James H. Southard

IMPORTANT COMPONENTS OF THE UW SOLUTION

The UW solution for preservation of the liver, kidney, and pancreas contains a number of components, and the importance of each of these has not been fully resolved. In the studies reported here the importance of glutathione and adenosine is demonstrated in isolated cell models (rabbit renal tubules and rat liver hepatocytes) of hypothermic preservation and reperfusion and in dog renal transplantation. Glutathione in the UW solution is necessary for the preservation of the capability of the cell to regenerate ATP and maintain membrane integrity. Adenosine in the UW solution provides the preserved cell with substrates for the regeneration of ATP during the reperfusion period following cold storage. The omission of GHS from the UW solution results in poorer renal function in the 48 hr dog kidney preservation-transplant model. The role of other components of the UW solution is discussed including lactobionic acid; other impermeants; and the colloid, hydroxyethyl starch. It is concluded that the development of improved preservation solutions will require a more detailed understanding of the mechanism of injury due to cold storage and, once obtained, solutions more complex than the UW solution may be required for improved long-term storage of organs.

72-hour preservation of the canine pancreas

A new flushout solution for preservation of the pancreas was tested in the dog model of segmental pancreas autotransplantation. The solution osmolality was 320 mOsm/L, K+=120 mM, Na+=30 mM, and it contained the anion, lactobionate, and raffinose as impermeants to the cell. Preservation times studied were 48 and 72 hr. The pancreas was flushed out with about 250 ml of the new solution and stored at 0°C. Dogs were monitored postoperatively for blood glucose and intravenous glucose tolerance (IVGTT). Results were compared with control (no preservation) segmental pancreas autotransplants. Dogs receiving pancreases stored for 48 or 72 hr were normoglycemic on day one and remained normoglycemic for at least 28 days, or until time of sacrifice. Two of four dogs with pancreases stored for 48 hr were sacrificed on day 14 with normal IVGTT for histology. The remaining two dogs had normal pancreatic function for 28 days. Two of eight dogs receiving pancreas grafts after 72-hr cold storage died of causes unrelated to the pancreas graft, which was still functioning at the time of death. Six dogs remained normoglycemic and had a normal IVGTT at least for 28 days. This study demonstrates the feasibility of preserving the pancreas for three days for transplantation.

Preservation of the canine liver for 24-48 hours using simple cold storage with UW solution.

The results of a series of 29 orthotopic liver transplants in the dog are described. The livers were preserved in a new cold storage fluid, UW solution, and were successfully transplanted after periods of storage of 24, 30, 36, and 48 hr. All six animals transplanted after 24 hr survived beyond 5 days after transplantation and had excellent graft function. Four of six survived for at least 5 days after 30 hr of cold storage, and five of five after 36 hr. Five of six consecutive dogs that received transplants that had been cold-stored for 48 hr survived for 5 or more days. This solution represents a substantial advance over all existing cold storage solutions for liver preservation.

Development of a cold storage solution for pancreas preservation

Canine pancreas tissue slices were incubated at 5 degrees C for 24 hr in solutions containing different saccharides (raffinose, sucrose, mannitol, or glucose). At the end of incubation tissue water (TW expressed as kg H2O/kg dry wt) was determined as a measure of tissue edema. Tissue edema was greatest in slices stored in Eurocollins (EC) solution (TW = 4.96 +/- 0.14) which contains glucose for osmotic pressure. The degree of edema was decreased by saccharides in proportion to their molecular mass: mannitol (MW = 180, TW = 3.84 +/- 0.08), sucrose (MW = 348, TW = 3.54 +/- 0.08), and raffinose (MW = 594, TW = 3.30 +/- 0.07). Tissue edema was also greatest in slices incubated in solutions containing the smallest molecular mass anions: Cl- (TW = 4.02 +/- 0.16), gluconate (TW = 3.69 +/- 0.10), and lactobionate (TW = 3.28 +/- 0.13). Cold storage of the intact pancreas in EC solution for 24 hr did not induce as much edema as in slices (TW = 2.88 +/- 0.10). However, on isolated reperfusion at normothermia (37 degrees C) the pancreas became edematous (TW = 3.33 +/- 0.12). Storage of the pancreas in a lactobionate-raffinose solution did not induce edema after 90 min of normothermic reperfusion. The suppression of tissue edema in the pancreas may be essential to obtaining long-term preservation (24-72 hr) of this organ which is currently limited to about 6-8 hr in EC solution. The newly developed lactobionate-raffinose solution appears to control tissue edema in both tissue slices and the intact-flushed out organ.

Seventy-two-hour preservation of the canine liver by machine perfusion

The UW solution effectively preserves the dog liver for up to 48 hr by simple cold storage. This solution contains lactobionate as the primary impermeant. Another solution developed for machine perfusion of the kidney is similar to the UW solution but contains gluconate in place of lactobionate. In this study the UW gluconate solution was used for the continuous hypothermic machine perfusion of dog livers for 72 hr. Dog livers were continuously perfused at 5 degrees C through the portal vein at a pressure of 16-18 mm Hg and transplanted. Seven of 8 dogs survived for 7 or more days following orthotopic transplantation. The livers functioned as well as those preserved for 48 hr by cold storage in the UW solution as indicated by various liver-function tests. Successful machine perfusion was only achieved when the perfusate contained a high concentration of potassium (125 mM) but not with a high concentration of sodium (125 mM). This study demonstrates the feasibility of machine-perfusion preservation of the liver that yields longer preservation of equal quality compared to simple cold storage. For the development of truly long-term preservation (5 or more days) and better quality short-term preservation, machine perfusion may be the method of choice.

An analysis of the components in UW solution using the isolated perfused rabbit liver.

The isolated perfused rabbit liver model has been used to determine the essential components of the UW solution for hepatic preservation by simple cold storage. Livers were stored on ice for 48 hr after initial flushing with the solution being tested, and then reperfused at 38 degrees C in an isolated perfusion circuit; bile flow and enzyme (SGOT, SGPT, and LDH) release during a 2-hr period were recorded. All solutions tested contained phosphate (25 mM) as a buffer and magnesium sulfate (5 mM). Sodium can be substituted for potassium without adverse effects. Lactobionate, raffinose and glutathione cannot be omitted; all other components can be eliminated without altering the effectiveness of the solution in this model.

Hypothermic preservation of hepatocytes. I. Role of cell swelling.

Hepatocytes from isolated rat livers were hypothermically incubated (5 degrees C) in an oxygenated environment with continuous shaking (to simulate organ perfusion preservation). The incubation solution was either a tissue culture medium (L-15), an organ preservation perfusate (UW gluconate), or a simple cold-storage solution used for organ preservation (UW lactobionate). Hepatocyte viability was assessed from the release of lactate dehydrogenase (LDH) into the incubation medium. Cell swelling (due to the uptake of water) was also measured. Within 24 hr, hepatocytes hypothermically stored in each of the three incubation solutions became swollen (30 to 40% water gain) and lost a significant amount of LDH (as much as 60%). The addition of polyethylene glycol (PEG; relative molecular mass 8000; 5 g%) to the solutions suppressed cell swelling and allowed the incubated hepatocytes to remain relatively well preserved (30% LDH release) for as long as 120 hr. Adding either dextran (relative molecular mass 10,000 to 78,000; 5 g%) or saccharides (100 mmol/liter) instead of PEG neither prevented cell swelling nor prevented the cells from dying. The results of this study suggest (i) there is a direct correlation (r = 0.873) between hypothermia-induced cell swelling and cell death (i.e., the suppression of cell swelling prevents cell death); (ii) the mechanism by which PEG prevents cell swelling (and thus maintains cell viability) is not related to the osmotic or oncotic properties of the molecule but instead is apparently related to some unknown interaction between PEG and the cell, an interaction that provides stability during hypothermic incubation; and (iii) hypothermia-induced cell swelling must be prevented if isolated hepatocytes are to be used as a model for studying the mechanism by which cell damage occurs during hypothermic organ preservation. By eliminating cell death due to cell swelling, the biochemical mechanisms of cell death can be studied.

The effects of fasting on the quality of liver preservation by simple cold storage

Although livers can be successfully preserved for 24 hr or more, often the transplanted livers have poor or no (primary nonfunction) function. The quality of the liver does not appear dependent upon the time of preservation but may be dependent upon the condition of the donor. In this study we have investigated the effects of fasting on the quality of livers for transplantation. Rabbits were fasted (48 hr) and livers preserved in the UW solution for 6-8 hr. Functions of the liver were analyzed by isolated perfusion for 2 hr. Also, pigs were fasted for 72 hr, livers preserved for 12 hr, and viability determined by orthotopic transplantation. Fasting depleted the liver glycogen by 85% but had no effect on ATP or glutathione concentrations. Rabbit livers from fasted animals produced similar amounts of bile, released similar concentrations of lactate dehydrogenase (LDH) and aspartate amino transaminase (AST) into the perfusate, maintained similar concentrations of ATP and glutathione in the tissue, and had a similar intracellular K:Na ratio after 24-hr preservation when compared to livers from fed animals. After 48-hr preservation, livers from fasted animals were less viable than livers from fed animals, including: reduced bile production (2.0 +/- 0.3 vs. 5.0 +/- 0.9 ml/2 hr, 100 g), greater release of LDH (3701 +/- 562 units vs. 1123 +/- 98 units) and AST, less ATP (0.326 +/- 74 vs. 0.802 +/- 160 nmol/g), less glutathione (0.303 +/- 13 vs. 0.933 +/- 137 nmol/g), and a lower K:Na ratio (1.5 +/- 0.9 vs. 7.4 +/- 0.6). Pigs receiving livers from fed animals preserved for 12 hr had better survival (5/6, 83%) than livers from fasted animals (3/6, 50%). The results show that the nutritional status of the donor can affect the outcome of liver preservation and transplantation. Increased injury in livers from fasted animals may be due to the loss of glycogen that may be an essential source of energy in the initial posttransplant period. In clinical liver transplantation the nutritional status of the donor may be an important factor in the initial function of the liver, and methods to increase the nutritional status of the donor may be important in increasing the quality of livers.

Successful five-day perfusion preservation of the canine kidney

Over 20 years ago, successful 3-day-perfusion preservation of canine kidneys was obtained. Since then, consistent 5-day preservation has not been reported. In this study, we investigated how the perfusate calcium concentration affected both mitochondrial function and posttransplant viability in dog kidneys preserved for 5 days. Dog kidneys were preserved by machine perfusion (5 degrees C) using a hydroxyethyl starch-gluconate solution that contained either 0.0, 0.5, 1.5, or 5.0 mM calcium. Mitochondria isolated from preserved kidneys has a loss of respiratory control when either 0.0, 1.5, or 5.0 mM calcium were present. However, the use of a perfusate with 0.5 mM calcium preserved the mitochondrial function at levels equivalent to controls for 5 days. Transplantation of kidneys preserved for 5 days with 0.0 or 1.5 mM calcium yielded poor survival (0% and 17%, respectively). The use of a 0.5-mM calcium perfusate increased posttransplant survival to 63% (5 of 8 transplanted). Donor pretreatment of kidneys with chlorpromazine (2.5 mg/kg i.v.) did not improve the function of mitochondria isolated from preserved kidneys but did increase survival in the 1.5-mM calcium group to 67% (4 of 6 transplanted) and in the 0.5 mM calcium group to 100% (7 of 7 transplanted). This is the first report to document consistently successful 5-day preservation of canine kidneys and clearly shows the importance of the perfusate calcium concentration in long-term kidney preservation. The specific mechanism by which calcium or chlorpromazine exert their effect is not known, but it is apparent that excessively high or low concentrations of calcium are damaging to the preserved organ, and an optimal calcium concentration combined with metabolic inhibition of calcium-dependent pathways can significantly improve the function of organs preserved for extended time periods.

Donor brain death reduces survival after transplantation in rat livers preserved for 20 hr.

Background. Eighty percent of donor organs come from donors who have suffered brain trauma (brain-dead donors). This unphysiological state alters the hemodynamic and hormonal status of the organ donor. This can cause organ injury, which has been suggested to alter the immunological or inflammatory status of the organ after transplantation, and may lead to increased sensitivity of the organ to preservation/transplantation injury. In this study we asked the question: does brain death cause injury to the liver that decreases successful liver preservation? Methods. The rat liver transplant model was used to compare survival in rats receiving a liver from a brain-dead donor versus a non-brain-dead donor. Brain death was induced by inflation of a cranially placed balloon catheter. The rats were maintained normotensive with fluid infusion for 6 hr. The livers were flushed with University of Wisconsin (UW) solution and immediately transplanted or cold stored for 20 hr before transplantation. Results. Recipient survival with immediately transplanted livers or those stored for 20 hr was 100% with livers from non-brain-dead donors. However, survival decreased when livers were procured from brain-dead donors. Survival was 75% (6/8) when storage time was 0 hr and 20% (2/10) when the liver was cold stored for 20 hr before transplantation. Conclusion. This study shows that brain death induces alterations in the donor liver that make it more sensitive to preservation/reperfusion injury than livers from donors without brain death. The mechanism of injury to the liver caused by brain death is not known. Because most livers used clinically for transplantation come from brain-dead donors, it is possible that poor function of these livers is due to the intrinsic condition of the donor organ, more than the quality of the preservation. Methods to treat the brain-dead donor to improve the quality of the liver may be needed to allow better preservation of the organ and to give better outcome after liver transplantation.