Hypothermic machine perfusion of rat livers preserves endothelial cell function.

This study was conducted to investigate the effect of starch in the preservation solution during hypothermic machine perfusion (HMP) on endothelial cell and hepatocyte functions in an isolated perfused rat liver model. Livers isolated from male Sprague-Dawley rats were perfused with the University of Wisconsin (UW) solution (HMP + starch group); modified UW solution (starch omitted) (HMP - starch group) at 0.4 mL/min per g liver; or simply stored in the UW solution (SCS group) at 4 degrees C for 24 hours. Following preservation, livers from HMP + starch, HMP - starch, SCS, and control group (without preservation) were perfused with Krebs-Henseleit Buffer solution at 37 degrees C for 30 minutes. Samples were taken every 10 minutes during 30-minute warm perfusion to assess hepatocyte and endothelial cell function and damage. After 24 hours of hypothermic preservation and 30 minutes rewarming, livers in the HMP + starch group displayed significantly lower lactate dehydrogenase levels and higher bile production. Endothelial cell function was also improved as indicated by hyaluronic acid uptake and shorter transient time for albumin observed in a multiple indicator dilution study. Liver wet and dry ratio and histological findings confirmed reduced edema formation in the tissue of the HMP + starch group livers compared with that of the HMP - starch and SCS group livers. These results suggest that HMP with the UW solution containing starch improve endothelial cell function and induce less hepatocellular damage following 24-hour preservation compared to SCS and HMP with the starch-free UW solution. These results also suggest that oncotic support may be an important component in preserving hepatic microcirculation in HMP.

The future of kidney preservation.

Both methods of hypothermic preservation of kidneys currently used clinically have been and continue to be useful procedures for the transplant surgeon. Each method has its limitations, benefits, and potential detriments. Which method is best is not a fully resolved question and the method of choice appears to be dependent upon, to a large extent, personal preference and circumstances. There is, however, a vital need for long-term kidney preservation. Obtaining this goal will most likely require carefully controlled experimental studies linking the biochemical and physiological aspects of organ metabolism at hypothermia to the design of perfusates, perfusion machine technology, and drug therapy. Improvements in perfusion preservation methodology based on sound basic data rather than by serendipity appears to offer the best opportunity for obtaining good quality long-term preservation.

Membrane stabilizing effects of calcium and taxol during the cold storage of isolated rat hepatocytes.

BACKGROUND: Calcium plays an important role in liver preservation and preservation induces depletion of cellular Ca. This may affect hepatocyte cytoskeleton integrity necessary for maintaining cell shape and organ viability. We tested the effects of a microtubular stabilizer (Taxol) in liver cell preservation. METHODS: Isolated rat hepatocytes were preincubated with or without a microtubule stabilizing agent, 100 microM Taxol, at 37 degrees C for 20 min, then stored in the University of Wisconsin (UW) solution +/-1.5 mM CaC12 at 4 degrees C for up to 48 hr. After storage, the cells were rewarmed in Krebs-Henseleit buffer with air at 37 degrees C for 1 hr. Morphological changes in the plasma membrane (scanning electron microscopy) and cell viability (percentage of lactate dehydrogenase [LDH] release) before and after rewarming were studied. RESULTS: Hepatocytes showed time-dependent increase in bleb formation (cytoskeleton disruption) during cold storage. Rewarming the cells caused even greater bleb formation and increased LDH release (cell death). Pretreatment of cells with Taxol and cold storage in the UW solution with 1.5 mM Ca suppressed both bleb formation and LDH release in 48-hr coldstored cells. CONCLUSIONS: Cold storage of hepatocytes leads to reperfusion injury and cell death. This can be suppressed with Taxol and Ca. This suggests that hypothermia induces changes in cellular Ca and a disruption of the microtubules, leading to loss of cell viability. Improved liver preservation may require suppression of Ca-dependent disruption of the cytoskeleton system of liver cells.

Alteration in cellular calcium and mitochondrial functions in the rat liver during cold preservation.

BACKGROUND: Preservation injury is multifactorial and its mechanism is still incompletely defined. Calcium may play an important role in preservation injury. METHODS: The effects of hypothermia on cytosolic free calcium concentration ([Ca2+]I) and total cellular calcium content in isolated rat hepatocytes were investigated by using fura-2 fluorescence and atomic absorption spectroscopy. Fura-2 loaded cells were placed into a prechilled (7 degrees C) cuvette equipped with a stirrer or preserved in the University of Wisconsin (UW) solution for up to 48 hr. In some experiments, cells were pretreated with inhibitors of Ca2+ release from mitochondria (m-iodobenzylguanidine [MIBG]) and from endoplasmic reticulum (ryanodine [RYA]) for 20 min at 37 degrees C. Mitochondrial functions after preservation were evaluated by measuring ATP and respiratory rates. RESULTS: Cooling to 7 degrees C caused a rapid increase in [Ca2+]I that was substantially blocked by MIBG and RYA pretreatment. The elevated calcium gradually leaked out of the cells into the Ca2+-free medium. In long-term storage of the cells in the UW solution, there was a marked decrease in both cytosolic free calcium and total cellular calcium. Pretreatment of the livers with MIBG before cold preservation in the UW solution resulted in a stimulation of ATP regeneration in tissue slices. MIBG pretreatment also improved mitochondrial respiratory functions after cold preservation. CONCLUSIONS: Thus, the loss of mitochondrial function after liver preservation in the UW solution may be related to the effects of hypothermia on calcium metabolism. Approaches to help maintain calcium homeostasis during storage may improve organ preservation.

Livers from fasted rats acquire resistance to warm and cold ischemia injury.

Successful liver transplantation is dependent upon many factors, one of which is the quality of the donor organ. Previous studies have suggested that the donor nutritional status may affect the outcome of liver transplantation and starvation, due to prolonged stay in the intensive care unit, may adversely affect the liver. In this study we have used the orthotopic rat liver transplant model to measure how fasting the donor affects the outcome of liver transplantation. Rat livers were preserved with UW solution either at 37 degrees C (warm ischemia for 45-60 min) or at 4 degrees C (cold ischemia for 30 or 44 hr). After preservation the livers were orthotopically transplanted and survival (for 7 days) was measured, as well as liver functions 6 hr after transplantation. After 45 min of warm ischemia 50% (3 of 6) animals survived when the liver was obtained from a fed donor about 80% (4 of 5) survived when the liver was obtained from a three-day-fasted donor. After 60 min warm ischemia no animal survived (0 of 8, fed group). However, if the donor was fasted for 3 days 89% (8 of 9) of the animals survived for 7 days. Livers cold-stored for 30 hr were 50% viable (3 of 6) and fasting for 1-3 days did not affect this outcome. However, if the donor was fasted for 4 days 100% (9 of 9) survival was obtained. After 44-hr preservation only 29% (2/7) of the recipients survived for 7 days. If the donor was fasted for 4 days, survival increased to 83% (5/6). Liver functions, bile production, and serum enzymes were better in livers from the fasted rats than from the fed rats. Fasting caused a 95% decrease in liver glycogen content. Even with this low concentration of glycogen, liver viability (animal survival) after warm or cold ischemia was not affected, and livers with a low glycogen content were fully viable. Thus liver glycogen does not appear to be important in liver preservation. This study shows that fasting the donor does not cause injury to the liver after warm or cold ischemia. In fact, the livers appeared to be better able to tolerate ischemia when obtained from fasted rats. Thus donor nutritional status may be an important factor for outcome of liver transplantation. Livers from fasted donors may be capable of tolerating long-term preservation better than livers from fed donors.

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.

Kupffer cells depress hepatocyte protein synthesis on cold storage of the rat liver.

The causes of liver failure after transplantation are multifactorial. An understanding of the mechanisms of injury to the liver could help to define methods to improve preservation and transplantation. We measured protein synthesis by 3H-leucine incorporation into acid precipitable protein in rat liver tissue slices, isolated hepatocytes, and isolated perfused liver (IPL) after cold storage for 24 or 48 hr in University of Wisconsin (UW) solution. Some rats were pretreated with dexamethasone prior to liver harvest. Protein synthesis was depressed in all in vitro models after 24 hr storage. The percent decrease was greater in tissue slices and IPL (about 70% decrease relative to fresh livers) than in isolated hepatocytes (about 30% decrease). Dexamethasone pretreatment improved protein synthesis significantly after 24 hr preservation in tissue slices and in IPL, but had no significant effect on protein synthesis in isolated hepatocytes. The greater loss of protein synthesis in tissue slices and IPL compared with that in isolated hepatocytes was considered in relation to the presence of Kupffer cells in the former systems and lack of Kupffer cells in the isolated cell suspensions. Kupffer cells generate cytotoxins that could cause injury to metabolically depressed hepatocytes or endothelial cells. Dexamethasone has been shown to modulate Kupffer cell inhibition of hepatocyte functions. The results suggest that preservation damage to hepatocytes sensitizes them to further damage on reperfusion by Kupffer cell-generated agents.

A comparison of histidine-lactobionate and UW solution in 48-hour dog liver preservation.

Many modifications of the UW solution have been reported to yield successful results in rat liver preservation and transplantation. One solution used histidine, in combination with lactobionate (HL-I), and gave superior preservation of the rat liver when compared with the UW solution. In this study we have compared the HL-I solution with 90 mM histidine, HL-II solution with 30 mM histidine, and the UW solution in dog liver preservation and transplantation. Dog livers were preserved for 48 hr in one of the three solutions and transplanted. The peak AST and ALT values were highest in livers preserved in HL-I, intermediate in UW solution, and lowest in HL-II. However, there were no significant differences among survival rates (average 5-7 days per group), posttransplant serum concentration of liver enzymes (AST, ALT, LDH, and alk-phos), clotting factors (PT and PTT), bilirubin, and fibrinogen concentration for each group. Dogs were sacrificed or died within 5-7 days due to rejection in nonimmunosuppressed dogs. Also, rat livers were preserved in the HL-II solution or in a solution in which histidine was replaced by isoleucine (IL-I). Isoleucine is an amino acid with a molecular mass similar to that of histidine, but is not as good a hydrogen ion buffer as histidine at the pH used for liver preservation (7.4). The buffer capacity of the IL-I solution was similar to the UW solution, but about one-half as much as the HL-II solution. Rats receiving a liver preserved for 30 hr in HL-II or IL-I were 100% viable. Rats receiving a liver preserved for 40-44 hr in HL-II or IL-I showed less survival (33% and 25%, respectively). This shows that histidine can be effectively replaced by isoleucine in a preservation solution and gives equivalent preservation results. Thus, the mechanism of improvement of liver preservation with histidine is not due to its action as a hydrogen ion buffer. These studies show that, although the HL solutions are superior for preservation of the rat liver, they are not superior to the UW solution for preservation of the dog liver. However, as others have shown in the rat liver transplant model, a simplified UW solution (HL-II) appears effective in dog liver preservation. The dog liver transplant model remains a more appropriate model for testing new preservation solutions prior to initiation of clinical trials.

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.

Combination perfusion-cold storage for optimum cadaver kidney function and utilization.

Dog kidneys were perfused with a newly developed perfusate containing adenosine and PO4 for 48 hr, stored in the same perfusate for an additional 24 hr without perfusion (total preservation time:72 hr), and transplanted with immediate contralateral nephrectomy. Posttransplant renal function and biochemical analysis of the kidneys revealed practically no additional preservation-induced damage during the cold storage period. Serum creatinine levels reached normal between the third and eighth day posttransplant. This preservation method may be the method of choice for patients receiving postoperative cyclosporine therapy--and, in addition, facilitate organ sharing between transplant centers, thus reducing wastage of cadaveric kidneys.

Effect of fasting on hepatocytes cold stored in University of Wisconsin solution for 24 hours.

Although there have been improvements in liver preservation, liver dysfunction still remains a serious consequence of liver transplantation. This may be related to cold ischemic injury since the incidence of dysfunction increases with longer preservation times. However, even some livers preserved for short periods of time (less than 15 hr) develop liver dysfunction. One possible cause may be the lack of adequate nutritional support, and the donor may be exposed to prolonged periods of hyponutrition. In this study, we have compared the effects of fasting on functions of hepatocytes isolated from the rat. Hepatocytes were cold stored in University of Wisconsin solution for 24 hr and analyzed at the end of preservation as well as at the end of rewarming in Krebs-Henseleit buffer for 120 min. The glycogen content of fed cells was 1.57 mumol/mg protein and this was reduced by 95% in cells from fasted rats. After cold storage and rewarming, hepatocytes from fasted rats lost 84.2 +/- 2.5% of the total cellular lactate dehydrogenase versus only 32.7 +/- 3.8% (P < 0.001) in cells from fed rats. Also, ATP and reduced glutathione content of fasted cells were significantly reduced, free fatty acids were higher (P = 0.0154), and protein synthesis was reduced to 41% of controls (versus only 88% in fed cells), although there were no differences in phospholipid content. When hepatocytes from fasted rats were rewarmed in Krebs-Henseleit buffer containing fructose (10 mM), lactate dehydrogenase release was reduced from 80% to 34.4 +/- 0.2% and ATP content was significantly higher with fructose than without. Hepatocytes from fasted rats, therefore, are more sensitive to cold ischemic injury than cells from fed rats. The increased sensitivity appears related to the lack of glycogen as a source of substrates for metabolism during rewarming. This is supported by the fact that addition of fructose, which is metabolized readily by hepatocytes through glycolysis, suppressed rewarming injury to cells from fasted rats. The nutritional status of the donor, therefore, may play a pivotal role in the results of liver preservation and transplantation. Effective donor nutritional management may reduce the incidence of liver dysfunction after transplantation.

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.

Successful 72-hour cold storage of dog kidneys with UW solution.

Effects of three cold-storage solutions on kidney function in dogs were examined with the isolated perfused (IPK) kidney model and the autotransplant model. EuroCollins' (EC) solution, phosphate-buffered sucrose solution, and a new solution developed at the University of Wisconsin (UW) were studied. Kidneys were cold-stored for 48 hr or 72 hr. With the IPK model, cold storage for 48 hr or 72 hr in each of the three solutions caused creatinine clearance to decrease by 80%-90%. More protein was excreted by kidneys stored for 48 hr in PBS solution than by kidneys stored in EC or UW solution; protein excretion after 72 hr of storage was similar for kidneys stored in EC or UW solution. Sodium reabsorption decreased after 48 hr or 72 hr of storage, but was higher in kidneys stored in UW solution (83% and 56%, respectively) than in EC solution (52% and 22%, respectively). With the autotransplant model, 40% of the kidneys were viable after 48-hr storage in PBS solution, but 80% viable when stored in EC solution and 100% were viable when stored in UW solution. All kidneys were viable when stored for 72 hr in UW solution; none were viable when stored for 72 hr in EC solution. These results suggest that UW solution effectively preserves kidneys for 72 hr. We previously reported successful 72-hr pancreas preservation. Recently UW solution was able to preserve canine livers for 30 hr. Thus, this single solution appears to be effective for preserving all intraabdominal organs and may simplify cold storage of organs for transplantation.

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.

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 degrees 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.

Limitations of heart preservation by cold storage.

Clinical heart preservation is currently limited to only 4-6 hr, while the kidney, liver, and pancreas can tolerate 24-48 hr of cold ischemia. A fundamental difference between these organs is that the heart is contractile, containing large quantities of actin and myosin, and is susceptible to contracture-induced injury caused by energy deprivation. We have quantified and correlated the onset of contracture with levels of ATP and glycogen during cold storage in rabbit hearts flushed with UW solution, with and without 1 mM calcium (Ca), or 3 mM iodoacetate (IAA). A fluid-filled left ventricular balloon was used to generate pressure-volume curves (compliance) at 1, 6, 12, 18, and 24 hr of cold storage. Onset of contracture occurred in UW stored hearts at 18 hr, contracture in hearts exposed to Ca occurred between 6 and 12 hr. Compliance was significantly less in hearts exposed to Ca at 12, 18, and 24 hr (P less than .01) than in hearts without Ca. ATP levels were well maintained for up to 18 hr in the hearts preserved in UW solution (78%), but fell more rapidly in the presence of Ca at 12 hr (P less than .005), 18 hr (P less than .005), and 24 hr (P less than .05). In comparison, the ATP supply of the liver and kidney was exhausted by only 4 hr of cold storage. Onset of myocardial contracture correlated with a decrease in ATP to less than 80% of control, and contracture accelerated ATP decline 3-6-fold. IAA caused nearly complete myocardial contracture and ATP depletion within 2 hr. Isolated heart function was 77% and 73% at 6 and 12 hr of storage, but fell to 54% and 42% at 18 and 24 hr, respectively, coinciding with development of contracture. We conclude that ischemic contracture in this model is a major cause of myocardial damage during cold storage, and is accelerated by the presence of Ca. Other organs can be successfully stored despite exhaustion of ATP reserves. Thus successful cold-storage of the heart is highly ATP-dependent. Since cold storage inevitably leads to ATP depletion, extension of myocardial ischemic tolerance will depend on either reversible inhibition of ATP hydrolysis during storage, reversible uncoupling of contracture development from ATP depletion, or maintaining ATP production by continuous hypothermic perfusion.

The use of myocytes as a model for developing successful heart preservation solutions.

The development of a successful method to preserve the heart for relatively long periods (24-48 hr) requires demonstrating successful orthotopic transplantation and long-term survival after preservation. There are, however, multiple variables that may affect the quality of heart preservation, and it is nearly impossible to systematically study all the variables in this complicated model. One model that may be useful to study how preservation parameters affect heart cell preservation is the isolated myocyte preparation. In this study myocytes were isolated from the rabbit heart and the effects of up to 24 hr cold storage on viability measured to determine if this would be a suitable preservation model. Myocytes were stored in various preservation solutions including; EuroCollins (EC), two cardioplegic solutions (Stanford [ST] and Bretschneider solution [HTK]) and the University of Wisconsin solution (UW) with or without the addition of polyethylene glycol. The viability of myocytes was judged by measuring the effects of preservation and rewarming after preservation on cellular morphology (percent rod-shaped cells), ATP concentration, and LDH release. Myocytes preserved in the cardioplegic solutions were least well preserved after 12 and 24 hr storage, as judged by the loss of rod-shaped morphology and lower ATP concentration. Preservation in EC resulted in a decrease in the percent rod-shaped cells after 12 hr and 24 hr storage that was greater than obtained in the UW solutions. The best preservation of myocyte morphology and highest content of ATP was obtained in myocytes stored in the UW solutions, especially those containing PEG. The myocyte model of heart preservation shows a loss of cell integrity that is related to the preservation solution (HTK greater than ST greater than EC greater than UW-PEG) and these results are similar to what has been shown in the past with other models of heart preservation. Thus the myocyte model appears to be a useful method to test how many preservation solutions and preservation variables affect heart cell metabolism. In the future, results from these types of studies may find use in developing improved heart preservation solutions for testing in the orthotopic transplant model.

Donor nutritional status--a determinant of liver preservation injury.

In liver transplantation, the quality of the liver is determined by a number of factors including donor nutritional status. Livers from fasted donors appear to tolerate long-term preservation better than livers from fed donors. In this study we repeated earlier results and obtained 31% (4/13) survival after 40-hr preservation of livers from fed donor Brown Norway rats and 67% (8/12) survivors with donor livers from 4-day-fasted rats (P = 0.154). The explanation for this improvement is not known but may be due to inactivation of Kupffer cells due to nutritional depletion of the liver. Kupffer cell activation has been one explanation advanced to explain how cold storage injuries livers during reperfusion (transplantation). In this study, we have measured how donor fasting affects Kupffer cell function (phagocytosis of colloidal carbon) after preservation of the rat liver. In addition, we measured how enhancing liver glycogen by feeding glucose to the rat donors affected outcome and liver functions tested by isolated perfusion after 24- and 40-hr cold storage of the liver. Preservation did not cause inactivation or activation of Kupffer cell phagocytosis of colloidal carbon. In livers with 0-hr preservation, colloidal carbon uptake was 3.1 +/- 0.2 mg/g/hr, after 40-hr preservation uptake was 3.8 mg/g/hr (P < 0.05 vs. 0 hr) (fed) and 2.7 +/- 0.3 mg/g/hr (fasted, P, 0.05 vs. 0-hr and 40-hr-fed). Thus, the improved survival obtained with livers from fasted donors does not appear related to inactivation of Kupffer cell phagocytosis. Although livers from fasted donors showed improved survival, there was extensive hepatocellular injury as indicated by large LDH release from the livers after 40-hr cold storage as tested by isolated perfusion. LDH released into the perfusate increased from 35.8 +/- 10.1 U/L (fed, 40-hr CS) to 301 +/- 65 U/L (fasted, 40-hr CS) after 1-hr reperfusion. AST release showed a similar pattern and bile production was suppressed more in livers from fasted donors than fed donors. Feeding rats glucose elevated liver glycogen and significantly reduced hepatocellular injury as measured by LDH release and AST release in the isolated perfused liver after 40-hr cold storage. Feeding rats glucose (40% in drinking water for 4 days) also improved survival: fed+glucose = 85% survival versus 31% survival with no glucose and fasted+glucose = 92% survival versus 67% survival with no glucose. These results show that both extensive donor fasting and glucose feeding enhanced outcome in orthotopic liver transplantation. This dilemma (both fasting and feeding improved survival) are discussed in terms of how the interactions between Kupffer cells and hepatocytes affect liver viability. Donor fasting is probably impractical clinically as a method to improve the donor liver, but elevating liver glycogen by glucose supplementation is possible and may lead to improved preservation and outcome in liver transplantation.

Toxicity of oxygen to mitochondrial respiratory activity in hypothermically perfused canine kidneys.

Respiratory activity of kidney cortex homogenates was measured after various periods of hypothermic pulsatile preservation of dog kidneys with cryoprecipitated plasma. There was a progressive loss of pyruvate plus malate-stimulated respiration (30 to 40% at 3 days and 70 to 80% at 5 days) and succinate-stimulated respiration (15 to 20% at 3 days and 50 to 60% at 5 days). Perfusion under conditions of low pO2 (30 to 40 mm Hg) or with inhibitors of the toxic effects of hyperbaric oxygen (CO2+ Mn2+) preserved homogenate respiratory activity better than with normal pO2 (150 mm Hg) or high pO2 (300 mm Hg). The results suggest that oxygen toxicity (lipid peroxidation) and the progressive loss of respiration in homogenates may be limiting factors in obtaining long-term preservation.

Biphasic mechanism for hypothermic induced loss of protein synthesis in hepatocytes.

BACKGROUND: A complication in liver transplantation is increased clotting times due to inhibition of protein synthesis resulting from prolonged hypothermic preservation. Protein synthesis is also blocked in cold preserved hepatocytes. In this study, the mechanism of inhibition of protein synthesis in cold preserved hepatocytes was investigated. METHODS: Hepatocytes prepared from rat liver were cold preserved in University of Wisconsin solution for 4, 24, and 48 hr. Protein synthesis was measured as incorporation of radiolabeled leucine into acid precipitable proteins. Hepatocytes were treated with antioxidants (dithiothreitol, trolox or deferoxamine, nitric oxide synthase inhibitor (N(G)-monomethyl-L-arginine monoacetate), steroids (dexamethasone or methylprednisolone), methods to keep adenosine triphosphate high (aerobic storage), and cytoskeletal disrupting agents (cytochalasin D or colchicine). RESULTS: There was a 26% decrease in protein synthesis after only 4 hr of cold storage and a further 25% decrease at 24 hr. Antioxidants, elevated adenosine triphosphate, and N(G)-monomethyl-L-arginine monoacetate did not affect the rate of loss of protein synthesis. Protein synthesis was not due to inhibition of amino acid transport or lack of amino acids in the storage medium. Steroid pretreatment of hepatocytes had no effect on the loss of protein synthesis occurring in the first 4 hr of storage but did suppress the loss occurring during the next 44 hr of storage. Cytoskeletal disrupting agents, added to freshly isolated cells, inhibited protein synthesis. CONCLUSION: The mechanism of loss of protein synthesis in cold preserved liver cells is not mediated by: (1) oxygen free radical generation or improved by antioxidant therapy, (2) nitric oxide generation in hepatocytes, (3) an adenosine triphosphate-sensitive destruction of cell viability, and (4) decreased permeability of amino acids or loss of amino acids from the cells. Loss of protein synthesis due to hypothermic storage appears biphasic. The first phase, occurring within 4 hr of storage, may be the result of the effects of hypothermia on the cell cytoskeletal system and may be untreatable. The second phase, which occurs during the next 24 to 48 hr is sensitive to steroid pretreatment. This phase may be amenable to improved preservation methodology. Improved preservation of the liver may require the use of steroids to conserve protein synthetic capabilities.