Original Paper Eur Surg Res 1992;24:333-338

Institute of Experimental Medicine, University of Cologne, FRG

Keywords Ischemia Liver Oxygen-free radicals Reperfusion Superoxide dismutase Transplantation

Evaluation of Antioxidant Treatment with Superoxide Dismutase in Rat Liver Transplantation after Warm Ischemia

Abstract In order to investigate the effects of the exogenously adminis­ tered radical scavenger superoxide dismutase (SOD) on the orthotopic liver graft, livers from male Wistar rats were trans­ planted after subjection to 40 min of warm ischemia and 30 min of storage at 4 °C. SOD was given at the onset of ischemia and before reperfusion as a supplement (6,000 IU) to the washout solutions. 30.000 IU were infused into the recipient. SOD reduced tissue levels of thiobarbituric acid-reacting sub­ stances at the end of ischemia (737 vs. 956 nmol/g; p < 0.01 ) and 60 min after the onset of reperfusion (629 vs. 947 nmol/g; p < 0.001) and preserved total adenine nucleotides after reperfusion (11.69 vs. 10.40 pmol/g; p < 0.01). Survival 2 weeks after transplantation was 18 % (2/11 ) in the SOD group versus 10% (1/10; nonsignificant) in untreated animals. It is concluded that SOD protects the ischemically altered liver from radical mediated peroxidation and preserves hepatic energy stores upon reperfusion. However, in our model no major improvement in organ viability could by achieved.

Introduction Oxygen-free radicals have been incrimi­ nated to mediate tissue alterations upon re­ perfusion of ischemically altered organs such

C.W. Chung was supported by a grant from the Frau Maria Pcsch Stiftung.

Received: April 29,1992 Accepted: June 2.1992

as heart, kidney and bowels [1-3]. However, conflicting results were reported with regard to the pathogenetic role of free radicals in postischemic reperfusion injury of the liver [4-6]. In these studies, the effects of radical

Th. Minor. MD Institute of Experimental Medicine U ni versity of Cologne Robert-Koeh-Strasse 10 D-W-500C Cologne 41 (FRG)

©1992 S. Karger AG. Basel 0 0 14—312X/92/ 0246-0333S2.75/0

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Th. Minor C. W. Chung Y. Yamamoto M. Ohara S. Saad W. Isselhard

U]. Hence, newer recombinant SOD prepara­ tions are well tolerated after intravenous ad­ ministration in man [12], do not require preischemic application and may thus be par­ ticularly interesting in clinical organ trans­ plantation. In this study we have attempted to evaluate the possible clinical impact of SOD treatment on hepatic energy metabolism and animal survival after orthotopic rat liver transplantation.

Material and Method All experiments were performed with permission according to German federal law concerning animal experiments. Male rats of the Wistar strain weighing 250-350 g were used. The animals had free access to food and water until the operation. All operations were done under ether anesthesia with Oi inhalation through a nasal catheter. Ortho­

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topic liver transplantation was performed by the modi­ fied 2-cuff technique of Kamada and Caine [ 13]. The donor livers were rinsed in situ via the portal vein with 10 ml of Ringer solution (RS) containing 500IU of heparin and 10 ml of Euro-Collins solution (ECS) containing 250 IU of heparin. They were there­ after preserved in a bath of RS for 40 min at 37 °C. Cuffs of the portal vein infrahepatic vena cava and common bile duct were inserted during the 40 min of warm ischemic storage. Then the livers were preserved in 4 °C ECS for 30 min, and the recipient operation was begun. The ECS was rinsed out with 10 ml RS prior to implantation. 5 ml of RS were continuously infused via the penile vein. The suprahcpatic inferior vena cava was sutured, the portal vein anastomosis and the continuity of the infrahepatic inferior vena cava were established by means of the cuff method. The common bile duct was connected to the recipient by a short polyethylene splint. The abdominal incision was sutured. The anhepatic time during implantation varied between 16 and 25 min. Arterial reconstruction was not performed as no benefit of the procedure could be proved [ 14] in the rat. Application o f SOD. 6.000 IU of CuZn-SOD (Gruncnthal. Aachen) were added to the rinsing solutions prior to ischemia and prior to implantation. Addition­ ally 30.000 IU of SOD were admixed to the 5 ml of RS given as an infusion to the recipient during the opera­ tion. Livers were freeze-clamped according to the Wollenbergcr technique [15] for the determination of the metabolic status in situ, at the end of the ischemic period (40 min at 37 °C and 30 min at 4 °C) or 60 min after transplantation. Survival rate was calculated from a group of animals which were observed for 14 days after transplantation. A control group was trans­ planted without warm ischemic storage of the livers. Frozen liver tissues were weighed and preserved for at least 5 days in a vacuum freezer(-45 °C, < 0.001 atm) to evaporate the tissue water. Assay o f Energy-Rich Phosphates in Liver Tissue. Freeze-dried tissue samples were homogenized and deproteinized with 0.33 M perchloric acid. The extract was centrifuged and the supernatant neutralized with 2 Ar K.OH and frozen to remove KC10.|. Adenine nucleo­ tides were determined enzymatically in the superna­ tant as described earlier [ 16], but using the hexokinase and glucose 6-phosphatc dehydrogenase reactions for the ATP assay [ 17], The sum of adenine nucleotides was calculated as ATP + ADP + AMP. Assay o f Lipid Peroxides in Liver Tissues. Forma­ tion of radical induced lipid peroxidation was approxi­ mated with the thiobarbituric acid method by spectro-

Minor/Chung/Yamamoto/Obara/Saad/ Isselhard

SOD Effects in Liver Transplantation

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scavenging compounds were evaluated in ex­ perimental models of liver ischemia in vivo or in vitro with varying success. In previous experiments in vitro using substrate-free or­ gan pcrsufflation with cither oxygen or nitro­ gen after liver ischemia, we found evidence that oxygen might have a toxic impact on the reoxygenated isolated rat liver and that this impact could be suppressed by exoge­ nous administration of superoxide dismutase (SOD) [7], Recently a crucial role of the vascular endothelial cell in the course of re­ perfusion injury has been suggested [8, 9], Actually we observed that the beneficial ef­ fect of SOD on the hepatic energy metabo­ lism after warm ischemia was associated with a significant reduction of hepatic vascu­ lar resistance during isolated perfusion of the liver [7], Yet there is little information about the usefulness of SOD administration in a trans­ plantation model of the liver especially con­ cerning adenylate energy metabolism [10,

Fig. 1. Survival of animals after transplantation of ischemic livers (40 min at 37 °C + 30 min at 4 °C) with or without treatment with SOD (control: transplantation after 30 min of ischemia at 4 °C).

Table 1. Metabolic data of fivers harvested in situ, at the end of ischemia (40 min at 37 °C + 30 min at 4 °C) or 60 min alter subsequent transplantation (Tx)

days

n

In situ Ischemia Untreated SOD Post-Tx Untreated SOD

ATP gmol/g

SAN gmol/g

TBRS nmol/g

6

10.30 ±0.64

15.44 ±0.88

460± 110

7 10

0.51 ±0.22 0.73 ±0.19

10.55 ±1.22 9.26 ±0.61

966 ±192 671 ±80***

10 9

5.81 ±1.05 5.94 ± 1.18

947± 144 10.40 ±1.02 11.69 ±0.92** 629+133***

** p < 0.0I: *** p < 0.001. vs. untreated. SAN =Sum of adenine nuc­ leotides.

photometric measurement of thiobarbituric acid­ reacting substances (TBRS) using tetramethoxypropanc as an external standard [ 18. 19]. Assay o f Enzyme Activities in Blood Plasma I h after Transplantation. Glutamate dehydrogenase (GLDH) and glutamate pyruvate transaminase (GPT) were determined photometrically using commercial standard kits (Boehringer. Mannheim, FRG). The results were expressed as mean ± standard deviation (SD). Differences between treated and un­ treated groups were considered of statistical signifi­ cance. ifp < 0.05. p < 0.01. or p < 0.001, determined by Student’s t lest (n 3* 6/group).

The survival rate after liver transplanta­ tion during the first 2 weeks is shown in figure 1. After 1 week 1/10 untreated animals (10%) survived as compared with 2/11 in the SODtreated group (18%). The difference was not significant and remained stable throughout the further observation period. Table 1 summarizes the results of the met­ abolic analyses. Liver ischemia led to a pro­ nounced decrease in tissue levels of ATP and a depletion of adenine nucleotides was seen in

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Results

G P T (U/ml)

G LD H (U/ml)

1

** p ■ 0.01 vs. untreated

0

SOD

untreated

SOD

both groups. Upon reperfusion after trans­ plantation SOD-treated livers revealed a sig­ nificantly more complete restauration of the sum of adenine nucleotides, while ATP levels remained similar in both groups. Lipid peroxidation, as evaluated from TBRS. was notable already during ischemia and significantly mitigated by SOD. After re­ perfusion the SOD-treated livers exhibited again significantly lower values of TBRS. Plasmatic enzyme activities of both GPT and GLDH were significantly less elevated after transplantation, when animals were treated with SOD (fig. 2).

Discussion The deleterious effects of warm ischemia on hepatic tissue are commonly recognized and energy-rich phosphates have been re­ ported to be quickly used up [20, 21]. On the other hand oxygen-free radicals might injure the liver by initiating radical lipid peroxida­ tion upon reperfusion or even during isch­ emic storage.

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Peroxidation has been observed even at rather low oxygen tensions in brain [22] and in rat liver [23]. As the regeneration of detoxi­ fying compounds in the liver is energy-depen­ dent [24], it seems quite understandable that we found enhanced lipid peroxidation in the ischemically stored liver. However, this radial-mediatcd impact was suppressed by ad­ ministration of SOD. and the protection of cellular membranes may contribute to the im­ proved outcome of the hepatocytes upon re­ perfusion. SOD reduced hepatocellular en­ zyme leakage after transplantation. While loss of GPT may reflect only reversible changes in membrane permeability without sustained in­ jury, the data on GLDH indicate mitochon­ drial damage and thus irreversible alterations [25]. Hepatic energy metabolism upon reperfu­ sion is particularly important to assess the viability of the graft [26, 27], In our study SOD improved the sum of adenine nucleo­ tides in the transplanted liver but tissue levels of ATP were comparable to the untreated control. These results are in opposition to pre­ vious data obtained in vitro, where ATP

Minor/Chung/Yamamoto/Obara/Saad/ Isselhard

SOD Effects in Liver Transplantation

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group).

stores were significantly increased after treat­ ment with SOD [7], It may be suggested that the liver graft in vivo is less vulnerable to radi­ cal-mediated injury than the isolated organ exposed to perfusion with an artificially high oxygen partial pressure. Furthermore red blood cells, containing large amounts of an­ tioxidant glutathione, may account for the comparably lower insult to the liver tissue in vivo [9], Our experiments were done without reconstrution of the hepatic artery. Arterialization of the transplanted liver graft could increase oxygen delivery to liver tissue and thus enhance oxygen-free radical generation upon reperfusion. This possibility has been investigated by Ramos et al. [28], who found that lipid peroxidation of the postischemic organ did not at all differ between arterialized grafts and those grafts where only the portal vein was anastomosed. Hence we can pre­ sume that hepatic arterial inflow would have no additional effect on free radical produc­ tion.

In contrast to Mizuta et al. [ 10], who trans­ planted livers after unique cold ischemic stor­ age, we could not reproduce a significant ame­ lioration of animal survival by SOD adminis­ tration. Possibly the benefit of SOD in our study would have been more accentuated in larger groups or differences in the experimen­ tal protocol might explain divergent results. Marzi et al. [11] reported that SOD re­ duced microcirculatory disturbances and granulocyte adherence in the transplanted liv­ er. As in our study he used a CuZn-enzyme preparation of SOD, which is known to be unable to penetrate the cell membrane [29], Therefore we conclude that the effects of SOD on the postischemic liver outcome will be due to a protective action exerted at the vascular endothelial site and/or at the outer cell surface of hepaiocytes and Kupffer cells in the sinu­ soides of the liver.

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Evaluation of antioxidant treatment with superoxide dismutase in rat liver transplantation after warm ischemia.

In order to investigate the effects of the exogenously administered radical scavenger superoxide dismutase (SOD) on the orthotopic liver graft, livers...
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