Respiratory activity of isolated rat hepatocytes following cold storage and subsequent rewarming: a comparison of sucrose-based and University of Wisconsin solutions

Investigations were carried out on the respiratory function of isolated rat hepatocytes after cold storage alone for periods up to 48 h in either sucrose-based solution (SBS) or University of Wisconsin (UW) solution and after subsequent normothermic preincubation. In both SBS and UW, cold storage for 24 h depressed respiratory function (to 21 +/- 3 and 23 +/- 3 nmol O(2)/min/10(6) cells, respectively) compared to control cell values (31 +/- 3 and 33 +/- 5 nmol O(2)/min/10(6) cells; P < 0.01 in each case). However, normothermic preincubation for 60 min returned respiratory activity to control values (for SBS and UW storage: 41 +/- 6 and 40 +/- 5 nmol O(2)/min/10(6) cells; for control cells: 43 +/- 5 and 46 +/- 6 nmol O(2)/min/10(6) cells). Storage for 48 h in both SBS and UW allowed further depression of respiratory activity, with no recovery after preincubation. Stimulation of respiration by succinate in hepatocytes stored for longer periods was suggestive of increased membrane permeability. Both SBS and UW are effective storage solutions for isolated hepatocytes for up to 24 h as judged by aerobic metabolism, but significant damage was expressed in both solutions when preservation was extended.     

A comparison of cold storage solutions for hepatic preservation using the isolated perfused rabbit liver

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 degrees 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/100 g) in livers stored in HC (8.8 +/- 2 ml), PPF (9.9 +/- 2.2 ml), and UW (10.3 +/- 1.9 ml); it was slightly depressed in EC (6.7 +/- 2.5 ml, P = 0.06), and markedly depressed in Ringer lactate (4.3 +/- 0.8 ml, P less than 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.     

Effect of cold storage on tissue and cellular glutathione

One of the mechanisms thought to cause injury in preserved organs is the formation of oxygen free radicals. The cell is protected from oxidative stress by many defense mechanisms. A major defense mechanism involves glutathione and glutathione-dependent enzymes. During organ preservation by simple cold storage the loss of glutathione may sensitize the organ to free radical damage after transplantation. In this study we show that glutathione is depleted from the rabbit liver, kidney, and heart cold-stored (5 degrees C) for up to 72 h in the UW solution without glutathione. In the first 24 h kidney glutathione decreased to 84 +/- 3% of control values, liver glutathione decreased to 49 +/- 3% of control values, and heart glutathione decreased to 73 +/- 3% of control values. After 48 h of storage the kidney and liver lost an additional 30 and 20%, respectively, whereas heart glutathione changed very little. By 72 h all three organs had lost more than 50% of the glutathione found in freshly obtained tissue. To determine if glutathione added to the UW solution can effectively prevent this loss of glutathione during preservation, hepatocytes were cold-stored for up to 72 h in a preservation solution with and without glutathione. We found that adding glutathione to the preservation solution slowed the rate of loss of glutathione from the cells. These data suggest that at hypothermia the cell may be permeable to GSH. Methods to suppress the loss of glutathione during preservation of organs may be an important factor in suppressing oxygen free radical injury.     

Effect of polyethylene glycol on lipid peroxidation in cold-stored rat hepatocytes

A mechanism suggested to cause injury to preserved organs is the generation of oxygen free radicals either during the cold-storage period or after transplantation (reperfusion). Oxygen free radicals can cause peroxidation of lipids and alter the structural and functional properties of the cell membranes. Methods to suppress generation of oxygen free radicals of suppression of lipid peroxidation may lead to improved methods of organ preservation. In this study we determined how cold storage of rat hepatocytes affected lipid peroxidation by measuring thiobarbituric acid reactive products (malondialdehyde, MDA). Hepatocytes were stored in the UW solution +/- glutathione (GSH) or +/- polyethylene glycol (PEG) for up to 96 h and rewarmed (resuspended in a physiologically balanced saline solution and incubated at 37 degrees C under an atmosphere of oxygen) after each day of storage. Hepatocytes rewarmed after storage in the UW solution not containing PEG or GSH showed a nearly linear increase in MDA production with time of storage and contained 1.618 +/- 0.731 nmol MDA/mg protein after 96 h. When the storage solution contained PEG and GSH there was no significant increase in MDA production after up to 72 h of storage and at 96 h MDA was 0.827 +/- 0.564 nmol/mg protein. When freshly isolated hepatocytes were incubated (37 degrees C) in the presence of iron (160 microM) MDA formation was maximally stimulated (3.314 +/- 0.941 nmol/mg protein). When hepatocytes were stored in the presence of PEG there was a decrease in the capability of iron to maximally stimulate lipid peroxidation. The decrease in iron-stimulated MDA production was dependent upon the time of storage in PEG (1.773 nmol/mg protein at 24 h and 0.752 nmol/mg protein at 48 h).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The effect of pH on the viability of hypothermically stored rat hepatocytes

Hepatocytes isolated from the rat liver were stored for up to 72 hr at 4 degrees C in a tissue culture medium (Liebovitz-15) at different pH values to determine how pH affects hepatocyte viability. This is a model to simulate cold storage of livers for transplantation and determine the optimal pH for maintenance of liver cell function. The cells were stored in the absence of oxygen. At the end of cold storage the percentage of the total cellular LDH released into the extracellular medium was used as a measure of hepatocyte viability. Also, lactate dehydrogenase (LDH) release was determined in hepatocytes incubated at normothermia (37 degrees C) for 90 min following 72 hr of cold storage. The results demonstrate that hepatocytes tolerate a wide range of pH values in the storage medium and that only about 10% of the total LDH was released from hepatocytes stored up to 72 hr at pH's from 5.0 to 8.0. Normothermic incubation, however, demonstrated that the pH of the storage medium affected viability. After 48 hr of storage only hepatocytes stored at pH values from 7.0 to 8.0 remained viable (LDH release similar to that of freshly incubated hepatocytes = 28 +/- 7.2%). After 72 hr of storage and 90 min of normothermic incubation, hepatocytes incubated at all pH values studied were nonviable (greater than 60% release of LDH). These results suggest that the optimal pH for storage of hepatocytes at 4 degrees C is near neutrality (7.0 to 7.4).     

Glycine prevention of cold ischemic injury in isolated hepatocytes

Isolated hepatocytes suspended in a liver preservation solution (University of Wisconsin (UW) solution) and exposed to cold (5 degrees C) ischemia lose viability (LDH release) after 3 (76.5 +/- 2.6% extracellular LDH) and 4 days (90.3 +/- 5.7% extracellular LDH) storage when rewarmed (37 degrees C) in Krebs-Henseleit buffer. However, if 3 mM glycine is added to Krebs-Henseleit buffer the loss of LDH on rewarming was suppressed (% LDH = 24.4 +/- 2.2% and 33.2 +/- 3.0%, at 3 and 4 days, respectively). The protection by glycine could also be obtained by storing the hepatocytes in the UW solution containing 15 mM glycine and rewarming in the absence of glycine in Krebs-Henseleit buffer. There did not appear to be a relationship between the protection by glycine and glutathione concentration of the hepatocytes as shown by the lack of effect of a glutathione synthetase inhibitor (butathionine sulfoximine) on the protective effects of glycine. Other amino acids did not provide protection to hepatocytes exposed to cold ischemia. The mechanism of action of glycine is not known, but this compound may be important in improving cold storage of livers for transplantation.     

Supercooling as a Viable Non-Freezing Cell Preservation Method of Rat Hepatocytes

Supercooling preservation holds the potential to drastically extend the preservation time of organs, tissues and engineered tissue products, and fragile cell types that do not lend themselves well to cryopreservation or vitrification. Here, we investigate the effects of supercooling preservation (SCP at -4^oC) on primary rat hepatocytes stored in cryovials and compare its success (high viability and good functional characteristics) to that of static cold storage (CS at +4^oC) and cryopreservation. We consider two prominent preservation solutions a) Hypothermosol (HTS-FRS) and b) University of Wisconsin solution (UW) and a range of preservation temperatures (-4 to -10 ^oC). We find that there exists an optimum temperature (-4^oC) for SCP of rat hepatocytes which yields the highest viability; at this temperature HTS-FRS significantly outperforms UW solution in terms of viability and functional characteristics (secretions and enzymatic activity in suspension and plate culture). With the HTS-FRS solution we show that the cells can be stored for up to a week with high viability (~56%); moreover we also show that the preservation can be performed in large batches (50 million cells) with equal or better viability and no loss of functionality as compared to smaller batches (1.5 million cells) performed in cryovials.     

Energy status of isolated hepatocytes after long-term cold storage in various types of media

Using of isolated hepatocytes for investigation of the effects of hypothermia, it has been demonstrated that sucrose-base solution provides of maintenance of the energetic parameters (level of ATP, glucose synthesis, rate of gluconeogenesis) within 48 hrs of storage at 4 degrees C. It efficiency was compared with effect on the energetic status of isolated hepatocytes widely used preservation solution--solution of University Wisconsin (UW). After long-term of cold storage of isolated hepatocytes (72 hrs) at 4 degrees C in both solutions, it has been shown sharp decrease of ATP level (on two time). Viability of the liver cells (in both cases) was practically without change.     

Hypothermia causes a marked injury to rat proximal tubular cells that is aggravated by all currently used preservation solutions

Cold preservation results in cell death via iron-dependent formation of reactive oxygen species, leading to apoptosis during rewarming. We aimed to study cold-induced damage (i.e., injury as a consequence of hypothermia itself and not cold ischemia) in proximal tubular cells (PTC) in various preservation solutions presently applied and to clarify the role of mitochondria in this injury. Primary cultures of rat PTC were incubated at 4 degrees C for 24 h in culture medium, UW, Euro-Collins or HTK solution with and without the iron chelator desferal and rewarmed at 37 degrees C in culture medium. Cell damage, morphology, and apoptosis were studied and mitochondrial membrane potential was assessed by fluorescence microscopy. Cold incubation of PTC in culture medium followed by rewarming caused marked cell damage compared to warm incubation alone (LDH release 39+/-10% vs. 1.6+/-0.3%). Cold-induced damage was aggravated in all preservation solutions (LDH release 85+/-2% for UW; similar in Euro-Collins and HTK). After rewarming, cells showed features suggestive for apoptosis. Desferal prevented cell injury in all solutions (e.g., 8+/-2% for UW). Mitochondrial membrane potential was lost during rewarming and this loss could also be inhibited by desferal. Trifluoperazine, which is known to inhibit mitochondrial permeability transition (MPT), was able to prevent cold-induced injury (LDH 85+/-5% vs. 12+/-2%). We conclude that cold-induced injury occurs in PTC and is aggravated by UW, Euro-Collins, and HTK solution. Iron-dependent MPT is suggested to play a role in this damage. Strategies to prevent cold-induced injury should aim at reducing the availability of "free" iron.     

Improvement of renal preservation by adding lidoflazine to University of Wisconsin solution. An experimental study in the rat.

The purpose of this study was to investigate the possibility of improving the organ preservation properties of the University of Wisconsin (UW) solution by adding the calcium entry blocker lidoflazine. We also investigated the possibility of decreasing the cold ischemia and reperfusion damage by pretreatment with lidoflazine of the donor and/or recipient. The protective effects of lidoflazine treatment were estimated by measuring the amount of trapped erythrocytes in the rat renal medulla after 48 h of cold storage, subsequent transplantation, and 20 min of reperfusion. Lidoflazine (20 mg/liter) added to the UW solution decreased the amount of erythrocyte trapping from 14.8 +/- 3.1% in controls to 8.6 +/- 1.7% (P less than 0.01). The flow rate of the flush-out solution during the harvesting procedure was also significantly (P less than 0.01) increased when lidoflazine was included in the UW solution (1.10 +/- 0.21 ml/min vs 0.75 +/- 0.22 ml/min). Administration of lidoflazine (0.28 mg/kg body wt) to the donor and/or the recipient did not further reduce the postischemia/reperfusion damage as estimated by the degree of erythrocyte trapping. In conclusion, the results indicate that the preservation properties of the UW solution can be significantly improved by adding lidoflazine to the solution.     

The response of liver to lactobionate/raffinose (University of Wisconsin--UW) solution during hypothermic preservation: a study using 31phosphorus nuclear magnetic resonance

31P nuclear magnetic resonance spectroscopy has been used to study rat livers following flushing with the University of Wisconsin (UW) lactobionate/raffinose solution (N. Jamieson, R. Sundberg, S. Lindell, J. Southard, and F.O. Belzer, Cryobiology 24, 573-574, 1987; M. Kalayoglu, H. Sollinger, R. Stratta, A. D'Alessandro, R. Hoffman, J. Pirsch, and F. O. Belzer, Lancet 1, 617-619, 1988). These studies have revealed that despite the improved storage properties that have been reported for this solution, hepatic ATP and ADP declined at a rate similar to that seen in the more commonly used Marshall's or Collins' solutions. However, there was a significant inhibition of the developing acidosis, such that by 5 hr postflush, the intracellular pH was 7.17 +/- 0.06 (mean +/- SD, n = 5) compared to 6.90 +/- 0.06 for Marshall's solution (4 hr postflush) and 6.94 +/- 0.04 for Collins' solution (4 hr postflush). This did not appear to be due to a buffering effect of the solution, as this was found to be relatively low, but was probably due to a modification of hepatic metabolism caused by the solution itself.     

The effect of human serum albumin on the extended storage of human oral keratinocyte viability under mild hypothermia

Isolated oral keratinocytes in suspension provide a number of advantages for use in maxillofacial surgery, however, the poor stability of this cell preparation at physiological temperatures is an apparent barrier preventing their use. The purpose of the present study was to evaluate whether human serum albumin (HSA) could serve as an effective constituent of a storage medium to enhance human oral keratinocyte (HOK) viability under conditions of mild hypothermia. Primary human oral keratinocytes were isolated from small pieces of the non-inflamed gingival tissues obtained during the extraction of the third molars of patients. HOK were cultured on collagen type I-coated culture dishes in keratinocyte growth medium (KGM). After the trypsinization of a culture dish (passage 2 or 3), freshly isolated HOK were stored for 24, 48, and 72 h at 4 degrees C or at room temperature in KGM, saline, Dulbecco's modified Eagle's medium (DMEM), saline supplemented with 10% HSA or DMEM supplemented with 10% (v/v) HSA under one atmosphere pressure. After storage, HOK cell survival was determined by dye exclusion using trypan blue and colony-forming assay and cell cycle change was obtained by flow cytometry. Highest cell viability was obtained in saline supplemented with 10% HSA and DMEM supplemented with 10% (v/v) HSA at 4 degrees C and at room temperature. Under these conditions no significant decline in keratinocyte viability was observed for at least 48 h. The cell cycle profiles of these cells were also maintained for at least 48 h at room temperature. These observations demonstrate that HSA might be better at preserving the viability of HOK stored under hypothermic and mild hypothermic conditions up to 48 h.     

Glutathione synthesis during the rewarming of rat hepatocytes preserved in the University of Wisconsin solution

In this study, we used isolated rat hepatocytes to investigate the effect of nucleoside content of the preserved cells on the ability to synthesize glutathione (GSH) during the rewarming process. We cold-stored hepatocytes in University of Wisconsin (UW) solution (72 h, 0 degrees C, N(2)) without nucleosides and with the addition of 5 mM adenosine or 10 mM ATP. After 72 h of cold storage, we determined the GSH synthesis rate and the ATP content of the cells. We found a GSH synthesis rate similar to that of freshly isolated hepatocytes only in the group of cells cold-stored with 10 mM ATP. When we tested the cellular ATP concentrations, we found that controls and preserved cells with 10 mM ATP showed a similar value of ATP during the rewarming step. Our results suggested that the incorporation of ATP in the UW solution increased the ATP content and the rate of GSH synthesis of cold-stored hepatocytes during rewarming.     

Extracellular calcium protects cultured rat hepatocytes from injury caused by hypothermic preservation

Effects of various preservation solutions were compared in an experimental hypothermic preservation model using cultured rat hepatocytes. Hepatocytes prepared by the collagenase perfusion method were cultured for 48 hr, then the medium in each culture dish was exchanged for various preservation solutions, and the dishes were hypothermically (0-2 degrees C) stored in a refrigerator for 12-72 hr. After the preservation period, the hepatocytes were cultured again at 37 degrees C for 2 hr. Hepatocytes' viability after 18-hr preservation and reculture was greater when they were preserved in "intracellular" rather than "extracellular" solutions. Even with Euro-Collins solution (intracellular solution), hepatocyte viability decreased to approximately 20% after 24-hr preservation, and an increase in the cellular lipid peroxide content was observed. However, when this solution contained a submillimolar concentration of calcium, lipid peroxidation was significantly suppressed and hepatocyte viability was dramatically improved. Vitamin E was almost equally effective and a marked synergistic effect was observed with calcium. Calcium was found to be capable of maintaining the cellular glutathione level during cold storage, which seems to suppress lipid peroxidation and consequently improve hepatocyte survival.     

Amino acid-rich medium (Leibovitz L-15) enhances and prolongs proliferation of primary cultured rat hepatocytes in the absence of serum.

Multiple rounds of cell division were induced in primary cultured rat hepatocytes in serum-free, modified L-15 medium supplemented with 20 mM NaHCO3 and 10 ng/ml EGF in a 5% CO2/95% air incubator. A 150% increase in cell number and DNA content was observed between day 1 and day 5. The time course of DNA synthesis of hepatocytes cultured in L-15 medium differed from that in DMEM/F12 medium in that there were four peaks of 3H-thymidine incorporation in the L-15 medium, at 60 h, 82 h, 96 h, and 120 h, but only one peak at 48 h in modified DMEM/F12 medium. Labeling studies of the hepatocytes indicated that more than 60% of the cells were stained with antibromodeoxyuridine (BrdU) antibody in the periods of 48-72 h and 72-96 h after plating at densities between 1.5 x 10(5) and 6.0 x 10(5) cells per 35-mm dish. Even at a density of 9.0 x 10(5) cells/dish, about 40% of the cell nuclei were stained with BrdU in the periods of 48-72 h and 72-96 h. In addition, about 20% of the hepatocytes in culture initiated a second round of the cell cycle between 48 and 96 h in culture. Proliferating cells, which were mononucleate with a little cytoplasm, appeared in small clusters or colonies in the culture from day 4. These proliferating cells produced albumin. The addition of essential amino acids to the DMEM/F12 medium enhanced the DNA synthesis of hepatocytes, thus indicating that the higher level of amino acids in L-15 medium may be an important factor in its enhanced ability to support the proliferation of primary cultured rat hepatocytes.     

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.     

Hypothermic preservation of hepatocytes. II. Importance of Ca2 and amino acids

The importance of the components of a tissue culture media, Leibovitz-15 (L-15), for maintaining viability of hypothermically preserved hepatocytes was analyzed. Hepatocytes isolated from rat livers were incubated at 5 degrees C in an oxygenated environment with continuous shaking (to simulate organ perfusion preservation). L-15 + 5 g% polyethylene glycol (PEG) or variants of this solution were used as the preservation media. After 48 hr of storage, hepatocyte viability was assessed by measuring the release of LDH into the incubation medium and cell volumes were determined. Following 90 min of normothermic incubation (to simulate organ reperfusion), mitochondrial function was measured. Hepatocytes stored in the complete L-15 solution were about 90% viable at the end of 48 hr of storage, while cells stored in a solution containing only the principle electrolytes (PE) lost viability (70% viable). Only the addition of a combination of divalent cations (Ca/Mg) and amino acids was sufficient to maintain viability equivalent to that obtained in the complete L-15 mixture. Hepatocytes suspended in L-15 maintained normal cell volumes (3.85 microliters/mg protein), while cells in the PE solution were swollen with cell volumes of 4.66 microliters/mg protein. Only the addition of Ca/Mg to the PE solution was effective at suppressing cell swelling similar to the complete L-15 media. Both basal and uncoupler-stimulated respiration were depressed in cells stored in the PE solution (15 and 28 nmol O2/min/mg protein) as compared to cells in L-15 (21 and 41 nmol O2/min/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)     

Ischemic preconditioning in a rodent hepatocyte model of liver hypothermic preservation injury

Ischemic preconditioning (IPC) is a phenomenon of protection in various tissues from normothermic ischemic injury by previous exposure to short cycles of ischemia-reperfusion. The ability of IPC to protect hepatocytes from a model of hypothermic transplant preservation injury was tested in this study. Rat hepatocytes were subjected to 30min of warm ischemia (37 degrees C) followed by 24 or 48h of hypothermic (4 degrees C) storage in UW solution and subsequent re-oxygenation at normothermia for 1h. Studies were performed with untreated control cells and cells treated with IPC (10min anoxia followed by 10min re-oxygenation, 1 cycle). Hepatocytes exposed to IPC prior to warm ischemia released significantly less LDH and had higher ATP concentrations, relative to untreated ischemic hepatocytes. IPC significantly reduced LDH release after 24h of cold storage before reperfusion and after 48h of cold storage and after 60min of warm re-oxygenation, relative to the corresponding untreated hepatocytes. ATP levels were also significantly higher when IPC was used prior to the warm and cold ischemia-re-oxygenation protocols. In parallel studies, IPC increased new protein synthesis and lactate after cold storage and reperfusion compared to untreated cells but no differences in the patterns of protein banding were detected on electrophoresis between the groups. In conclusion, IPC significantly improves hepatocyte viability and energy metabolism in a model of hypothermic preservation injury preceded by normothermic ischemia. These protective effects on viability may be related to enhanced protein and ATP synthesis at reperfusion.     

Effects of chlorpromazine and methylprednisolone on perfusion preservation of rabbit livers

The isolated perfused rabbit liver was used to determine how continuous hypothermic perfusion affected liver function. Rabbit livers were perfused for 0, 24, 48, and 72 hr at 5 degrees C with the UW perfusate containing hydroxyethyl starch (5 g%) dissolved in a solution containing gluconate (80 mM), adenosine (5 mM), glutathione (3 mM), phosphate (25 mM), and additives as described previously, and they were used successfully for kidney preservation. At the end of preservation the livers were perfused in an isolated circuit with a Krebs-Henseleit solution with addition of 4 g% bovine serum albumin and 10 mM glucose at 38 degrees C for 120 min. Bile was collected from the cannulated common duct. Biliary excretions of indocyanine green and liver enzymes lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase, were determined both in the cold perfusate and the normothermic perfusate. Livers were also studied after pretreatment of the donor with chlorpromazine (CPZ) and/or methylprednisolone (MP). Bile production (ml/120 min, 100 g liver) upon reperfusion produced the most interesting data and decreased from a control value of 10.3 +/- 2.6 to 9.3 +/- 1.0 (24 hr), 5.3 +/- 0.7 (48 hr), and 4.1 +/- 1.5 (72 hr). Enzyme release was not predictive of the degree of preservation-induced damage. Pretreatment of rabbits with a combination of CPZ/MP improved bile flow at 48 and 72 hr (8.3 +/- 3.0 and 7.0 +/- 1.3, P less than 0.05). Pretreatment with either drug alone also improved function after 72 hr of preservation (7.1 +/- 1.8, CPZ; 8.2 +/- 3.5, MP).(ABSTRACT TRUNCATED AT 250 WORDS)