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A comparison of cold storage solutions for hepatic preservation using the isolated perfused rabbit liver
CRYOBIOLOGY
25, 30&310 (1988)
A Comparison of Cold Storage Solutions for Hepatic Preservation Using the Isolated Perfused Rabbit Liver’ N. V. JAMIESON,2 R. SUNDBERG, S. LINDELL, J. H. SOUTHARD, AND F. 0. BELZER Department of Surgery, University of Wisconsin, 600 Highland Avenue, Madison, Wisconsin 53792 Rabbit livers were stored cold for periods of 6 or 24 hr and tested using the isolated perfused liver model. Five solutions were tested: Eurocollins (EC), Ross and Marshall’s hypertonic citrate (HC), modified plasma protein fraction (Cambridge PPF), Ringer lactate, and the recently developed “University of Wisconsin” (UW) solution. After storage livers were perfused with an erythrocyte-free oxygenated Krebs-Henseleit solution containing 4% bovine serum albumin at 38°C for 2 hr. Bile production proved to be the most sensitive index of liver function for discriminating between the various storage solutions and the different preservation times. After 6 hr of cold storage, bile production was similar to control liver bile production (9.8 * 2.4 ml/2 hr/lOO g) in livers stored in HC (8.8 2 2 ml), PPF (9.9 + 2.2 ml), and UW (10.3 2 1.9 ml); it was slightly depressed in EC (6.7 2 2.5 ml, P = 0.06), and markedly depressed in Ringer lactate (4.3 + 0.8 ml, P < 0.05). After 24 hr of cold storage bile production in UW-stored livers was near normal (9.3 * 0.7 ml) but significantly depressed (3.5-6.2 ml) in all other solutions tested. Release of enzymes into the normothermic perfusate was also measured (aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase). In this small series the differences between cold storage solutions did not always reach statistical significance although the trend was for less enzyme release in livers stored in UW solution. This technique permits rapid assessment and refinement of new storage methods and new solutions for liver preservation prior to testing in a large animal transplant model. The results suggest that UW solution is superior to other preservation solutions and would permit successful 24-hr storage of livers. 8 1988 Academic press, hc.
Clinical liver transplantation is now an established form of treatment for end-stage liver disease and some inborn errors of metabolism (21). Preservation of livers for transplantation remains unsatisfactory, with a safe upper limit of 6-8 hr using the currently available cold storage solutions. There has been little progress in this field over the last decade. A major difficulty in developing new solutions and preservation techniques is the complexity and expense of performing experimental liver grafts in the large animal model where survival is the main index of success. Received November 17, 1987; accepted January 22, 1988. r Supported in part by Grant No. DK 35143from the NIH. * Supported in part by the The Michael McGough Foundation against Liver Disease in Children and the Ralph Shackman Trust.
Failure of such experiments may be due to many factors other than poor liver preservation (22, 26) and considerations of cost and time limit the number of different techniques or solutions which can be assessed. Donors of livers for transplantation often become available in institutions many miles from the recipient center, occasionally even in another country. After harvesting, the liver is transported by road or air to the hospital where it is to be implanted. Because of the relative lack of complexity simple cold storage remains the chosen technique for clinical liver preservation during such transportation. The time constraints imposed by current preservation techniques require that the donor and recipient operations be closely coordinated in order to minimize the hypothermic ischemit period to which the liver is exposed. In the event of unanticipated time-
300 OOll-2240/88 $3.00 Copyright AU rights
8 1988 by Academic Press, Inc. of reproduction in any form reserved.
COLD
STORAGE
consuming difficulty in recipient hepatectomy or delays in transportation the result may be that due to the prolonged storage time the patient receives a compromised organ. Hypothermic perfusion has been applied experimentally with remarkable success for periods of up to 48 hr (24) but is technically complex in the case of the liver which has a dual blood supply and is difficult to combine with the requirements of distant procurement and transportation. In combination with hyperbaric oxygen a perfusion technique was used for some time by the Denver group (7) but was abandoned in favor of simpler cold flush techniques. Most transplant groups use solutions devised originally for renal preservation (9, 20), although one solution has been developed exclusively for the liver (30). The isolated perfused liver was probably first used by Claude Bernard over 100 years ago. Since then it has been used extensively in biochemical, physiological, and pharmacological studies (11). The technique has also been applied to the investigation of normothermic ischemic injury (4, 15, 17) and to the assessment of livers following periods of hypothermic preservation. Many of these experiments have used limited numbers of large animal livers (pig, dog, and calf), although multiple parameters have been studied in each case (1, 12, 16, 27). Others have employed a simpler system using rat livers (10, 18). We felt that there was a place for such a simple system to be used in the rapid assessment of new solutions for hypothermic preservation, thus allowing multiple variations to be tested before proceeding to large animal experiments. We describe here a series of experiments, using rabbit livers, comparing the three cold storage solutions in current use in hepatic transplantation, Eurocollins (9), modified plasma protein fraction (Cambridge PPF) (30), and Ross and Marshall’s hypertonic citrate (20). In addition, Ringer lactate and UW solution, a new cold stor-
301
SOLUTIONS
age solution recently described for successful 72-hr pancreas preservation (28), have been evaluated. MATERIALS
AND
METHODS
Organ Harvesting Livers were harvested from 60 nonfasted New Zealand White rabbits weighing 3.3 + 0.9 kg using intravenous barbiturate anesthesia. The abdomen was opened using a long midline incision, the bile duct was cannulated with a 5 French-gauge polyethylene catheter which was secured in place by a silk ligature, and the bile duct was then divided distally. The portal vein was dissected free and the hepatic artery was ligated. A perfusion cannula, 4-mm in diameter, was inserted into the portal vein, secured in place with a silk ligature, and, after dividing the infra hepatic vena cava, the liver was flushed in situ with 10&150 ml of the preservation fluid being assessed. The period of warm ischemia before commencing the washout was always less than 1 min. The solutions were at 4°C and a perfusion pressure of 50 cm of water was used. The compositions of the solutions used are given in Table 1. The liver was then excised leaving a small cuff of diaphragm around the suprahepatic vena cava. The cystic duct was ligated, the gallbladder was removed, and the organ was stored in 150 ml of the preservation fluid on ice. Storage Control livers were flushed with cold Ringer lactate and then immediately assessed on the isolated perfusion system. Three livers were flushed with warm Ringer lactate and then stored at 38°C for 2 hr to show the effects of severe injury. The remaining livers were stored on ice for periods of either 6 hr (a period common in clinical liver transplantation with satisfactory preservation) or 24 hr (after which time the liver would not normally be expected to have life sustaining function).
302
JAMIESON ET AL. TABLE 1 Composition of the Solutions Used for Preservation
Solutions tested (mkfkter)
Ringer lactate
Na Lactate K Lactobionate KH,P04 K,HPO, KC1 K Citrate Na Citrate Na HC03 NaCl CaCl, Mg SO, Glucose Mannitol Raffinose Adenosine Glutathione Allopurinol Insulin Bactrim Ampicillin Heparin HCl HES PPF Hydrocortisone PH mOsmol/liter
28
4
Eurocollins
Hypertonic citrate
1.5 42 15
Cambridge PPF
uw solution
15
100 25
27.5 27.5 10 100 1.5 35 194
4.0 1.4
5
187 30 5 3 1 100 U/liter 0.5 ml/liter 500 mg/liter 2000 u. 6 ml 0.1 N 5% 6.5 273
7.0 355
7.1 400
1 liter 250 mg 6.8 33&340
7.4 320-330
Note. The pH of the UW solution is adjusted to 7.4 using sodium hydroxide giving a final sodium concentration in this solution of 30 mM.
using Krebs-Henseleit bicarbonate solution with 4% bovine serum albumin (FracA specially designed polycarbonate per- tion V) and 50 mgkter of streptomycin sulfusion chamber was used. It is a cylinder fate and penicillin G (Sigma Chemical Co., 16-cm in diameter and 9 cm deep with a St Louis, MO). A Masterflex peristaltic cover and inlet and outlet ports for the per- pump (Cole-Palmer Instruments, Barfusate. To help maintain the perfusate tem- rington, IL) was used to perfuse the livers perature there is a heating coil in the lower at 3 ml/g/min (as an erythrocyte-free medipart of the chamber. Following cold storage um was being used (11)) via the portal vein the livers were flushed with 100 ml of in a recirculating system. The perfusate Ringer lactate at ambient temperature to re- was oxygenated with a 95% 0,/5% CO* move the preservation fluid. Each liver was mixture at a flow rate of 100-200 ml/min via then placed in the perfusion chamber where a membrane oxygenator (Model OSOO-2A it floated in warm perfusate and the portal SciMed, Minneapolis, MN), giving oxygen cannula was connected to the circuit inlet. tensions of 450-550 mm Hg. The pH was maintained at 7.4 + 0.1 by varying the 02/ The system is illustrated in Fig. 1. The livers were perfused for 2 hr at 38°C CO;! ratio (19) in accordance with the values
Isolated Perfusion
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COLD STORAGE SOLUTIONS
FIG. 1. Isolated perfused rabbit liver apparatus.
determined using an in line pH electrode (Cole-Palmer). Bile was collected in a graduated tube and the volume was recorded at 15min intervals. Perfusate samples were taken for enzyme (aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH)) and electrolyte estimations at 30-min intervals. Samples were also taken at 30-min intervals from the portal inflow and caval outflow for estimation of pH, PO,, pCO,, and base excess. Following a 30-min period to permit equilibration 5 mg of indocyanine green (ICG) (Sigma) was added to the perfusate and the rate of disappearance was determined using samples taken at 5-min intervals; in a similar fashion the percentage of the administered dose excreted in the bile was measured. The concentration of ICG was determined by measuring absorbance at 805 nm in diluted samples (13) using a uv-visible spectrophotometer (Model 9420 IBM Instrument Co.). At the end of the 2-hr period, samples of tissue were snap frozen in dry ice/acetone for estimation of adenine nucleotides using
high-performance liquid chromatography (32). Enzyme assays were performed using standard calorimetric techniques as follows, LDH kit 500, AST kit 505, and ALT kit 505-P (Sigma Diagnostics). Pet&sate oxygen and carbon dioxide tensions were measured immediately after the samples were taken using a Corning 158 pH/blood gas analyzer (Corning Medical, Medtield, MA). Liver weights were recorded before and after storage and at the end of the perfusion period. There were 5-g livers in each group. Statistical analysis was made using the MannWhitney U test. All results were expressed per 100 g of liver tissue and are shown as mean values with standard deviations. RESULTS
Bile flow commenced 5-10 minutes after the livers were placed in the circuit and was well maintained during the 2-hr perfusion. The results of total bile flow in the 2-hr period are summarized in Fig. 2 for all solu-
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JAMIESON
ET AL.
A 12
-1
T
TT
10 6 $4 6 s:p 6 5% 26
RINGER LACTATE
q q
4
HYPERTONIC CITFIATE CAMRIDGEPPF
2
FIG. 2. Bile production during reperfusion. (A) After depressed compared to those of controls but this did Ringer lactate differed significantly from controls (P < (P < 0.05). (B) After 24 hr of storage UW solution was each case) and did not differ significantly from control
tions following 6 and 24 hr of cold storage. After 6 hr of cold storage bile production was similar to control liver bile production (9.8 k 2.4 ml/2 hr/lOO g) in livers stored in HC (8.8 2 2 ml), PPF (9.9 k 2.2 ml), and UW (10.3 + 1.9 ml); slightly depressed in EC (6.7 k 2.5 ml p = 0.06); and significantly depressed in Ringer lactate (4.3 + 0.8 ml). After 24 hr of cold storage, bile production in UW-stored livers was near normal (9.3 k 0.7 ml) but significantly depressed (mean values 3.5-6.2 ml) in all other solutions tested. The results of the livers subjected to 2 hr of warm ischemia are included in the 24-hr storage results to show the effect of severe injury. ICG clearance from the perfusate was similar in all groups and appeared to reflect only the rate of flow through the liver, since it was removed almost completely in a sin-
6 hr of storage the values for Eurocollins were not reach statistical significance (P = 0.06). 0.05). UW solution is superior to Eurocollins superior to all the other solutions (P < 0.05 in values.
gle pass. Total ICG excreted in the bile during the 90 min following its addition to the circuit was also measured and is summarized in Fig. 3. After 6 hr of cold storage only Ringer lactate differed significantly from controls and after 24 hr Ringer lactate and PPF were inferior to controls (P < 0.05), but the difference for the other solutions did not reach statistical significance due to the variations seen within these small experimental groups. Enzyme release (AST, ALT, and LDH) into the perfusate was maximal during the first 30 min of perfusion and then reached a plateau, reflecting principally release from cells damaged during storage and initial reperfusion, rather than continuing damage during perfusion. The values reported here are those measured after 120 min of reperfusion and are summarized in Figs. 4-6.
COLD
STORAGE
305
SOLUTIONS
RINOEA LACTATE HYPERTONIC C
FIG. 3. Indocyanine green excretion during reperfusion. (A) After 6 hr of storage only Ringer lactate diiered significantly from control values (P < 0.05). (B) After 24 hr of storage Ringer lactate and PPF were significantly worse than controls (P < 0.05 in both cases). The differences for the other solutions did not reach statistical significance.
Standard deviations were relatively large, and although there was a trend for less enzyme release in livers stored in UW solution, the differences did not reach statistical significance in most cases. The statistical data is summarized in the legends accompanying these figures. Once again the results of the livers subjected to 2 hr of warm ischemia are included in the 24-hr storage results for comparison to show the effect of severe injury. All livers were able to resynthesize ATP during the course of the 2-hr perfusion period, levels varied between 1.74 k 0.64 and 2.05 + 0.4 FM/g wet weight. There was no statistically significant difference between the different storage solutions. There were no significant differences in the weights of the livers before and after storage or after completion of the perfu-
sion. The range of liver weights was similar in all groups (69-185 g). Perfusate potassium levels were measured at 15-min intervals; there were no significant changes in potassium concentration during the course of the perfusion in any of the groups. Oxygen tension differences between the portal inflow and caval outflow were measured as an index of oxygen usage and once again there was no difference between groups, the decrease in ~0, being approximately 20&250 mm Hg. DISCUSSION
Of the multiple parameters recorded for these experimental groups the most consistent and sensitive appeared to be the rate of bile production on reperfusion. This is in accord with the observation in clinical liver transplantation that well-preserved livers
306
COLD
STORAGE
SOLUTIONS
q
RINGER LACTATE ElJR3XXLlN.S HYPERTONIC CITRATE CAMBRIDGE PPF
j?d Et 0 65
RINGER LACTATE EUKZCU#S HYPERTONIC CITRATE CAMBRIDGE PPF
q 0
FIG. 4. AST release after preservation and reperfusion. (A) Following 6 hr of cold storage, UW solution was superior to Eurocollins (P CO.05) but did not differ significantly from the other storage solutions. (B) Following 24 hr of cold storage, UW solution did not differ significantly from the other storage solutions although it wa superior to Ringer lactate.
resume bile production promptly after re- tained in the absence of an infusion of bile vascularization and is in agreement with the salts although this would presumably have findings of other authors that bile produc- been necessary if we had continued the pertion is an important index in assessment of fusion for more than 120 min. Although all livers during isolated perfusion (2, 4, 15, livers were able to synthesize ATP during 27). Bile production is a complex, energy- the perfusion period, indicating satisfactory requiring process with two components, mitochondrial function, this was not reone bile salt dependent and one bile salt flected by a resumption of normal bile proindependent (5, 25). It is not a simple tiltra- duction. Bile flow is thus a complex protion process like urine formation; the rate cess requiring the integrity of multiple sysof flow is independent of perfusate flow tems and processes within the liver, rate over a relatively wide range (25) and is including those of anatomical, physiological, and biochemical nature. Bile producunaffected by increasing the hydrostatic pressure in the biliary tree by 4-9 cm of tion is thus the most complex of the paramwater above the perfusion pressure (5). It is eters recorded and its discriminant value is an integrated function requiring both the not unexpected. Indocyanine green is a water soluble tripreservation of the ultrastructural integrity of the liver and the maintenance of the abil- carbocyanine dye introduced in 1957 which ity of hepatocytes to synthesize ATP. Un- is excreted almost totally in an unchanged like rat livers (11) we found that in the rab- form in the bile (31). It has been used in bit liver a satisfactory bile flow was main- man to assess hepatic function by measur-
JAMIESON
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ET AL.
B 900 600
I 700 j ZHRS WARM ISCHAEMI
;$ St
500
45
600 i
2E
400 300
FIG. 5. ALT release after preservation and reperfusion. (A) Following 6 hr of cold storage, UW solution was superior to Ringer Lactate and Marshalls hypertonic citrate solution (P i 0.05 in each case) but did not differ significantly from the other storage solutions. (B) Following 24 hr of cold storage, UW solution was superior to Marshalls hypertonic citrate solution (P < 0.05) but did not differ significantly from the other storge solutions.
ing rate of clearance from the blood after a age to hepatocytes. In agreement with the single intravenously administered bolus findings of other authors (2, 4, 27), these (13). In our present study the results were measurements added little to the informadisappointing; the rate of disappearance tion given by the bile production. Even affrom the perfusate depended only on the ter 6 hr of cold storage when livers would perfusate flow rate and the measurement of be viable on transplantation, there were sigthe amount excreted in the bile was simply nificant rises in enzyme levels in all cases a poor and variable reflection of the rate of except for LDH levels in the UW group. bile production. Other authors, using Brom- When storage time was increased to 24 hr sulphthalein (which is handled in a fash- there were further increases in all groups, ion similar to ICG), have obtained compa- including the livers stored in UW solution. rable results (2, 27). It is possible that fur- Although trends could be detected with ther information could have been obtained lower enzyme release in livers stored in by investigating the kinetics of ICG excre- UW, the variability of the values within tion. groups was such that statistical significance The levels of intracellular enzymes was seldom achieved. Thus in these small (AST, ALT, and LDH) released into the groups, AST, ALT, and LDH were not able perfusate are an index of leakiness of the to predict the substantially improved procell membrane and hence indirectly of dam- tective effect of UW over the other solu-
308
COLD STORAGE SOLUTIONS
0 0
HYPERTCWC CITRATE CAMWDGEPPF
FIG. 6. LDH release after preservation and reperfusion. (A) Following 6 hr of cold storage, UW solution was superior to all the other solutions (P < 0.05 in all cases) and did not differ significantly from control values. (B) Following 24 hr of cold storage, values for UW solution did not differ significantly from those of other storage solutions, although they were superior to those for Ringer
tions suggested by bile flow values. Larger groups might show statistically significant differences but in our present study, bile flow was considered to be the most useful index of function. Numerous other parameters have been used to monitor the viability of livers during isolated perfusion for biochemical or pharmacological purposes (1 l), and of these additional investigations we chose to measure inflow/outflow oxygen difference to monitor oxygen utilization and potassium release as a marker of membrane damage and cell lysis. Neither of these proved of any value in discriminating between the storage solutions being tested. They appear to become abnormal only when the organ has sustained gross damage far in excess of that which would render it incapable of support-
ing life after transplantation. As such they are of no value in detecting small differences in performance of solutions for organ preservation. Using this model there was little difference between the currently applied cold storage solutions after 6 hr of preservation. This is not unexpected as they do provide relatively satisfactory clinical liver preservation for this length of time (3, 30). Following 24 hr of preservation all livers stored in these solutions performed poorly; this again was expected as livers stored in these solutions for this period are not viable on transplantation. Indeed after only 18 hr of preservation in these conventional preservation fluids poor survival rates have been reported (3, 23). By comparison the new UW solution appeared superior to all of the
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309
other solutions and after 24 hr of cold pres- simply because of considerations of time ervation livers were able to produce quan- for transportation. Using this model it is possible simply, tities of bile similar to those of control livrapidly, and inexpensively to carry out mulers. Wahlberg, in the initial tissue slice exper- tiple experiments investigating new or modiments performed in developing this new ified storage solutions or new storage techsolution based on lactobionate, raffinose, niques. The results can satisfactorily preof large animal and hydroxyethyl starch for pancreas pres- dict the outcome ervation, also studied liver slices and experiments which are much more timeshowed that lactobionate and raffinose consuming, complex, and expensive. Soluwere effective impermeants in the liver, un- tions and techniques can thus be screened, like glucose and mannitol which fulfill this developed, and brought to the large animal role in Eurocollins and Marshall’s solutions model only after they have been fully inves(29). Glucose and mannitol are effective im- tigated in this isolated perfusion system. permeants for renal preservation but will Further advances and improvements simireadily enter liver cells (8) resulting in lar to those displayed by the new solution swelling and cell damage. The role of the will thus be simplified and encouraged. other constituents in the new solution is not clear, they are included for theoretical reaREFERENCES sons and a study is planned to “dissect” the 1. Abouna, G. M. Pig liver perfusion with human solution using this model to see which are blood. The effect of preparing and flushing the necessary and which are not. liver with various balanced solutions on its subThe ultimate test of successful liver pressequent viability and function. Brit. J. Surg. 55, 761-768 (1968). ervation remains the ability to support life 2. Abouna, G. M., Ashcroft, T., Hull, C., Hodson, after orthotopic transplantation and, enA., Kirkley, J., and Walder, D. N. The assesscouraged by the excellent performance of ment of function of the isolated perfused porthis new solution in the isolated perfusion cine liver. Brit. J. Surg. 56, 289-295 (1969). 3. Benichou, J., Halgrimson, C. G., Weil, R., Koep, model, we have performed a series of transL. J., and Starzl, T. E. Canine and human liver plants in the dog. These confirmed the represervation for 6-18 hours by cold infusion. sults obtained in this simple perfusion sysTransplantation 24, 407-411 (1977). tem and demonstrated successful preserva4. Bowers, B. A., Branum, G. D., Rotolo, F. S., tion after 24 hr and 30 hr of cold storage Watters, C. R., and Meyers, W. C. Bile flow(14). A trial in human liver transplantation An index of ischemic injury. .I. Surg. Res. 42, 565-569 (1987). is now under way. If this solution lives up 5. Boyer, J. L. Canalicular bile information in the to its initial promise in prolonging safe storisolated perfused rat liver. Amer. J. Physiol. age times it will allow a substantial change 221, 1156-1163 (1971). in the clinical practice of liver transplanta6. Brauer, R. W., Leong, G. F., and Holloway, R. J. tion. Instead of being performed as emerMechanics of bile secretion: Effect of perfusion pressure and temperature on bile flow and bile gency procedures such transplants will be secretion pressure. Amer. J. Physiol. 177, 103performed as elective procedures under op112 (1954). timal conditions, and sharing of organs for 7. Brettschneider, L., Daloze, M. P., Huguet, C., urgent cases between distant centers will Porter, K. A., Groth, C. G., Kashiwagi, N., become possible. It will be of particular Hutchinson, D. E., and Starzl, T. E. The use of combined preservation techniques for extended value in pediatric practice where suitable storage of orthotopic liver homografts. Surg. donors are in short supply and matching of Gynecol. Obstet. 263-274 (1968). donor and recipient for size is of great im8. Cahill, G. F., Jr., Ashmore, J., Scott Earle, A., portance. At present otherwise suitable doand Zottu, S. Glucose penetration into liver. nors in a distant hospital may not be used Amer. J. Physiol. 192, 491496 (1958).
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20. Ross, R. H., Marshall, V. C., and Escott, M. L. 72-Hr Canine kidney preservation without continuous perfusion. Transplantation 21, 498-501 (1976). 21. Starzl, T. E., Iwatsuki, S., Shaw, B. W., Jr., and Gordon, R. D. Orthotopic liver transplantation in 1984. Transplant. Proc. 17, 250-258 (1985). 22. Starzl, T. E., Marchioro, T. L., Porter, K. A., Taylor, P. D., Faris, T. D., Herrmann, T. J., Hlad, C. J., and Waddell, W. R. Factors determining short- and long-term survival after orthotopic liver homotransplantation in the dog. Surgery 58, 131-155 (1965). 23. Tamaki, T., Kamada, N., and Pegg, D. E. Hypothermic preservation of the rat liver assessed by orthotopic transplantation-A comparison of flush solutions. Transplantation 41, 396-397 (1986). 24. Tamaki, T., Kamada, N., Wight, D. G., and Pegg, D. E. Successful 48-hr preservation of the rat liver by continuous hypothermic perfusion with Haemacel-isotonic citrate solution. Transplantation 43, 468-471 (1987). 25. Tavoloni, N., Reed, J. S., and Boyer, J. L. Hemodynamic effects on determinants of bile secretion in isolated rat liver. Amer. J. Physiol. 234, E584-E592 (1978). 26. Todo, S., Kam, I., Lynch, S., and Starzl, T. E. Animal research in liver transplantation with special reference to the dog. Semin. Liver Dis. 5, 309-317 (1985). 27. Vladovic-Relja, T. Some functional parameters in the isolated calf liver after hypothermic preservation. Cryobiology 21, 511-520 (1984). 28. Wahlberg, J. A., Love, R., Landegaard, L., Southard, J. H., and Belzer, R. 0. Successful 72 hour preservation of the canine pancreas. Transplantation 43, 5-8 (1987). 29. Wahlberg, J. A., Southard, J. H., and Belzer, F. 0. Development of a cold storage solution for pancreas preservation. Cryobiology 23,477482 (1986). 30. Wall, W. J., Calne, R. Y., Herbertson, B. M., Baker, P. G., Smith, D. P., Underwood, J., Kostakis, A., and Williams, R. Simple hypothermic preservation for transporting human livers long distances for transplantation. Transplantation 23, 21&216 (1977). 31. Wheeler, H. O., Cranston, W. I., and Meltzer, J. I. Hepatic uptake and biliary excretion of indocyanine green in the dog. Proc. Sot. Exp. Biol. Med. 99, 11-14 (1958). 32. Zakaria, M., and Brown, P. R. High-performance liquid column chromatography of nucleotides, nucleosides and bases. J. Chromatogr. 226, 267-290 (1981).
25, 30&310 (1988)
A Comparison of Cold Storage Solutions for Hepatic Preservation Using the Isolated Perfused Rabbit Liver’ N. V. JAMIESON,2 R. SUNDBERG, S. LINDELL, J. H. SOUTHARD, AND F. 0. BELZER Department of Surgery, University of Wisconsin, 600 Highland Avenue, Madison, Wisconsin 53792 Rabbit livers were stored cold for periods of 6 or 24 hr and tested using the isolated perfused liver model. Five solutions were tested: Eurocollins (EC), Ross and Marshall’s hypertonic citrate (HC), modified plasma protein fraction (Cambridge PPF), Ringer lactate, and the recently developed “University of Wisconsin” (UW) solution. After storage livers were perfused with an erythrocyte-free oxygenated Krebs-Henseleit solution containing 4% bovine serum albumin at 38°C for 2 hr. Bile production proved to be the most sensitive index of liver function for discriminating between the various storage solutions and the different preservation times. After 6 hr of cold storage, bile production was similar to control liver bile production (9.8 * 2.4 ml/2 hr/lOO g) in livers stored in HC (8.8 2 2 ml), PPF (9.9 + 2.2 ml), and UW (10.3 2 1.9 ml); it was slightly depressed in EC (6.7 2 2.5 ml, P = 0.06), and markedly depressed in Ringer lactate (4.3 + 0.8 ml, P < 0.05). After 24 hr of cold storage bile production in UW-stored livers was near normal (9.3 * 0.7 ml) but significantly depressed (3.5-6.2 ml) in all other solutions tested. Release of enzymes into the normothermic perfusate was also measured (aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase). In this small series the differences between cold storage solutions did not always reach statistical significance although the trend was for less enzyme release in livers stored in UW solution. This technique permits rapid assessment and refinement of new storage methods and new solutions for liver preservation prior to testing in a large animal transplant model. The results suggest that UW solution is superior to other preservation solutions and would permit successful 24-hr storage of livers. 8 1988 Academic press, hc.
Clinical liver transplantation is now an established form of treatment for end-stage liver disease and some inborn errors of metabolism (21). Preservation of livers for transplantation remains unsatisfactory, with a safe upper limit of 6-8 hr using the currently available cold storage solutions. There has been little progress in this field over the last decade. A major difficulty in developing new solutions and preservation techniques is the complexity and expense of performing experimental liver grafts in the large animal model where survival is the main index of success. Received November 17, 1987; accepted January 22, 1988. r Supported in part by Grant No. DK 35143from the NIH. * Supported in part by the The Michael McGough Foundation against Liver Disease in Children and the Ralph Shackman Trust.
Failure of such experiments may be due to many factors other than poor liver preservation (22, 26) and considerations of cost and time limit the number of different techniques or solutions which can be assessed. Donors of livers for transplantation often become available in institutions many miles from the recipient center, occasionally even in another country. After harvesting, the liver is transported by road or air to the hospital where it is to be implanted. Because of the relative lack of complexity simple cold storage remains the chosen technique for clinical liver preservation during such transportation. The time constraints imposed by current preservation techniques require that the donor and recipient operations be closely coordinated in order to minimize the hypothermic ischemit period to which the liver is exposed. In the event of unanticipated time-
300 OOll-2240/88 $3.00 Copyright AU rights
8 1988 by Academic Press, Inc. of reproduction in any form reserved.
COLD
STORAGE
consuming difficulty in recipient hepatectomy or delays in transportation the result may be that due to the prolonged storage time the patient receives a compromised organ. Hypothermic perfusion has been applied experimentally with remarkable success for periods of up to 48 hr (24) but is technically complex in the case of the liver which has a dual blood supply and is difficult to combine with the requirements of distant procurement and transportation. In combination with hyperbaric oxygen a perfusion technique was used for some time by the Denver group (7) but was abandoned in favor of simpler cold flush techniques. Most transplant groups use solutions devised originally for renal preservation (9, 20), although one solution has been developed exclusively for the liver (30). The isolated perfused liver was probably first used by Claude Bernard over 100 years ago. Since then it has been used extensively in biochemical, physiological, and pharmacological studies (11). The technique has also been applied to the investigation of normothermic ischemic injury (4, 15, 17) and to the assessment of livers following periods of hypothermic preservation. Many of these experiments have used limited numbers of large animal livers (pig, dog, and calf), although multiple parameters have been studied in each case (1, 12, 16, 27). Others have employed a simpler system using rat livers (10, 18). We felt that there was a place for such a simple system to be used in the rapid assessment of new solutions for hypothermic preservation, thus allowing multiple variations to be tested before proceeding to large animal experiments. We describe here a series of experiments, using rabbit livers, comparing the three cold storage solutions in current use in hepatic transplantation, Eurocollins (9), modified plasma protein fraction (Cambridge PPF) (30), and Ross and Marshall’s hypertonic citrate (20). In addition, Ringer lactate and UW solution, a new cold stor-
301
SOLUTIONS
age solution recently described for successful 72-hr pancreas preservation (28), have been evaluated. MATERIALS
AND
METHODS
Organ Harvesting Livers were harvested from 60 nonfasted New Zealand White rabbits weighing 3.3 + 0.9 kg using intravenous barbiturate anesthesia. The abdomen was opened using a long midline incision, the bile duct was cannulated with a 5 French-gauge polyethylene catheter which was secured in place by a silk ligature, and the bile duct was then divided distally. The portal vein was dissected free and the hepatic artery was ligated. A perfusion cannula, 4-mm in diameter, was inserted into the portal vein, secured in place with a silk ligature, and, after dividing the infra hepatic vena cava, the liver was flushed in situ with 10&150 ml of the preservation fluid being assessed. The period of warm ischemia before commencing the washout was always less than 1 min. The solutions were at 4°C and a perfusion pressure of 50 cm of water was used. The compositions of the solutions used are given in Table 1. The liver was then excised leaving a small cuff of diaphragm around the suprahepatic vena cava. The cystic duct was ligated, the gallbladder was removed, and the organ was stored in 150 ml of the preservation fluid on ice. Storage Control livers were flushed with cold Ringer lactate and then immediately assessed on the isolated perfusion system. Three livers were flushed with warm Ringer lactate and then stored at 38°C for 2 hr to show the effects of severe injury. The remaining livers were stored on ice for periods of either 6 hr (a period common in clinical liver transplantation with satisfactory preservation) or 24 hr (after which time the liver would not normally be expected to have life sustaining function).
302
JAMIESON ET AL. TABLE 1 Composition of the Solutions Used for Preservation
Solutions tested (mkfkter)
Ringer lactate
Na Lactate K Lactobionate KH,P04 K,HPO, KC1 K Citrate Na Citrate Na HC03 NaCl CaCl, Mg SO, Glucose Mannitol Raffinose Adenosine Glutathione Allopurinol Insulin Bactrim Ampicillin Heparin HCl HES PPF Hydrocortisone PH mOsmol/liter
28
4
Eurocollins
Hypertonic citrate
1.5 42 15
Cambridge PPF
uw solution
15
100 25
27.5 27.5 10 100 1.5 35 194
4.0 1.4
5
187 30 5 3 1 100 U/liter 0.5 ml/liter 500 mg/liter 2000 u. 6 ml 0.1 N 5% 6.5 273
7.0 355
7.1 400
1 liter 250 mg 6.8 33&340
7.4 320-330
Note. The pH of the UW solution is adjusted to 7.4 using sodium hydroxide giving a final sodium concentration in this solution of 30 mM.
using Krebs-Henseleit bicarbonate solution with 4% bovine serum albumin (FracA specially designed polycarbonate per- tion V) and 50 mgkter of streptomycin sulfusion chamber was used. It is a cylinder fate and penicillin G (Sigma Chemical Co., 16-cm in diameter and 9 cm deep with a St Louis, MO). A Masterflex peristaltic cover and inlet and outlet ports for the per- pump (Cole-Palmer Instruments, Barfusate. To help maintain the perfusate tem- rington, IL) was used to perfuse the livers perature there is a heating coil in the lower at 3 ml/g/min (as an erythrocyte-free medipart of the chamber. Following cold storage um was being used (11)) via the portal vein the livers were flushed with 100 ml of in a recirculating system. The perfusate Ringer lactate at ambient temperature to re- was oxygenated with a 95% 0,/5% CO* move the preservation fluid. Each liver was mixture at a flow rate of 100-200 ml/min via then placed in the perfusion chamber where a membrane oxygenator (Model OSOO-2A it floated in warm perfusate and the portal SciMed, Minneapolis, MN), giving oxygen cannula was connected to the circuit inlet. tensions of 450-550 mm Hg. The pH was maintained at 7.4 + 0.1 by varying the 02/ The system is illustrated in Fig. 1. The livers were perfused for 2 hr at 38°C CO;! ratio (19) in accordance with the values
Isolated Perfusion
303
COLD STORAGE SOLUTIONS
FIG. 1. Isolated perfused rabbit liver apparatus.
determined using an in line pH electrode (Cole-Palmer). Bile was collected in a graduated tube and the volume was recorded at 15min intervals. Perfusate samples were taken for enzyme (aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH)) and electrolyte estimations at 30-min intervals. Samples were also taken at 30-min intervals from the portal inflow and caval outflow for estimation of pH, PO,, pCO,, and base excess. Following a 30-min period to permit equilibration 5 mg of indocyanine green (ICG) (Sigma) was added to the perfusate and the rate of disappearance was determined using samples taken at 5-min intervals; in a similar fashion the percentage of the administered dose excreted in the bile was measured. The concentration of ICG was determined by measuring absorbance at 805 nm in diluted samples (13) using a uv-visible spectrophotometer (Model 9420 IBM Instrument Co.). At the end of the 2-hr period, samples of tissue were snap frozen in dry ice/acetone for estimation of adenine nucleotides using
high-performance liquid chromatography (32). Enzyme assays were performed using standard calorimetric techniques as follows, LDH kit 500, AST kit 505, and ALT kit 505-P (Sigma Diagnostics). Pet&sate oxygen and carbon dioxide tensions were measured immediately after the samples were taken using a Corning 158 pH/blood gas analyzer (Corning Medical, Medtield, MA). Liver weights were recorded before and after storage and at the end of the perfusion period. There were 5-g livers in each group. Statistical analysis was made using the MannWhitney U test. All results were expressed per 100 g of liver tissue and are shown as mean values with standard deviations. RESULTS
Bile flow commenced 5-10 minutes after the livers were placed in the circuit and was well maintained during the 2-hr perfusion. The results of total bile flow in the 2-hr period are summarized in Fig. 2 for all solu-
304
JAMIESON
ET AL.
A 12
-1
T
TT
10 6 $4 6 s:p 6 5% 26
RINGER LACTATE
q q
4
HYPERTONIC CITFIATE CAMRIDGEPPF
2
FIG. 2. Bile production during reperfusion. (A) After depressed compared to those of controls but this did Ringer lactate differed significantly from controls (P < (P < 0.05). (B) After 24 hr of storage UW solution was each case) and did not differ significantly from control
tions following 6 and 24 hr of cold storage. After 6 hr of cold storage bile production was similar to control liver bile production (9.8 k 2.4 ml/2 hr/lOO g) in livers stored in HC (8.8 2 2 ml), PPF (9.9 k 2.2 ml), and UW (10.3 + 1.9 ml); slightly depressed in EC (6.7 k 2.5 ml p = 0.06); and significantly depressed in Ringer lactate (4.3 + 0.8 ml). After 24 hr of cold storage, bile production in UW-stored livers was near normal (9.3 k 0.7 ml) but significantly depressed (mean values 3.5-6.2 ml) in all other solutions tested. The results of the livers subjected to 2 hr of warm ischemia are included in the 24-hr storage results to show the effect of severe injury. ICG clearance from the perfusate was similar in all groups and appeared to reflect only the rate of flow through the liver, since it was removed almost completely in a sin-
6 hr of storage the values for Eurocollins were not reach statistical significance (P = 0.06). 0.05). UW solution is superior to Eurocollins superior to all the other solutions (P < 0.05 in values.
gle pass. Total ICG excreted in the bile during the 90 min following its addition to the circuit was also measured and is summarized in Fig. 3. After 6 hr of cold storage only Ringer lactate differed significantly from controls and after 24 hr Ringer lactate and PPF were inferior to controls (P < 0.05), but the difference for the other solutions did not reach statistical significance due to the variations seen within these small experimental groups. Enzyme release (AST, ALT, and LDH) into the perfusate was maximal during the first 30 min of perfusion and then reached a plateau, reflecting principally release from cells damaged during storage and initial reperfusion, rather than continuing damage during perfusion. The values reported here are those measured after 120 min of reperfusion and are summarized in Figs. 4-6.
COLD
STORAGE
305
SOLUTIONS
RINOEA LACTATE HYPERTONIC C
FIG. 3. Indocyanine green excretion during reperfusion. (A) After 6 hr of storage only Ringer lactate diiered significantly from control values (P < 0.05). (B) After 24 hr of storage Ringer lactate and PPF were significantly worse than controls (P < 0.05 in both cases). The differences for the other solutions did not reach statistical significance.
Standard deviations were relatively large, and although there was a trend for less enzyme release in livers stored in UW solution, the differences did not reach statistical significance in most cases. The statistical data is summarized in the legends accompanying these figures. Once again the results of the livers subjected to 2 hr of warm ischemia are included in the 24-hr storage results for comparison to show the effect of severe injury. All livers were able to resynthesize ATP during the course of the 2-hr perfusion period, levels varied between 1.74 k 0.64 and 2.05 + 0.4 FM/g wet weight. There was no statistically significant difference between the different storage solutions. There were no significant differences in the weights of the livers before and after storage or after completion of the perfu-
sion. The range of liver weights was similar in all groups (69-185 g). Perfusate potassium levels were measured at 15-min intervals; there were no significant changes in potassium concentration during the course of the perfusion in any of the groups. Oxygen tension differences between the portal inflow and caval outflow were measured as an index of oxygen usage and once again there was no difference between groups, the decrease in ~0, being approximately 20&250 mm Hg. DISCUSSION
Of the multiple parameters recorded for these experimental groups the most consistent and sensitive appeared to be the rate of bile production on reperfusion. This is in accord with the observation in clinical liver transplantation that well-preserved livers
306
COLD
STORAGE
SOLUTIONS
q
RINGER LACTATE ElJR3XXLlN.S HYPERTONIC CITRATE CAMBRIDGE PPF
j?d Et 0 65
RINGER LACTATE EUKZCU#S HYPERTONIC CITRATE CAMBRIDGE PPF
q 0
FIG. 4. AST release after preservation and reperfusion. (A) Following 6 hr of cold storage, UW solution was superior to Eurocollins (P CO.05) but did not differ significantly from the other storage solutions. (B) Following 24 hr of cold storage, UW solution did not differ significantly from the other storage solutions although it wa superior to Ringer lactate.
resume bile production promptly after re- tained in the absence of an infusion of bile vascularization and is in agreement with the salts although this would presumably have findings of other authors that bile produc- been necessary if we had continued the pertion is an important index in assessment of fusion for more than 120 min. Although all livers during isolated perfusion (2, 4, 15, livers were able to synthesize ATP during 27). Bile production is a complex, energy- the perfusion period, indicating satisfactory requiring process with two components, mitochondrial function, this was not reone bile salt dependent and one bile salt flected by a resumption of normal bile proindependent (5, 25). It is not a simple tiltra- duction. Bile flow is thus a complex protion process like urine formation; the rate cess requiring the integrity of multiple sysof flow is independent of perfusate flow tems and processes within the liver, rate over a relatively wide range (25) and is including those of anatomical, physiological, and biochemical nature. Bile producunaffected by increasing the hydrostatic pressure in the biliary tree by 4-9 cm of tion is thus the most complex of the paramwater above the perfusion pressure (5). It is eters recorded and its discriminant value is an integrated function requiring both the not unexpected. Indocyanine green is a water soluble tripreservation of the ultrastructural integrity of the liver and the maintenance of the abil- carbocyanine dye introduced in 1957 which ity of hepatocytes to synthesize ATP. Un- is excreted almost totally in an unchanged like rat livers (11) we found that in the rab- form in the bile (31). It has been used in bit liver a satisfactory bile flow was main- man to assess hepatic function by measur-
JAMIESON
307
ET AL.
B 900 600
I 700 j ZHRS WARM ISCHAEMI
;$ St
500
45
600 i
2E
400 300
FIG. 5. ALT release after preservation and reperfusion. (A) Following 6 hr of cold storage, UW solution was superior to Ringer Lactate and Marshalls hypertonic citrate solution (P i 0.05 in each case) but did not differ significantly from the other storage solutions. (B) Following 24 hr of cold storage, UW solution was superior to Marshalls hypertonic citrate solution (P < 0.05) but did not differ significantly from the other storge solutions.
ing rate of clearance from the blood after a age to hepatocytes. In agreement with the single intravenously administered bolus findings of other authors (2, 4, 27), these (13). In our present study the results were measurements added little to the informadisappointing; the rate of disappearance tion given by the bile production. Even affrom the perfusate depended only on the ter 6 hr of cold storage when livers would perfusate flow rate and the measurement of be viable on transplantation, there were sigthe amount excreted in the bile was simply nificant rises in enzyme levels in all cases a poor and variable reflection of the rate of except for LDH levels in the UW group. bile production. Other authors, using Brom- When storage time was increased to 24 hr sulphthalein (which is handled in a fash- there were further increases in all groups, ion similar to ICG), have obtained compa- including the livers stored in UW solution. rable results (2, 27). It is possible that fur- Although trends could be detected with ther information could have been obtained lower enzyme release in livers stored in by investigating the kinetics of ICG excre- UW, the variability of the values within tion. groups was such that statistical significance The levels of intracellular enzymes was seldom achieved. Thus in these small (AST, ALT, and LDH) released into the groups, AST, ALT, and LDH were not able perfusate are an index of leakiness of the to predict the substantially improved procell membrane and hence indirectly of dam- tective effect of UW over the other solu-
308
COLD STORAGE SOLUTIONS
0 0
HYPERTCWC CITRATE CAMWDGEPPF
FIG. 6. LDH release after preservation and reperfusion. (A) Following 6 hr of cold storage, UW solution was superior to all the other solutions (P < 0.05 in all cases) and did not differ significantly from control values. (B) Following 24 hr of cold storage, values for UW solution did not differ significantly from those of other storage solutions, although they were superior to those for Ringer
tions suggested by bile flow values. Larger groups might show statistically significant differences but in our present study, bile flow was considered to be the most useful index of function. Numerous other parameters have been used to monitor the viability of livers during isolated perfusion for biochemical or pharmacological purposes (1 l), and of these additional investigations we chose to measure inflow/outflow oxygen difference to monitor oxygen utilization and potassium release as a marker of membrane damage and cell lysis. Neither of these proved of any value in discriminating between the storage solutions being tested. They appear to become abnormal only when the organ has sustained gross damage far in excess of that which would render it incapable of support-
ing life after transplantation. As such they are of no value in detecting small differences in performance of solutions for organ preservation. Using this model there was little difference between the currently applied cold storage solutions after 6 hr of preservation. This is not unexpected as they do provide relatively satisfactory clinical liver preservation for this length of time (3, 30). Following 24 hr of preservation all livers stored in these solutions performed poorly; this again was expected as livers stored in these solutions for this period are not viable on transplantation. Indeed after only 18 hr of preservation in these conventional preservation fluids poor survival rates have been reported (3, 23). By comparison the new UW solution appeared superior to all of the
JAMIESON ET AL.
309
other solutions and after 24 hr of cold pres- simply because of considerations of time ervation livers were able to produce quan- for transportation. Using this model it is possible simply, tities of bile similar to those of control livrapidly, and inexpensively to carry out mulers. Wahlberg, in the initial tissue slice exper- tiple experiments investigating new or modiments performed in developing this new ified storage solutions or new storage techsolution based on lactobionate, raffinose, niques. The results can satisfactorily preof large animal and hydroxyethyl starch for pancreas pres- dict the outcome ervation, also studied liver slices and experiments which are much more timeshowed that lactobionate and raffinose consuming, complex, and expensive. Soluwere effective impermeants in the liver, un- tions and techniques can thus be screened, like glucose and mannitol which fulfill this developed, and brought to the large animal role in Eurocollins and Marshall’s solutions model only after they have been fully inves(29). Glucose and mannitol are effective im- tigated in this isolated perfusion system. permeants for renal preservation but will Further advances and improvements simireadily enter liver cells (8) resulting in lar to those displayed by the new solution swelling and cell damage. The role of the will thus be simplified and encouraged. other constituents in the new solution is not clear, they are included for theoretical reaREFERENCES sons and a study is planned to “dissect” the 1. Abouna, G. M. Pig liver perfusion with human solution using this model to see which are blood. The effect of preparing and flushing the necessary and which are not. liver with various balanced solutions on its subThe ultimate test of successful liver pressequent viability and function. Brit. J. Surg. 55, 761-768 (1968). ervation remains the ability to support life 2. Abouna, G. M., Ashcroft, T., Hull, C., Hodson, after orthotopic transplantation and, enA., Kirkley, J., and Walder, D. N. The assesscouraged by the excellent performance of ment of function of the isolated perfused porthis new solution in the isolated perfusion cine liver. Brit. J. Surg. 56, 289-295 (1969). 3. Benichou, J., Halgrimson, C. G., Weil, R., Koep, model, we have performed a series of transL. J., and Starzl, T. E. Canine and human liver plants in the dog. These confirmed the represervation for 6-18 hours by cold infusion. sults obtained in this simple perfusion sysTransplantation 24, 407-411 (1977). tem and demonstrated successful preserva4. Bowers, B. A., Branum, G. D., Rotolo, F. S., tion after 24 hr and 30 hr of cold storage Watters, C. R., and Meyers, W. C. Bile flow(14). A trial in human liver transplantation An index of ischemic injury. .I. Surg. Res. 42, 565-569 (1987). is now under way. If this solution lives up 5. Boyer, J. L. Canalicular bile information in the to its initial promise in prolonging safe storisolated perfused rat liver. Amer. J. Physiol. age times it will allow a substantial change 221, 1156-1163 (1971). in the clinical practice of liver transplanta6. Brauer, R. W., Leong, G. F., and Holloway, R. J. tion. Instead of being performed as emerMechanics of bile secretion: Effect of perfusion pressure and temperature on bile flow and bile gency procedures such transplants will be secretion pressure. Amer. J. Physiol. 177, 103performed as elective procedures under op112 (1954). timal conditions, and sharing of organs for 7. Brettschneider, L., Daloze, M. P., Huguet, C., urgent cases between distant centers will Porter, K. A., Groth, C. G., Kashiwagi, N., become possible. It will be of particular Hutchinson, D. E., and Starzl, T. E. The use of combined preservation techniques for extended value in pediatric practice where suitable storage of orthotopic liver homografts. Surg. donors are in short supply and matching of Gynecol. Obstet. 263-274 (1968). donor and recipient for size is of great im8. Cahill, G. F., Jr., Ashmore, J., Scott Earle, A., portance. At present otherwise suitable doand Zottu, S. Glucose penetration into liver. nors in a distant hospital may not be used Amer. J. Physiol. 192, 491496 (1958).
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