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.

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.

Preservation of metabolic reserves and function after storage of myocytes in hypothermic UW solution.

Isolated rat myocytes cold stored anaerobically up to 24 h in University of Wisconsin solution lost 95% of their ATP and 100% of their glycogen. They underwent contracture when rewarmed in a Krebs-Henseleit (KH) medium that contained Ca unless Ca addition was delayed. In the latter case, cell function, measured by stimulation-induced cell shortening, was surprisingly well retained. Aerobically stored cells were resistant to Ca on rewarming, although 96% of glycogen was still lost, along with 46% of ATP. Cells that were incubated for 48 h aerobically with the substrates glucose and pyruvate at pH 6.2 retained 77% of their ATP and 59% of their glycogen, with good cell morphology. At pH 6.2, the demand for ATP was only 55% of that at pH 7.4. However, after rewarming, these cells functioned no better than anaerobically stored cells, although their inotropic response to isoproterenol was improved. We conclude that 1) aerobic conditions with substrates at low pH preserve myocyte metabolic reserves well for 48 h, partly by reducing the demand for ATP; 2) rewarming conditions are critical for anaerobically stored cells with metabolic stores that are severely depleted; and 3) unloaded cell function is surprisingly insensitive to a period of severe metabolic deprivation.

Effect of phospholipase A2 inhibitors on the release of arachidonic acid and cell viability in cold-stored hepatocytes.

We investigated the effect of phospholipase A(2) (PLA(2)) inhibitors on PLA(2) activity and cell viability in cold-stored rat hepatocytes. The cells were radiolabeled with [(3)H] arachidonic acid (AA) and cold stored in the University of Wisconsin (UW) solution containing various PLA(2) inhibitors. PLA(2) activity was determined by measuring the total free (cellular + supernatant) AA by thin-layer chromatography after inhibiting reacylation of free AA with inhibitors of energy production (carbonyl cyanide m-chlorophenylhydrazone + iodoacetate). Aristolochic acid, chlorpromazine, and quinacrine in the UW solution showed a significant inhibitory effect throughout 48 h cold storage but only at relatively high concentration. PLA(2) activity was also suppressed (58% of control) by trifluoperazine (50 microM), but its effect was limited to only 24 h. In contrast, pretreatment of the cells prior to hypothermic preservation with trifluoperazine (10 to 100 microM) suppressed PLA(2) activity during 48 h storage. Inclusion of calmodulin antagonist W-7 did not affect PLA(2) activity. Thus, the inhibitory activity of these agents appears unrelated to Ca-calmodulin-phospholipid interaction but to have an inhibitory effect on PLA(2) activity. To study the effects of PLA(2) inhibitors on cell viability, lactate dehydrogenase (LDH) release was measured in the presence or absence of inhibitors upon rewarming cold-stored cells in Krebs-Henseleit buffer for 2 h at 37 degrees C. None of the inhibitors tested improved cell viability after 48 h storage. Thus, although PLA(2) inhibitors blocked PLA(2) activity, there was no suppression of LDH release. PLA(2) may play a minor role in preservation/reperfusion injury to cold-stored hepatocytes.

Phospholipid metabolism of hypothermically stored rat hepatocytes.

Isolated rat hepatocytes were suspended and stored in either Liebovitz-15 medium (37 degrees C or 4 degrees C) or University of Wisconsin (UW) solution (4 degrees C) containing [(3)H] arachidonic acid (AA). At varying times, membrane phospholipids were separated by thin layer chromatography. AA labeled phospholipids similarly at both 4 degrees C and 37 degrees C. Analysis of the ratios of [(3)H] AA and [(14)C] glycerol incorporated into phosphatidic acid or other phospholipids in dual-labeled cells indicated that the deacylation/reacylation cycle was the major route of AA incorporation at hypothermia. This was supported by showing that blocking phospholipase A(2) (PLA(2)) activity by trifluoperazine suppressed AA incorporation into phospholipids. PLA(2) activity, measured by determining the release of AA, was slow during 48-hour cold storage, but increased significantly when ATP was depleted by inhibition of mitochondria and glycolysis. In the whole rat liver, there was no significant loss of phospholipids during 48-hour storage (total phospholipids [micromol phosphorus/L/mg] : 0.197 +/-. 001 at 0 hours) unless energy blockers were used (0.155 +/-.005 at 48 hours) or glycogen depleted by fasting the rat (0.167 +/-.001 at 48 hours). This study shows that a net PLA(2) stimulated hydrolysis of phospholipids is seen only when ATP is depleted and its generation from anaerobic glycolysis inhibited. Thus, PLA(2) hydrolysis of phospholipids is not a significant cause of liver cell injury during cold storage when livers are obtained in optimal condition. However, conditions affecting the generation of ATP during cold storage could alter PLA(2) leading to membrane damage.

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.

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.

University of Wisconsin solution with butanedione monoxime and calcium improves rat lung preservation.

BACKGROUND: A limitation to fully using lung transplantation for patients with end-stage lung diseases is short, safe preservation time (4 to 6 hours). Our goal is to extend this to 24 hours or more, which would greatly improve clinical lung transplantation. METHODS: We used the isolated perfused rat lung to test how two preservation solutions (low potassium dextran and University of Wisconsin solution) affected quality of lungs after 6, 12, and 24 hours of preservation. Also, we tested modifications of the University of Wisconsin solution, including reversing the ratio of Na/K, the addition of 1.5 mmol/L calcium, and the combination of calcium and butanedione monoxime, agents that improve cardiac preservation. After preservation at 4 degrees C, lungs were reperfused at 37 degrees C with a physiologically balanced solution. Pulmonary artery flow rate, airway peak inspiratory pressure, and tissue edema were used to assess degree of preservation and reperfusion injury. RESULTS: Low potassium dextran solution gave poor preservation (decreased pulmonary artery flow, tissue edema) after 12 hours of cold storage. There were no differences between regular and reversed Na/K ratio University of Wisconsin solutions at 12 or 24 hours of preservation. Addition of calcium had no beneficial effect on lung preservation. However, University of Wisconsin solution with calcium and butanedione monoxime gave excellent 24-hour cold storage, with pulmonary artery flow rate, tissue edema, and airway peak inspiratory pressure equal to control (0 hours of preservation) lungs. CONCLUSIONS: The University of Wisconsin solution appears capable of lung preservation for up to 24 hours if modified to contain calcium and butanedione monoxime. The mechanism of action of butanedione monoxime may be related to the suppression of smooth muscle contraction resulting in vasodilation of the cold-stored lung on reperfusion.

TNF-alpha and heat-shock protein gene expression in ischemic-injured liver from fasted and non-fasted rats. Role of donor fasting in the prevention of reperfusion injury following liver transplantation.

We have previously shown that livers from long-term-fasted rats acquire tolerance to warm ischemic injury following transplantation, despite the fact that fasting depletes glycogen and ATP from the liver. The precise mechanism of the protective effect induced by donor fasting, however, is still a matter of controversy. In this experiment we determined heat-shock protein (GRP78) mRNA expression in livers during long-term fasting and TNF-alpha mRNA expression in transplanted livers exposed to warm ischemia. We also measured the concentration of TNF-alpha by ELISA in the ascitic fluid of fed and fasted rats injected intraperitoneally with zymosan to investigate why livers from fasted rats tolerate ischemic injury better. There seemed to be a positive correlation between GRP78 mRNA expression and survival. TNF-alpha secretion into the ascitic fluid of fasted rats was markedly suppressed, and fasting donor animals induced cytoprotective substances, such as GRP78, in the liver. These factors may contribute to the tolerance to ischemic injury produced by donor fasting.

Optimal pH for simple cold storage or machine perfusion of dog kidneys with UW solution.

Metabolic suppression by temperature is a key to successful organ preservation. Additional methods for inducing metabolic suppression may further improve organ preservation. Extracellular acidosis has been shown to suppress warm anoxic injury to various isolated cells. Acidosis may suppress enzymes with a pH optimum at the pH of the cytosol (pH 7.3). In this study, the combination of hypothermia and acidosis was used to determine if it would improve renal preservation. Dog kidneys were cold-stored (CS) for 48 h in University of Wisconsin (UW) solution with the pH adjusted to 6.4, 6.8, 7.4, or 7.8. Kidneys were also machine-perfused (MP) for 3 days with the gluconate perfusion solution (Belzer's machine perfusion solution, MPS) at pHs similar to those tested for CS. Renal function (serum creatinine, SCr) and survival were recorded in immediate contralateral nephrectomized recipients. On the basis of maximum SCr values, kidneys preserved by CS or MP were best preserved at pHs of 7.4 or 7.8. At a pH of 6.8, SCr values were elevated and returned to normal at a slower rate than in those preserved at higher pHs. This study shows that acidosis is not cytoprotective to cold-stored dog kidneys and causes preservation/reperfusion injury.

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.

Improved preservation of rat hindlimbs with the University of Wisconsin solution and butanedione monoxime.

The purpose of this study was to compare the effects of the University of Wisconsin solution plus butanedione monoxime, the University of Wisconsin solution without butanedione monoxime, and saline on the preservation of muscle tissue. Forty-nine rat hindlimbs were amputated and replanted. The study population was subdivided into four groups according to flushing solution, storage, and replantation protocols. The limbs of the control group (n = 12) were flushed with 20 ml University of Wisconsin solution and immediately replanted onto the same rat. In the remaining three groups, the limbs were immersed in solution, stored in a refrigerator at 4 degrees C for 24 hours, and then replanted onto a fresh rat. The limbs in the no flushout group (n = 7) were placed into storage in cold saline solution without being flushed. The limbs in the University of Wisconsin solution group (n = 17) were flushed with 20 ml of University of Wisconsin solution prior to storage, and those in the University of Wisconsin solution plus butanedione monoxime group (n = 13) were flushed with 20 ml University of Wisconsin solution plus 20 mM butanedione monoxime. Limb survival rate was 100 percent for the control and University of Wisconsin solution plus butanedione monoxime groups, 87 percent for the University of Wisconsin solution group, and 71 percent for the no flushout group. Seven days after replantation, ATP levels were 71 percent of control in the University of Wisconsin solution plus butanedione monoxime group, 33 percent in the University of Wisconsin solution group, and 29 percent in the no flushout group. Tissue K+/Na+ ratio showed that the University of Wisconsin solution plus butanedione monoxime group maintained electrolyte balance, whereas the balance was significantly lowered in University of Wisconsin solution and no flushout groups. The University of Wisconsin solution plus butanedione monoxime limbs did not exhibit cell swelling, whereas total tissue water values for the University of Wisconsin solution and no flushout groups increased significantly. Serum creatinine kinase, measured 24 hours after replantation, was 120 percent of control in the University of Wisconsin solution plus butanedione monoxime group, 550 percent in the University of Wisconsin solution group, and 772 percent in the no flushout group. Limbs in the University of Wisconsin solution plus butanedione monoxime group had more flexible ankle joints and pliable muscle (i.e., less contracture) than those in the University of Wisconsin solution and no flushout groups. In conclusion, rat hindlimbs can be preserved hypothermically for 24 hours using the University of Wisconsin solution, the University of Wisconsin solution plus butanedione monoxime, or saline. However, the University of Wisconsin solution plus butanedione monoxime limbs had better ATP levels and less cellular injury after replantation. Based on these results, we believe that, biochemically, flushing and storage of muscle tissue in the University of Wisconsin solution plus butanedione monoxime are the most effective means of those studied for preserving composite tissue grafts for 24 hours.

Cold storage sensitizes hepatocytes to oxidative stress injury.

Liver cold storage leads to oxygen free radical production and reperfusion injury. Antioxidants are effective in suppression reperfusion injury in rat livers when used in the reperfusion medium. However, in clinical liver transplantation their effectiveness is not clear, which may be due to the way they are used (in the recipient). In this study we compare the effectiveness of antioxidants when used in the reperfusion medium versus the cold storage solution in isolated hepatocytes and the isolated perfused liver. Hepatocytes were cold stored in UW solution for 24 h. Oxidative stress, induced by t-butyl hydroperoxide (tBHP), was measured in the presence of one of five different antioxidants--deferoxamine (DFO), dithiothreitol (DTT), trolox, tocopherol, dimethylthiourea (DMTU)--in the reperfusion buffer or UW solution. Efficacy was judged by reduction in membrane damage (LDH release) during rewarming. Also, rat livers were cold stored for 48 h in UW solution (+/- antioxidant) and reperfused (+/- antioxidants). Efficacy was judged by the effect on enzyme release and bile production. Cold storage of hepatocytes for 24 h sensitized them to oxidative stress. The concentration of tBHP required to induce maximal cell death (80%-90% LDH release) was reduced from 1.3 mM (fresh cells) to 0.37 mM (LD-50 values). All antioxidants except DMTU suppressed oxyradical-induced LDH release when used in the reperfusion medium, but only DFO was effective when used in the UW solution. In the isolated perfused liver, DFO, DTT, and trolox were effective and suppressed enzyme release when added to the reperfusion buffer, but none were effective when used in the UW solution. We conclude that cold storage sensitizes liver cells to oxidative stress. The most effective antioxidant was the iron chealator, DFO, which was effective in the reperfusion buffer (isolated perfused sliver or hepatocytes) but not in the UW solution when tested in the isolated perfused liver. Suppression of reperfusion injury in liver transplantation could be obtained by antioxidant therapy. However, it is unclear how best to deliver the antioxidants to the site of oxyradical generation.

Inhibition of warm ischemic injury to rat liver, pancreas, and heart grafts by controlling the nutritional status of both donor and recipient.

In this study, we tested the effect of donor fasting with or without the use of an essential fatty acids deficiency (EFAD) diet in the recipient using rat heart, pancreas, and liver transplant models. We then compared the survivals, tumor necrosis factor alpha (TNF-alpha) response, and white cell accumulation in rats in order to clarify the mechanisms of the beneficial effect of donor fasting and recipient EFAD. It was found that when the grafts were obtained from fasted donors and then transplanted into fed recipients, the survival rate was significantly higher for all three grafts than for those obtained from fed rats and transplanted into fed rats. The best survival was seen for pancreas grafts obtained from fasted donors and then transplanted into EFAD recipients. TNF-alpha secretion was significantly suppressed in both fasted and EFAD rats, and both the total cell count and neutrophil count were suppressed in EFAD rats. These results clearly indicate that in addition to liver grafts, both heart and pancreas grafts obtained from fasted animals are more tolerant to warm ischemic injury. Furthermore, the combination of donor fasting and recipient EFAD acts synergistically to inhibit the post-transplantation inflammatory reaction (through decreased TNF-alpha secretion and white cell accumulation), thus resulting in an improved survival.