A novel way of liver preservation improves rat liver viability upon reperfusion

A novel way of liver preservation improves rat liver viability upon reperfusion 1 first-author 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:marian.kukan@szu.sk †E-mail:marian.kukan@szu.sk marian.kukan@szu.sk 1 2 Laboratory of Perfused Organs, Slovak Center for Organ Transplantation, Slovak Medical University, Limbová 12, 83303 Bratislava, Slovakia Laboratory of Perfused Organs, Slovak Center for Organ Transplantation, Slovak Medical University, Limbová 12, 83303 Bratislava, Slovakia Department of Pathology, School of Medicine, Comenius University, 81372 Bratislava, Slovakia Department of Pathology, School of Medicine, Comenius University, 81372 Bratislava, Slovakia Background/aim: 2 Currently, the liver is cold-preserved at 0~4 °C for experimental and clinical purposes. Here, we investigated whether milder hypothermia during the initial phase of the preservation period was beneficial for liver viability upon reperfusion. Methods: 2 In the first set of experiments, rat livers were preserved either conventionally in clinically used histidine-trypthopan-ketoglutarate (HTK) solution (Group A: 45 min and Group B: 24 h) or by slow cooling HTK solution (from 13 °C to 3 °C) during the initial 45 min of preservation (Group C: 24 h). In the second set of experiments, additional groups of livers were evaluated: Group BB—preservation according to Group B and Group CC—preservation according to Group C. Further, some livers were preserved at 13 °C for 24 h. Livers were then reperfused using a blood-free perfusion model. Results: 2 Bile production was approximately 2-fold greater in Group C compared to Group B. Alanine transaminase (ALT) and aspartate transaminase (AST) release into perfusate were 2~3-fold higher in Group B compared to Group C. No significant differences were found in ALT and AST release between Group C and Group A. Livers in Group CC compared to Group BB exhibited significantly lower portal resistance, greater oxygen consumption and bromosulfophthalein excretion into bile and lower lactate dehydrogenase (LDH) release into perfusate. Histological evaluation of tissue sections in Group BB showed parenchymal dystrophy of hepatocytes, while dystrophy of hepatocytes was absent in Group CC. Livers preserved at 13 °C for 24 h exhibited severe ischemic injury. Conclusion: 2 These results suggest that the conventional way of liver preservation is not suitable at least for rat livers and that slow cooling of HTK solution during the initial phase of cold storage can improve liver viability during reperfusion. Rat Hepatic graft Cold ischemia Liver protection Histidine-trypthopan-ketoglutarate solution (HTK) INTRODUCTION 1 A major factor determining morbidity and mortality after liver transplantation therapy is preservation injury of hepatic grafts (Lemasters and Thurman, 1997 1997 ; Kukan and Haddad, 2001 2001 ). Prevention of liver preservation injury may decrease the health care burden of liver transplant recipients as well as the need for retransplantation and thus increase the availability of donor livers for patients on waiting lists (Lemasters and Thurman, 1997 1997 ; Kukan and Haddad, 2001 2001 ). At present, during their procurement, human livers are washed out with cold preservation solution (4 °C) and then immediately submerged into plastic bags containing preservation solution (4 °C). The plastic bag is then placed into iced slush, where the liver is preserved at temperature of 0~4 °C. It is supposed that this procedure is advantageous for the liver, since it allows a rapid decrease of its temperature and minimizes organ metabolism (Hertl et al., 1996 1996 ). However, Drews et al.( 1998 1998 ) showed that in rat livers their rapid cooling by iced water during procurement led to a marked deterioration of microperfusion in peripheral and subcapsular regions during cold reperfusion. This interesting work (Drews et al., 1998 1998 ) remained neglected by scientists working in the area of liver preservation. Furthermore, we found that in our previous experiments surface liver temperature after standard washing out of rat livers with University of Wisconsin preservation solution (4 °C) was approximately 19 °C (Lutterová et al., 2001 2001 ). Thus, if one takes into consideration the difference between the temperature of liver surface and the preservation solution, it is not clear whether the current technique of liver preservation is appropriate. Indeed, we found very recently that preservation of rat livers in physiologic saline at the initial temperature of 10 °C was better than preservation at the initial temperature of 4 °C (Kebis and Kukan, 2006 2006 ). Therefore, this study was designed to test the hypothesis that the initial hypothermia close to liver surface temperature as well as slow cooling of histidine-trypthopan-ketoglutarate (HTK) solution (which is in clinical practice for human liver preservation) during the initial phase of cold storage is beneficial for liver viability during reperfusion. MATERIALS AND METHODS 1 Animals 2 Male Wistar rats, weighing 280~320 g, obtained from Velaz Co. (Prague, Czech Republic) were used. The animals were housed in an air-conditioned room at 22 °C and fed Velaz rat chow and water ad libitum up to the time of the experiments. The study was approved by the local animal welfare committee. Liver procurement 2 The rats were given ketamine/xylazine [(100/10) mg/kg] intraperitoneally to induce anesthesia before surgery and the liver was prepared as described (Kukan, 1999 1999 ). Briefly, the bile duct was cannulated with polyethylene PE 50 tubing and 250 IU of heparin was injected intravenously. To ensure that global liver function was comparable between the groups, bile production was measured for 5 min during liver procurement. The portal vein was then cannulated with PE 240 tubing and the hepatic artery was ligated. The liver was flushed out in situ through the portal vein with 30 ml of cold (4 °C) HTK solution (Dr. Franz Köhler Chemie GmbH, Alsbach-Hähnlein, Germany) at constant pressure of 12 cmH 2 O. Then, as rapidly as possible, the livers were excised and transferred to the preservation/perfusion chamber made from plexiglass containing 70 ml of HTK solution (Kebis and Zvrškovec, 2002 2002 ). Liver preservation 2 In the first set of experiments, rat livers were preserved either conventionally in HTK solution [Group A: 45 min ( n =6) and Group B: 24 h ( n =6)] or by slow cooling HTK solution (from 13 °C to 3 °C) during the initial 45 min of preservation [Group C: 24 h ( n =6)]. In the second set of experiments, additional groups of livers were evaluated: Group BB ( n =5)—preservation according to Group B and Group CC ( n =5)—preservation according to Group C). Finally, some livers were preserved at 13 °C for 24 h ( n =3). Liver perfusion 2 The livers were perfused through the portal vein from a height of 12 cm in a recirculating perfusion system at 37 °C (Kukan, 1999 1999 ). As a perfusion medium, Krebs-Henseleit bicarbonate buffer (KHB) (200 ml, pH=7.4) containing glucose (10 mmol/L) and saturated with 95% oxygen and 5% carbon dioxide (inflow P O 2 =400~500 mmHg) was used. In the first set of experiments, the following parameters were assessed: portal flow, bile production, alanine transaminase (ALT) and aspartate transaminase (AST) release into perfusate. In the second set of experiments, we measured portal resistance, oxygen consumption, bromosulfophthalein (BSP) excretion, (BSP was added into the reservoir at 30 min of reperfusion as a 6 μmol bolus), and lactate dehydrogenase (LDH) release into perfusate. Temperature measurements 2 We measured the temperature of HTK solution inputing rat livers, surface liver temperature, and HTK solution in the preservation/perfusion chamber (for the first 60 min of preservation) by a thermometer (HI 8751 Minitherm, Hanna Instruments, Kahl am Rhein, Germany). Histological analysis by hematoxylin and eosin (HE) staining 2 After each perfusion of the second set of experiments, the middle part of the great left lateral lobe (lobus sinister lateralis) was fixed in 10% formalin. Fixed samples were cut and stained by HE. Pathologist (Ján Jakubovský) performed morphological evaluation of liver tissues. Morphological evaluation was carried out on HE-stained sections at original magnification×100. Assays 2 The activities of perfusate ALT, AST, and LDH were measured with standard reagent kits, which were purchased from Pliva-Lachema Diagnostica (Brno, Czech Republic). BSP concentrations were measured in 0.1 mol/L NaOH spectrophotometrically at 580 nm after appropriate dilution of bile samples. Oxygen consumption was assessed by 248 pH/Blood Gas Analyzer (Ciba Corning Diagnostics Limited, UK). Treatment of data and statistics 2 Oxygen consumption was calculated from the equation: O 2 consumption=( C in − C out ) ml/(min·g liver), where C in and C out are oxygen perfusate levels at input and output. Percentage change in liver weight during the reperfusion period was calculated as follows: (Δliver weight/initial liver weight)×100%. Values are given as mean± SEM . The data from three groups of livers were analyzed by ANOVA followed by Bonferroni correction test for multiple comparisons. Statistical evaluation between two groups was performed using Student’s t test. A difference was considered significant when P < 0.05. RESULTS 1 The first set of experiments 2 1. Animal weight, liver weight and bile production prior to preservation 3 There were no significant differences in animal weight (g) between Group A, Group B, and Group C (Group A: 309±6; Group B: 306±3; Group C: 302±2). Similarly, no significant differences were found in liver weight (g) (Group A: 10.8±0.2; Group B: 10.7±0.1; Group C: 10.6±0.1). Bile production [µl/(min·g liver)] in vivo, as a sign of global hepatic function, was similar prior to cold preservation (Group A: 1.95±0.1; Group B: 2.2±0.2; Group C: 2.19±0.2). 2. The temperature of the preservation solution and of liver surface after liver procurement 3 There were no significant differences in the temperature (°C) of HTK solution inputing livers between the groups (Group A: 4.0±0.3; Group B: 4.5±0.1; Group C: 4.8±0.3). Likewise, surface liver temperatures (°C) after washing out of the liver were similar between the groups (Group A: 16.8±0.3; Group B: 16.8±0.9; Group C: 17.7±0.3). 3. The temperature of the preservation solution during the initial phase of cold storage 3 Fig. 1 1 shows temperature of HTK solution during the first 60 min of preservation. It can be seen that there were significant differences in the temperature of HTK solution between Group C and Group A or Group B during the first 35 min of preservation. After 45 min of preservation temperatures were similar. 4. Effects of the initial phase of cold storage on liver viability during reperfusion in Group A, Group B, and Group C 3 Viability parameters of isolated reperfused rat livers are shown in Figs. 2a~2d 2a~2d . Portal flow was comparable between Group A and Group C. Although nonsignificant, but better flow was recorded in Group C compared to Group B (Fig. 2a 2a ). Livers were perfused after standard cold preservation for 45 min (Group A), after standard cold preservation for 24 h (Group B), or following slow cooling of the preservation solution from 13 °C to 3 °C during the initial 45 min of preservation (Group C). Data represent mean± SEM (6 livers per group) and were expressed per gram liver weight. Significantly different at P < 0.05: * Group B vs Group A or Group C, # Group C vs Group B Bile production, as a sign of global hepatic function, was severely suppressed in 24-h preserved livers. Slow cooling of HTK solution at the initial phase of cold preservation (Group C) improved significantly bile production after 30 min reperfusion compared to Group B (Fig. 2b 2b ). ALT and AST release into perfusate, as measures of hepatocellular membrane integrity, were nonsignificantly different between the groups at 5 min of reperfusion, but they were 2~3 times higher in Group B compared to Group A at 60 min of reperfusion (Figs. 2c and 2d 2c and 2d ). Release of ALT and AST into perfusate of Group C after 60 min of reperfusion was comparable to control livers (Group A). The second set of experiments 2 1. Effects of the initial phase of cold storage on liver viability during reperfusion in Group BB and in Group CC 3 To prove that slow cooling of the preservation solution is indeed better than the conventional way of preservation, we performed further experiments and measured other viability parameters of livers during reperfusion (Note: livers in Group BB were preserved according to Group B and livers in Group CC were preserved according to Group C). Portal resistance during reperfusion of Group BB and Group CC is shown in Table 1 1 . It can be seen that after 30 min of reperfusion portal resistance was significantly lower in Group CC compared to Group BB. Data represent mean± SEM (5 livers per group) Significantly different at P < 0.05 Reperfusion time (min) Group BB Group CC 5 0.516±0.058 0.518±0.029 15 0.460±0.046 0.464±0.025 30 0.438±0.031 0.406±0.015 45 0.450±0.026 0.384±0.007 * * 60 0.450±0.026 0.374±0.008 * * Likewise, oxygen consumption was greater after 30 min of reperfusion in Group CC compared to Group BB (Table 2 2 ). Data represent mean± SEM (5 livers per group) Significantly different at P < 0.05 Reperfusion time (min) Group BB Group CC 5 0.734±0.076 0.760±0.072 15 0.826±0.104 0.882±0.081 30 0.854±0.106 1.056±0.067 45 0.800±0.070 1.088±0.058 * * 60 0.842±0.073 1.108±0.058 * * Bile production was nonsignificantly better in Group CC compared to Group BB during the first 30 min of reperfusion (Table 3 3 ). However, BSP-induced bile flow was significantly greater in Group CC compared to Group BB. Similarly, BSP excretion into bile was significantly greater in Group CC compared to Group BB (Table 3 3 ). Data represent mean± SEM (5 livers per group) Bile flow during the first 30 min of reperfusion Bile flow after addition of BSP into the reservoir during the last 30 min of reperfusion Significantly different at P < 0.05 Treatment groups a a Bile flow [μl/(min·g liver)] b b BSP-induced bile flow [μl/(min·g liver)] BSP excretion [μmol/(30 min·liver)] Group BB 0.30±0.06 0.43±0.04 3.63±0.52 Group CC 0.42±0.04 0.62±0.05 * * 4.62±0.56 * * LDH release into perfusate, as a measure of hepatocellular membrane integrity, was (240±52) units/L in Group BB (after 60 min of reperfusion), while in Group CC LDH release was only (116±25) units/L. 2. Histological analysis by HE staining 3 Fig. 3 3 shows histological findings of HE staining. Compared to the control liver, livers in Group BB (by conventional preservation) and Group CC (by novel way of preservation) showed typical widening of sinusoids and dettachments of endothelial cells. Parenchymal dystrophy of hepatocytes was absent in the control liver and in Group CC, while in Group BB parenchymal dystrophy of hepatocytes was observed (Fig. 3 3 ). Thus, it can be concluded that the above novel way of preserving hepatocytes is consistent with measurements of viability parameters. 3. Preservation of livers at 13 °C for 24 h 3 Liver swelling (calculated as percentage change of liver weight during reperfusion) was 44.6%±4.1%, indicating severe ischemic injury. Compared to Group BB, portal resistance was approximately doubled and oxygen consumption halved, bile production during the first 30 min of reperfusion was one tenth that of normal, BSP-induced bile flow was approximately one twentieth that of normal and BSP excretion into bile was negligible. Finally, LDH release into perfusate was (1840±591) units/L. DISCUSSION 1 The objective of the present study was to investigate the hypothesis that slow cooling of the preservation solution during the initial phase of cold storage may improve liver viability during reperfusion and thus decrease liver reperfusion injury. We used for these experiments our preservation/perfusion chamber (Kebis and Zvrškovec, 2002 2002 ) since our previous study showed that preservation of rat livers is more suitable in a preservation chamber than in plastic bags (Kebis et al., 2004 2004 ). In addition, from the technical point of view a controlled decrease of the preservation solution can be achieved more precisely in plexiglass chamber than in plastic bag (Note: in the plastic bag the temperature of the preservation solution may interfere with ice slush surrounding the bag). The data presented in Figs. 2b~2d 2b~2d , Fig. 3 3 and Tables 1 1 ~ 3 3 strongly suggest that slow cooling is indeed beneficial for liver viability during reperfusion and that the current procedure used in liver preservation is not suitable at least for the rat model. Of all viability parameters studied during liver reperfusion, bile flow is considered to be the most reliable indicator of ischemic and preservation damage (Bowers et al., 1987 1987 ; Kukan and Haddad, 2001 2001 ). Bile flow correlated with ATP tissue levels after liver transplantation and rapid resumption of bile flow in both rat liver transplantation (Sumimoto et al., 1988 1988 ) and clinical liver transplantation (Lemasters and Thurman, 1997 1997 ) is considered to be an early sign of a successful graft. Data on improved bile flow and BSP excretion into bile, as an index of the hepatic organic anion transport, indicate that milder initial hypothermia and slow cooling of the preservation solution may improve transplantation prognosis. Interestingly, Fujita et al.( 1993 1993 ) showed that moderate hypothermia of 10 °C to 20 °C was more appropriate for maintaining structure and function of rat livers than temperature at 5 °C when the continuous perfusion of livers was used for preservation. However, for static liver preservation, temperatures close to 0 °C were found to be more appropriate than 4 °C (Hertl et al., 1994 1994 ; Yoshida et al., 1999 1999 ). In line with the latter findings, we observed severe liver injury, when livers were preserved at 13 °C for 24 h. The question now arises as to whether results presented in this study can be extrapolated to human livers. On one hand, surface-to-volume ratio of the rat liver is much higher than that of the human liver. Therefore, the injury resulting from human hepatic grafts may be much smaller than that resulting from rat liver hepatic grafts. On the other hand, the percentage of apoptotic hepatocytes in human hepatic grafts was the highest (30%) in the subcapsular region of the grafts (Borghi-Scoazec et al., 1997 1997 ), i.e. in the region mainly exposed to the effects of cold environment during the procurement phase. Further, apoptosis of hepatocytes was correlated with postoperative serum levels of AST (Borghi-Scoazec et al., 1997 1997 ). CONCLUSION 1 The results of the present study strongly suggest that slow cooling of the preservation solution during the initial phase of cold storage is more appropriate procedure for liver preservation than the currently used method. Whether these results hold true in humans is not clear. 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DOI: 10.1006/jsre.1998.5505 http://doi.org/10.1006/jsre.1998.5505 . A novel conception for liver preservation at a temperature just above freezing point The authors sincerely thank Mr. Eduard Mališ for his excellent technical assistance. 10.1631/jzus.2007.B0289 Project supported by the Ministry of Health of the Slovak Republic under the project of Modulation of Heat Transfer in Isolated Liver (No. 2005/32-SZU-10) and the VEGA Grant (No. 1/1158/04), Slovakia 2006-08-29 2007-03-05 2022-01-05 Copyright © 2007, Journal of Zhejiang University Science 2007 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 05 08