platelet sequestration to be a reliable marker of injury as well as subsequent organ viability after transplantation. This was confirmed by Iu et al. (14) while investigating allograft viability in a rat liver model.

Platelet adherence to the altered sinusoidal lining cell may contribute to the microvascular changes that lead to subsequent ischemic damage in the liver. Platelet activating factor production within the liver within 12 hr of such an insult has been demonstrated (23). Preliminary data from our experience with porcine isolated liver perfusions has suggested that an increase in red cell lysis (i.e., level of free hemoglobin) may reflect increases in reperfusion injury, a finding not previously reported. This may represent resident sinusoidal Kupffer cell activation and subsequent phagocytosis of red cells.

One of the major mechanisms involved in reperfusion injury is nucleotide metabolism. During ischemia, inefficient anaerobic metabolism leads to ATP breakdown into adenosine. This is converted to hypoxanthine, which is normally metabolized by xanthine dehydrogenase (XD) in an NAD dependent process. However, under ischemic conditions, XD is converted to xanthine oxidase (XO). Upon reperfusion, the accumulated XO utilizes oxygen to degrade the hypoxanthine stores into urate, which goes on to create free radicals (respiratory burst). During hypothermia, the injurious ischemic mechanism is compounded by an accelerated breakdown of ATP. Only ongoing normothermic oxygen delivery to tissue can allow for constant regeneration of ATP and NAD, allow for normal nucleotide metabolism, and prevent the accumulation of potentially toxic metabolites.

Livers preserved in cold storage have an abundance of hypoxanthine compared with warm-perfused livers before reperfusion. Therefore, upon reperfusion, higher levels of urea and urate should be expected, because those represent products of the two pathways of hypoxanthine metabolism. In addition, the ischemic conversion of XD to XO in the cold group should shunt hypoxanthine toward the injurious path (urate production) more so than in the warm group. Therefore, urea levels should be elevated in the cold group, but one would expect a much greater disparity in urate levels between the two groups. Our results exemplify this well.

Synthetic liver function in terms of bile and factor V production have been used by many groups to assess liver viability in an extracorporeal model (22,24). Several mechanisms have been proposed to explain the reduced production of bile after ischemia reperfusion injury. ATP depletion reduces the active biliary secretion of bile acids (25). Bile-acid dependent canalicular bile excretion may decrease, and biliary tight junction permeability may also increase after ischemia-reperfusion injury, leading to dissipation of canalicular osmotic gradients. This relationship was clearly borne out in this study, with significantly less bile production in the cold-preserved group of livers, despite both groups receiving bile salt supplementation as a replacement for the enterohepatic circulation.

In view of the fact that the circuit is fully heparinized, the commonly used measure of synthetic liver function, the prothrombin time, would not be applicable. Therefore, an individual component of the clotting pathway, Factor V, was measured. This protein, when activated, enhances the conversion of prothrombin to thrombin in the presence of phospholipid and calcium ions. The normal range is 50–150 IU/dl. It has the particular benefit of having a short half-life and is therefore sensitive to alterations in liver function. Our data showed significantly better function in the normothermic group in respect to both of these indices.

Weight change during the reperfusion phase of the experiment was very different in the two groups. Whereas the cold-preserved livers developed gross edema, the normothermic group lost a small amount of weight during the reperfusion period. It would seem logical to surmise that sinusoidal endothelial damage allows excess water to leak into the liver and that edema is therefore a surrogate marker for the extent of reperfusion injury.

A distinct sequence of histopathologic changes occurs in ischemic liver tissue after reperfusion. This sequence has some variability, depending on the length of ischemia, mechanism of reperfusion, and length of time after reperfusion. In general, on reperfusion, cellular changes can be readily seen and includes cellular swelling, vacuolization, endothelial cell disruption and fenestration, and neutrophilic infiltration. Kupffer cells exhibit progressive rounding, ruffling of the cell surface polarization, appearance of worm-like densities, vacuolization, and degranulation (26).

In this study, histological analysis readily separated the two groups of livers. The cold-stored group showed large (>30%) amounts of necrosis, implying the tissue was nonviable. The warm-preserved group had significantly less necrosis, although some sinusoidal dilatation, and diffuse vacuolization. To avoid the risk of observer bias, these assessments were made using a blinded technique.

The best perfusate for continuous perfusion of an organ has not been defined. However, the perfusate should enable the delivery of oxygen to achieve the full benefit of perfusion to be realized (27–31). In this study we chose whole blood as the circuit perfusate, because studies have shown that oxygenated-buffer solutions may require higher flows for adequate oxygen delivery and create degenerative changes in the perfused tissues not seen when red blood cells are used (32,33). A hematocrit of 20% has been suggested to provide optimum combination of blood and oxygen-carrying capacity at physiologic flows and pressures (34). It was recently discovered that red blood cells attenuate sinusoidal damage by scavenging XO-dependent radicals in perfused rat livers (35). To whole blood we added prostacyclin (as a vasodilator/free-radical scavenger), bile salt (taurocholate) to mimic the enterohepatic circulation, thereby replacing depleted bile acid stores, and total parenteral nutrition as a source of vital nutrients.

The cold-preserved organs were stored in UW solution and then received a colloid rinse before reperfusion on the circuit. This is in contrast to the warm group, which received a 60-min flush with cold Eurocollins solution to separate their preservation period from their reperfusion. This may be seen as a potential criticism of our data. UW is more viscous and thus is less well washed out after a 1 L colloid rinse. This could potentially lead to greater contamination of the perfusate with built up of metabolic toxins during the reperfusion phase in the cold-preserved livers and bias the comparison of subsequent function.

This however should be seen as a disadvantage of hypothermic UW preservation in the clinical situation, leading to greater exposure of a recipient to the contaminates of anaerobic metabolism and the subsequent enormous risks of acute reperfusion injury. We believe that the preferential results in group W livers are not due to the fact that Collins solution facilitated the removal of contaminates before reperfusion but that a continued supply of oxygen and substrates during the preservation period in group W enabled restoration of high-energy phosphates, hence preventing the production of harmful contaminates.

The shortage of healthy organs for transplantation demands optimal utilization of the available donor pool. Transplanted livers must function well immediately to give the recipient a realistic chance of survival. At present the decision as to whether an organ should be discarded is made both on objective donor criteria and a subjective overall impression made on macroscopic appearance, perhaps with a biopsy. This may result in an incorrect decision either to wastefully discard a healthy liver or to utilize an organ with a very high risk of primary nonfunction. This means of assessment allows livers to be transplanted that fail to function and almost certainly excludes other livers that would have performed adequately.

Machine preservation offers a window of opportunity during which assessment of an organ’s viability can be made. Perfusion characteristics as well as sampling of perfusate together yield useful information that can be correlated to transplant outcomes. This may not only lead to a more rational use of the current donor pool but lead to the accurate assessment of marginal organs, such as those procured from non-heart-beating donors and livers displaying severe fatty change; at present these are not transplanted clinically for fear of primary nonfunction.

Although simple and effective, cold storage may have reached its limitations in terms of preservation length and ability to maintain a viable organ devoid of ischemic injury. In addition this technique does not allow for viability assessment, and therefore the use of marginal organs remains difficult. By mimicking the organs natural physiological environment and providing oxygen and other substrates necessary for normal metabolism, normothermic machine perfusion may offer the next step toward the development of the ideal organ preservation technique.

CONCLUSION

We have clearly demonstrated that normothermic machine perfusion provides preservation superior to cold storage over a 24-hr period in terms of synthetic and metabolic serum markers, hemodynamic parameters, and histological appearances.

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