reperfusion injury of rat liver

GASTROENTEROLOGY1995;109:189-197

Role of Kupffer Cells in Cold Ischemia/Reperfusion Injury of Rat Liver HIROSHI IMAMURA,* FANNY SUTTO,* ANTOINE BRAULT,* and PIERRE-MICHEL HUET*'* *Andr6-Viallet Clinical Research Center and *Department of Medicine, H6pital Saint-Luc and Universit6 de Montr6al, Montreal, Quebec, Canada

Background & Aims: Kupffer cell activation is hypothesized to play an etiopathogenic role in storage-related graft failure after liver transplantation. The aim of this study was to verify whether the elimination of Kupffer cells modifies the magnitude of cold ischemia/reperfusion injury of the liver. Methods: Rat Kupffer cells were eliminated by an intravenous injection of liposome-encapsulated dichloromethylene diphosphonate. Livers from control and treated rats were isolated and perfused before and after 24-hour cold ischemia in the University of Wisconsin solution (4°C). Hepatocyte and sinusoidal endothelial cell functions were evaluated by taurocholate and hyaluronic acid elimination, respectively. Liver transplantation was also performed using control and treated donor livers stored under identical conditions. Results: Compared with baseline values, similar alterations were found in both groups after cold ischemia for hepatocyte function (intrahepatic resistance, bile secretion, lactate dehydrogenase release, oxygen consumption, and taurocholate intrinsic clearance) and for sinusoidal endothelial cell function (hyaluronic acid intrinsic clearance). The lO-day survival rate of animals undergoing transplantation was not different between the groups (6 of 15 vs. 4 of 15, control vs. treated donor livers, respectively). Conclusions: The presence or absence of Kupffer cells does not modify the effect of 24-hour cold ischemia/reperfusion on the rat liver.

iver transplantation has become an accepted therapy for end-stage liver disease,* yet injury from cold ischemic storage and the resulting primary graft nonfunction remain major clinical problems. 2'3 In spite of the development of the University of Wisconsin (UW) solution, liver preservation time is usually limited to 12 hours. 4'~ An increasing number of reports have shown that the cellular target of cold ischemia/reperfusion injury is the sinusoidal endothelial ceils (SECs). 6-** Although their alteration and ensuing microcirculatory disturbances are thought to be the primary cause of early graft failure after transplantation, the precise mechanism underlying storagerelated graft nonfunction is not well understood.

Kupffer cells (KCs), resident macrophages of the liver, are known to produce a wide variety of biologically toxic mediators such as protease, reactive oxygen intermediates, leukotrienes, and tumor necrosis factor 12-14 and have been strongly implicated in the pathogenesis of hepatic injury in various animal models) 5-*7 In liver transplantation, it is currently hypothesized that KC activation plays a causal role in the cold ischemia/reperfusion injury leading to primary graft nonfunction. 18-2° Recently, van Rooijen 21 developed a method to selectively eliminate KCs in vivo using liposome-encapsulated dichloromethylene diphosphonate (MDP). KC phagocytosis of liposome-encapsulated MDP injected intravenously results in the complete elimination of KCs within 2 4 - 4 8 hours without apparent damage to other cell types. 21 In the present study, we investigated whether KC activation is a major contributing factor to cold ischemia/ reperfusion injury of the liver. In a first set of experiments, we compared KC-depleted rats and control rats by assessing the hepatocyte and SEC functions in an isolated perfused tat liver model before and after cold preservation in U W solution. In a second set of experiments, we used a rat liver transplantation model to compare the survival rate of animals receiving KC-depleted livers with that of animals receiving control livers. Materials

and Methods

Experiment 1 consisted of an isolated perfused rat liver model; male Wistar rats (Charles River, St-Constant, Quebec, Canada) weighing 250-300 g were used. The rats were housed in Plexiglas cages and had free access to normal rat chow except for the 12 hours preceding the study, when they had free access to 5% dextrose only. Rats were randomly assigned to MDP treatment (n = 8) or to control (n = 8) groups. Experiment Abbreviations used in this paper: HA, hyaluronic acid; HA.Cli, hyaluronic acid intrinsicclearance; HA.E, hyaluronic acid hepatic extraction ratio; KC, Kupffer cell; MDP, dichloromethylene diphosphonate; SEC, sinusoidal endothelial cell; TC, taurocholate; TC.Cli, taurocholate intrinsic clearance; TC.E,taurocholateextractionratio; UW, Universityof Wisconsin. © 1995 by the American Gastroenterological Association 0016-5085/95/$3.00

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2 consisted of an orthotopic rat liver transplantation. Inbred male Lewis rats (Charles River, Canada) weighing 2 8 0 - 3 2 0 g were used to avoid immunologic interference. Rats had free access to normal rat chow. Twelve hours before organ harvesting, donor rats had access only to 5% dextrose. Recipient rats had free access to normal rat chow until implantation. Rats were randomly assigned to MDP treatment (n = 15) or control (n = 15) groups.

KC Elimination Liposome-encapsulated MDP was prepared using the method of van Rooijen. 2~ Briefly, 75 mg phosphatidylcholine and 11 mg cholesterol (Sigma Chemical Co., St. Louis, MO) were dissolved in 20 mL methanol-chloroform (1:1) in a roundbottomed flask. The thin film formed on the interior of a flask after low-vacuum rotary evaporation at 37°C was dispersed in 10 mL phosphate-buffered saline (PBS; 10 mmol/L, p H 7.4) containing 1.89 g MDP (courtesy of Boehringer Mannheim, Laval, Quebec, Canada) by gently rotating for 10 minutes. Free MDP was removed by rinsing the liposomes with PBS and centrifuging for 30 minutes at 100,000g at 16°C. Liposomes were resuspended in 4 mL PBS, and 2 mL was injected intravenously 42 hours before the perfusion study or donor hepatectomy.

Experiment 1: Isolated Perfused Rat Liver Animals were anesthetized with pentobarbital (50 mg/ kg intraperitoneally). After cannulation of the bile duct, portal vein, and suprahepatic inferior vena cava, livers were flushed with 100 mL oxygenated Krebs-Henseleit buffer (37°C) as previously described. 22 Heparin was not used to avoid the competitive inhibition of hyaluronic acid (HA) metabolism. 23 Hepatic artery and infrahepatic inferior vena cava were ligated. The liver was then removed and perfused in a closed recycling system using a perfusion apparatus (Mx/Ambex Two/ten; Mx International Inc., Aurora, CO). The perfusion medium (250 mL of total volume) consisted of Krebs-Henseleit buffer, p H 7.4, containing 20% prewashed bovine erythrocytes (vol/vol), 20 g/L albumin (wt/vol), and 1 glL dextrose (wt/vol). The perfusate was saturated by equilibration with 95% 02 and 5% CO2, and its temperature was kept at 37°C. The perfusion flow was measured volumetrically and set at 20 mL/min. Livers were perfused for 40 minutes (baseline perfusion period), flushed with 60 mL U W solution (4°C; Dupont Critical Care, Mississauga, Ontario, Canada), clamped, and kept at 4°C in the U W preservation solution for 24 hours, until being reperfused under the same experimental conditions as used for the baseline period for an additional 40-minute period. Hepatocyte and SEC functions were assessed by measurements of taurocholate (TC) and H A elimination. During both perfusion periods, loading doses of unlabeled TC (mixed with tracer doses of [14CITe) and H A were added to the reservoir to attain a theoretical plasma concentration of 11.5 gg/mL for TC and 150 ng/mL for HA, followed by continuous infusion to main-

GASTROENTEROLOGY Vol. 109, No. 1

tain these levels. HA, prepared from rooster comb, was obtained from Sigma Chemical Co. (catalog no. H 5388) and infused at a rate of 2.7 btg/min as previously reported. 24 Liver viability was evaluated during both periods by assessing gross appearance, lactate dehydrogenase release, 02 consumption, bile production, and perfusion pressure. 25 After the reperfusion period, the liver was perfused for an additional 10 minutes with Krebs-Henseleit buffer (37°C). Then liver biopsy specimens from the main four lobes were obtained, snapfrozen in liquid nitrogen, and kept at -70°C. Assessment of TC elimination. [I4C]TC plasma levels were determined in duplicate using a beta counter (Beck-

mann, Montreal, Quebec, Canada). Plasma samples were obtained during baseline and reperfusion periods (20, 25, 30, 35, and 40 minutes). TC elimination was evaluated by its hepatic extraction ratio (TC.E) and intrinsic clearance (TC.Cli) using the following formulas: TC.E = (Ci - Co)/Ci and TC.Cli = - Q × In(1 - E), in which Ci and Coare TC plasma levels at inflow and outflow and Q is the total flow. TC.Cli is calculated using the sinusoidal model. 26 Assessment of HA elimination. H A plasma levels were determined in duplicate using a radiometric assay (Pharmacia, Montreal, Quebec, Canada) as previously described = at the same times as for TC determination. H A hepatic extraction ratio (HA.E) and H A intrinsic clearance (HA.Cli) were calculated using the same formula as for TC. 26

Experiment 2: Orthotopic Rat Liver Transplantation Liver transplantation was performed according to the technique of Kamada and Calne 27 as modified by Harihara et al. 28 All surgical procedures were performed under halothane and nitrous oxide anesthesia. Before hepatectomy, the donor liver was perfused in situ via the portal vein with 10 mL chilled U W solution. The excised liver was placed in a bath of cold U W solution for cuff preparation, followed by perfusion with another 10 mL cold U W solution. After cuff preparation, a biopsy specimen was obtained from the caudate lobe ( 0 . 5 1 g) for immunohistochemical study. The liver was then stored in a beaker containing 20 mL U W solution at 4°C for 24 hours. After cold preservation, the donor liver was flushed with 20 mL Ringer's lactate solution and transplanted orthotopically into the recipient rat. Ampicillin (50 mg/kg intramuscularly) was administered for 3 days after the operation. Surgery required 6 0 - 7 0 minutes, and portal vein clamping never exceeded 16 minutes. Results are expressed by comparing the 10-day survival rate of control and MDP-treated animals. To confirm the validity of the transplantation technique and to verify the effect of KC depletion on the survival outcome after the transplantation under nonpreserved condition, livers from both control (n -- 5) and MDP-treated (n = 5) animals were stored for 1 hour in cold normal saline (0.9%) and trans-

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KUPFFER CELLS IN COLD ISCHEMIA/REPERFUSION

Ontario, Canada) followed by avidin-biotin interaction technique using an avidin-biotin complex kit (Vector, Burlingame, Canada) as described previously. 29 Biotin-labeled horse antimouse immunoglobulin was used as a second antibody, and diaminobendine tetrahydrochloride (Sigma) was used as a substrate to detect the avidin-biotinylated peroxidase complex.

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Data are expressed as mean _+ SEM. Paired t tests, unpaired Student's t tests, or Fisher's Exact Test were used to compare the two groups. We also used two-way analysis of variance (ANOVA) with posttest correction by the Bonferroni method for multiple comparisons. A P value of <0.05 was considered statistically significant.

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Experiment 1: Isolated Perfused Rat Liver

Figure 1. Intrahepatic resistance values were measured in the control (D; n = 8) and MDP-treated (B; n = 8) groups during baseline and at different times during reperfusion. Mean intrahepatic resistances within each group differed significantly (P < 0.001), but no significant difference was found between the two groups by two-way ANOVA analysis. Corrected Bonferroni P values: *P < 0.05, **P < 0.01, ***P < 0.001 compared with basal values.

planted. A liver biopsy specimen was also obtained after organ harvesting. The hepatic artery was not reconstructed because the results of our preliminary study showed that short-term survival rate was not decreased using this transplantation model (100% at 10 days) in nonpreserved conditions (1 hour in cold normal saline) and that complications such as hyperbilirubinemia and fibrosis with bile duct proliferation occurred later (approximately the third week). These complications are currently thought to be caused by the lack of vascularization of the biliary tree.

Immunohistochemical Staining for Liver Biopsy Specimens Frozen biopsy specimens were cut with a cryostat into 8-btm sections, air dried, and fixed in acetone for 10 minutes at room temperature. KCs were detected with mouse antirat macrophage monoclonal antibody ED2 (Serotec, Toronto,

Because no significant differences were found in body weight (280 vs. 275 g), liver weight (8.6 vs. 9.0 g), or liver/body weight ratio (0.031 vs. 0.033) between control and MDP-treated animals, respectively, data were expressed per gram of liver. Data were calculated as the mean values obtained between the 20th and 40th minute of each perfusion period, unless otherwise stated. Viability parameters. The livers had a normal, uniform, bright color without signs of perfusion defects in all experiments during the baseline and reperfusion periods. Viability parameters for these same periods are shown in Figure 1 and Table 1. Lactate dehydrogenase release was very low during baseline, increased significantly and similarly in both groups 5 minutes after reperfusion, and remained stable thereafter until the 40th minute, being 19 times greater than baseline values in controls and treated rats (P value, NS). Bile production was approximately 2 btL • min -l • g-1 during baseline and decreased in a similar way in both groups ( - 3 5 % and - 2 7 % in control and treated rats, respectively; P value, NS). The intrahepatic resistance, as calculated by perfusion pressure/perfusion rate (mm Hg • min -1 mL -1) increased 5 minutes after reperfusion (+33% and

Table &, Viability P a r a m e t e r s o f I s o l a t e d P e r f u s e d Livers From Control Rats and MDP-Treated Rats Lactate dehydrogenase release

Oxygen consumption

Bile production

( x l O 2 IU. min l . g - ~ )

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(#L. min -1. g - l )

Rat group

Baseline

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Baseline

Reperfusion

Baseline

Reperfusion

Control MDP-treated

1.0 ± 0.17 0.9 ± 0.26 NS

17.1 +_ 3.03 a 12.9 _+ 1.93 a NS

3.49 _+ 0.19 2.79 ± 0.16 P < 0.005

2.89 + 0.13 b 2.22 + 0.11 c P < 0.005

1.97 _+ 0.11 2.17 ± 0.10 NS

1.23 + 0.38 a 1.59 ± 0.32 c NS

NOTE. Control rats, n = 8; MDP-treated rats, n = 8. ap < 0.005, bp < 0.05, °P < 0.01 VS. baseline value.

192 IMAMURA ET AL.

GASTROENTEROLOGYVol. 109, No. 1

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Figure 2. (A) TC.E and (B) TC.Cli in the control (r~; n = 8) and MDP-treated (B; n = 8) groups during baseline and reperfusion periods.

+34% in control and treated rats, respectively; P value, NS), then declined progressively but remained elevated in both groups until the 40th minute (Figure 1). Finally, the mean oxygen consumption was significantly lower in the MDP-treated group than in the control group during the baseline period ( - 2 0 % ; P < 0.05) and decreased significantly but similarly during reperfusion in both groups ( - 2 3 % for control, P < 0.05; - 2 3 % for MDPtreated, P < 0.05 ) (Table 1). TO elimination. Neither TC.E nor TC.Cli showed a significant difference between groups during baseline (Figure 2). Although both parameters decreased significantly after reperfusion, the decreases were similar in the two groups (Figure 2), with TC.CIi decreasing by 45% in controls and by 39% in the treated rats (P value, NS). HA elimination. No significant differences were found between the groups in either HA.E or HA.Cli during baseline (Figure 3). These parameters decreased markedly after reperfusion in both groups (Figure 3). However, once again, no significant difference was found in the decrease of HA.E or HA.Cli when the groups were compared (Figure 3). HA.Cli decreased by 94% and 93% in control and treated animals, respectively (P value, NS). Experiment 2: Orthotopic Rat Liver Transplantation

All rats undergoing transplantations using livers obtained from control (n = 5) and MDP-treated (n = 5) animals and stored for 1 hour in cold normal saline

lived for more than 10 days. After 24-hour preservation in U W solution, 6 of 15 animals (40%) receiving livers from control rats and 4 of 15 animals (27%) receiving livers from MDP-treated rats lived for more than 10 days. The difference between the groups was not statistically significant (P value, NS; Fisher's Exact Test). All rats recovered from the anesthesia and were conscious within 25 minutes after surgery. However, the clinical status of nonsurvivors began to deteriorate within 4 - 6 hours, and all rats died within 24 hours. Postmortem examination showed 2 - 3 mE ascitic fluid without frank blood and 1 - 2 mL serous pleural effusion. Immunohistochemical Studies

In normal rats, KCs (fixed liver macrophages) are positive for the monoclonal antibody ED2. 3° The ED2positive KCs, which were clearly visible in control rats (Figure 4A), completely disappeared in all lobes of livers from MDP-treated rats (Figure 4B). Liver biopsy specimens, also processed for light microscopy, showed no morphological change in liver architecture or any inflammatory infiltration.

Discussion In the present study, we evaluated the role of KCs in cold ischemia/reperfusion injury of the liver. First, we compared hepatocyte and SEC functions of KC-deplered rats and control rats in an in vitro isolated perfused liver model before and after 24-hour cold preservation in U W solution. Subsequently, in an in vivo orthotopic rat liver transplantation model, we compared the survival rate after

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KUPFFER CELLS IN COLD ISCHEMIA/REPERFUSION

transplantation after 24-hour cold preservation in U W solution in two other groups of KC-depleted and control rats. It is currently accepted that SECs are the main targets of cold ischemia/reperfusion injury and that their deterioration, with the ensuing microcirculation disturbance, is one of the primary factors leading to storage-related graft nonfunction following liver transplantation. 6-.1 However, the precise mechanism underlying the preservation/ reperfusion injury remains obscure. It has been hypothesized that KCs play a role in this injury, particularly because morphological features of KC activation have been observed after cold ischemia/reperfusion. 2°'31 It is well known that activated KCs can produce highly toxic mediators, such as reactive oxygen intermediates, protease, and cytokines, 12-~4 thought to be involved in the pathogenesis of various animal hepatotoxicity models ~5-.7 and hepatic hypoxia/reoxygenation.32-37 On the other hand, KC activation can induce SEC cell apoptosis in isolated cell preparations 38 and can suppress SEC function, as evaluated by HA elimination in isolated perfused rat liver. 39 Conversely, a suppression of KC activation by nisoldipine, a calcium channel blocker, and pentoxifylline has been advocated in the increased survival of transplanted rats after prolonged cold liver preservation in the presence of these two drugs. 18'19 However, during cold preservation, morphological alterations of SECs have been reported to precede reperfusion and the maximal expression of KC activation. 4° Also, conflicting findings have been reported, not supporting a causal role for reactive oxygen intermediates in the cold ischemia/reperfusion injury of the liver, particularly of SEes. 11,41,42

jury by selectively eliminating KCs from rat liver in vivo. Several techniques for inactivating KCs have been reported43'44; however, these methods may not result in complete depletion or inactivation of KCs. 33'45 W i t h the method used in the present study, liposomes with entrapped MDP are ingested by macrophages located in the spleen and particularly in the liver (KCs). Once phagocytosed, the liposomal membranes are disrupted by KC's lysosomal phospholipase and the drug is released intracellularly. This results in KC death, probably achieved by the calcium-binding activity of MDP. 2I After KC destruction, MDP is immediately diluted in the circulation, 46 with a half-life of only a few minutes without further toxicity to other cell types. 21 KC depletion is generally attained within the first 2 days after MDP injection. 21'47 In rats, KC repopulation starts by day 3 and is completed by day 8. 47 In the present study, complete depletion of KCs was confirmed 2 days after MDP injection by immunohistochemical staining, without any other structural alteration or inflammatory response (Figure 4). In the present investigation, rat livers were preserved for 24 hours in cold (4°C) U W solution. Using this preservation condition, a survival rate of about 30% has been reported after liver transplantation in Wistar-toWistar rats 48 and between 30% and 50% in Lewis-toLewis rats32'49; these values are in good accordance with the values of the present study in Lewis-to-Lewis rats (40%). Thus, 24-hour cold storage in cold U W solution, although a severely compromising condition, should allow an adequate evaluation of the effect of KC depletion on preservation/reperfusion injury, particularly to SECs, because about one third of the transplanted animals will finally recover and survive.

To address this controversy, we decided to evaluate directly the role of KCs in cold ischemia/reperfusion in-

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IMAMURA ET AL.

Figure 4. Frozen liver biopsy specimens from (A) a control rat and (B) an MDP-treated rat stained using a mouse ED2 monoclonal antibody. (A) In the control rat, strong staining can be seen in cells located in the sinusoidal lumen (fixed liver macrophages or KCs), (B) whereas there is no staining in the MDP-treated rat (original magnification 200x).

In the first set of experiments, we assessed the effect of KC depletion on liver function after 24-hour cold preservation in isolated perfused rat livers specifically in relation to SEC injury, because recent studies have shown the particular vulnerability of SECs to cold ischemia/reperfusion with the relative sparing of other parenchymal and nonparenchymal cells. 6-1° These observations were based on morphological changes using light, transmission, or scanning electron microscopy9'1° and on semiquantitative evaluation of cell death by nuclear staining by trypan blue. 6-8 However, possible sublethal alterations can be underestimated when assessing irreversible cell death using the trypan blue staining method. 6 In addition, differentiation between SECs and other nonparenchymal cells may be difficult under light microscopy. In the present study, we assessed the alteration of SEC function by HA elimination. HA is eliminated almost exclusively by SEC, 5°

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and HA eliminating capacity was reported as an accurate quantitative indicator to evaluate SEC function in isolated perfused rat livers. 23 Also, we have recently shown that HA elimination is a useful tool to estimate SEC damage after cold ischemia/reperfusion in the rat. 24 During the baseline perfusion period, oxygen consumption was the only parameter of liver viability that showed a significant difference between the control and MDP-treated groups. This decrease in oxygen consumption can be attributed to KC depletion because KCs represent 15% of the total cell population of the rat liver) 1 Furthermore, because there was no difference in TC or HA elimination between the groups, we conclude that the KC depletion method used in the present study was specifically directed to KCs without any negative effect on hepatocyte or SEC function, at least in terms of the isolated perfused rat liver. During the reperfusion period, parameters of liver viability showed moderate impairments in both groups. However, no significant differences were found between the two groups except for oxygen consumption, once again with similar decreases from baseline in both groups (Table 1 and Figure 1). Likewise, hepatocyte function as evaluated by TC elimination also showed similar impairment in both groups (Figure 2), indicating that the alteration in hepatocyte function was probably independent of KC activation. SEC function, assessed by HA elimination, showed profound impairment in both control and MDP-treated groups (Figure 3). The striking deterioration in SEC function, which contrasts with the moderate impairment of hepatocyte function, is in line with former investigations. 6'7'1°'52 However, the differences between control and MDP-treated groups were not significant (Figure 3). These alterations were present as early as 5 minutes after reperfusion and remained stable until the end of the study in both groups (data were not shown). These data, obtained using an isolated perfused rat liver model, clearly show that the presence or absence of KCs does not modify the initial phase of injury induced by 24hour cold ischemia/reperfusion and, consequently, that their possible activation should not play a crucial role in the marked SEC alteration and the moderate hepatocyte impairment in these conditions. These data are in line with those reported by Clavien et al. 53 using a similar model and showing that blockade of KC by gadolinium chloride did not influence the early lymphocyte adhesion in reperfused liver after a 30-hour cold ischemia in U W solution, a phenomenon thought to contribute to reperfusion injury. The question still remains whether the KC activation

July 1995

further aggravates the later phase of injury induced by 24-hour cold ischemia/reperfusion leading to primary graft nonfunction. To address this question, we also undertook orthotopic rat liver transplantations in a Lewisto-Lewis rat model, chosen to avoid immunologic interference. In a first series of transplantations, donor livers, either with or without KCs, were stored for only 1 hour in cold saline. All animals survived beyond 10 days. These results not only confirm the validity of the transplantation procedure but also show that KC depletion is not associated with any negative effect on the survival outcome after a short cold preservation. In a separate study, we found that 12 hours after transplantation of KC-depleted livers (n = 4), no ED2-positive ceils were observed in grafted livers, suggesting that no recipient macrophage had moved to donor livers, at least during the first few hours after transplantation. In this rat model, after a 24-hour cold preservation in U W solution, survival was markedly reduced but no significant difference could be found between control and MDP-treated groups, all deaths occurring within the first 24 hours. The findings of the present in vivo study indicate that the presence or absence of KCs does not modify the overall time course of cold ischemia/reperfusion injury leading to storage-related graft failure. The results of our study on the role of KCs in the pathophysiology of liver graft failure from cold storage injury differ from the results of the North Carolina group. 54 Contrary to our study, most studies by this group, reporting a significant role for KCs, were indirect, based mainly on morphological signs of KC activation occurring in parallel with endothelial cell death during reperfusion. However, if it is well accepted that marked alteration of SECs begins before reperfusion, 9'~°'54 our study clearly shows that SEC death during reperfusion is independent of morphologically stimulated KCs. On the other hand, the improvement of graft survival using various agents, such as nisoldipine, pentoxifylline, adenosine and prostaglandin El, thought to down-regulate KC function, 18'19'54 does not infer that their effect is KCrelated; these drugs have several other known and probably unknown effects. Interestingly, Marzi et al. 45 using methyl palmitate pretreatment of donor rats, an inhibitor of KC phagocytosis, reported a significant delay in graft failure, although all pretreated and control rats finally died within the first 3 days. In conclusion, we show that the technique of in vivo KC elimination can be safely applied to an isolated perfused rat liver without a negative effect on hepatocyte or SEC function. Likewise, transplantation of KC-depleted livers does not have a negative outcome on survival rate

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195

under nonpreserved conditions. Using the isolated perfused rat liver preparation, we showed that the elimination of KCs did not modify the initial phase of 24-hour cold ischemia/reperfusion injury, particularly the marked SEC alteration found in these conditions. Moreover, elimination of KCs does not alter the overall survival rate of animals after transplantation following 24-hour cold ischemia in U W solution. These data stongly suggest that the presence or absence of KCs does not modify the effect of cold ischemia/reperfusion on rat liver at least after a 24-hour preservation in cold U W solution.

References 1. NIH. Consensus Development Conference statement. Hepatology 1984;4:107S-110S. 2. Greig PD, Woolf GM, Sinclair SB. Treatment of primary liver graft nonfunction with prostaglandin El. Transplantation 1989;48: 447-453. 3. PIoeg RJ, D'alessandro AM, Knechtle SJ, Stegall MD, Pirsch JD, Hoffmann RM, Sasaki T, Sollinger HW, Belzer FO, Kalayoglu M. Risk factors for primary dysfunction after liver transplantation-a multivariate analysis. Transplantation 1993;55:807-813. 4. Todo S, Nery J, Yanaga K, Podesta L, Gordon RD, Starzl TE. Extended preservation of human liver grafts with UW solution. JAMA 1989;261:711-714. 5. Kalayoglu M, Hoffmann RM, D'Alessandro AM, Pirsch JD, Sollinger HW, Belzer FO. Results of extended preservation of the liver for clinical transplantation. Transplant Proc 1989; 21:34873488. 6. Marzi I, Zhong Z, Lemasters JJ, Thurman RG. Evidence that graft survival is not related to parenchymal cell viability in rat liver tranplantation. Transplantation 1989;48:463-468. 7. CaldwelI-Kenkel JC, Thurman RG, Lemasters JJ, Selective loss of nonparenchymal cell viability after cold ischemic storage of rat livers. 1988;45:834-837. 8. CaldwelI-Kenkel JC, Currin RT, Tanaka Y, Thurman RG, Lemasters JJ. Reperfusion injury to endothelial cells following cold ischemic storage of rat livers. Hepatology 1989; 10:292-299. 9. Myagkaya GL, van Veen HA, James J. Ultrastructural changes in the rat liver during Euro-Collins storage, compared with hypothermic in vitro ischemia. Virchows Arch [B] 1987; 53:176-182. 10. McKeown CMB, Edwards V, Phillips M J, Harvey PRC, Petrunka CN, Strasberg SM. Sinusoidal lining cell damage: the critical injury in cold preservation of liver allografts in the rat. Transplantation 1988;46:178-191. 11. Holloway CMB, Harvey PRC, Mullen JBM, Strasberg SM. Evidence that cold preservation-induced microcirculatory injury in liver allografts is not mediated by oxygen-free radicals or cell swelling in the rat. Transplantation 1989;48:179-188. 12. Arthur MJP, Kowalski-Saunders P, Wright P. Effect of endotoxin on release of reactive oxygen intermediates by rat hepatic macrophages. Gastroenterology 1988; 95:1588-1594. 13. Decker K. Biologically active products of stimulated liver macrophages (Kupffer cells). Eur J Biochem 1990;192:245-261. 14. Michie HR, Manogue KR, Spriggs DR, Revhaug A, O'Dwyer S, Dinarello CA, Cerami A, Wolff SM, Wilmore DW. Detection of circulating tumor necrosis factor after endotoxin administration. N Engl J Med 1988;318:1481-1486. 15. Laskin DL, Pilaro AM. Potential role of activated macrophages in acetaminophen hepatotoxicity. Toxicol Appl Pharmaco11986; 86: 204-215.

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Received December 19, 1994. Accepted March 14, 1995. Address requests for reprints to: Pierre-Michel Huet, M.D., Andr~Viallet Clinical Research Center, HSpital Saint-Luc, 264 Ren6-L6-

KUPFFER CELLS IN COLD ISCHEMIA/REPERFUSION 197

vesque Boulevard East, Montreal, quebec H2X 1P1 PQ, Canada. Fax: (514) 281-2492. Supported by a grant from the Medical Research Council of Canada, a fellowship from Tsumura & Co. Ltd., Japan (to H.I.), and a studentship from the Canadian Liver Foundation (to F.S.). Presented at the 44th annual meeting of the American Association for the Study of Liver Diseases, Chicago, Illinois, in November 1993. The authors thank Boehringer Mannheim Canada Inc., particularly D. Mantha, for the gift of dichloromethylene diphosphonate and Fran~oise Trotier for assistance in preparing the manuscript.