A better way to do small-for-size liver transplantation in rats

Abstract

Establishing a model for small-for-size liver transplantation is the basis for this study of partial and living donor graft liver transplantation. This study aims to explore a simpler and more effective way of establishing a 30% small-for-size liver transplantation in rats. Sprague-Dawley rats were selected as the donors and recipients. Small-for-size orthotopic liver transplantation was performed using Kamada’s two-cuff method. The donor’s liver was flushed via the abdominal aorta and hepatectomy was performed in situ. The animals were divided into three groups depending on the graft selected, with 40 pairs of rats in each group. In group I, the median lobe of the liver was used as graft; in group II, the right half of the median lobe and the right lobe were used as graft; and in group III, the median and right lobes were used as graft. In groups I and II, the bodyweights of donors were the same as those of recipients; however, in group III the bodyweights of donors were 100–120 g less than those of the recipients. The duration needed for transplantation, the 7-day survival rates, and the technical complication rates were compared among these three groups. The time required for hepatectomy was shorter in group III compared with groups I and II (8.8€±€0.7 min vs. 11.5€±€1.1 min and 10.1€±€1.0 min, P = 0.001). The cold ischemia time for the grafts, the anhepatic times, and the transplantation times for the recipients were not significantly different among the three groups. Compared with groups I and II, the incidence of bleeding, bile leakage, and inferior vena caval strictures were significantly decreased in group III (P<0.05). No significant differences between the three groups were found based on other complications after the operation (P>0.05). Group III had better 7-day survival rates and longer median survival times but the differences were not statistically significant. The method of small for donor bodyweight using the median and right lobes for grafting may be a more effective and simpler way of establishing a 30% small-for-size liver transplantation in rats, as shown by the shorter hepatectomy time and the occurrence of fewer complications after the operation.

Keywords

liver transplantation / small-for-size / rats

Introduction

Improvements in surgical and anesthetic techniques, and the introduction of new immunosuppressive regimens have increased patient survival rate after liver transplantation, resulting in a 5-year survival rate of more than 90% in experienced transplant centers [1-3]. However the donor liver shortage has been the rate-limiting step in the expansion of hepatic transplantation. The techniques of partial liver transplantation using a living donor graft expand the supply of organs and partially overcome the graft shortage. But these benefits are limited when the volume of the grafts was small [4]. The adequate size of the graft is the major concern of adult-to-adult living donor liver transplantation. Harvesting a larger graft leads to a higher risk for the living donor. A small-for-size graft might not only be functionally inadequate for the recipient, but also sustain injury characterized by cholestasis and histological features of ischemia after implantation. So the appropriate model is the basis for the study mentioned above. The aim of the present study is to establish a model of small-for-size liver transplantation in a simple and effective way. Three methods for achieving 30% small-for-size orthotopic liver transplantation (SOLT) were compared, resulting in a simple and effective way for achieving a repeatable 30% SOLT in rats. The methods are described in detail in this study.

Materials and Methods

Animals

Inbred Sprague-Dawley rats (210-380 g), purchased from the Model Animal Research Center of Kunming Medical College, were housed with free access to water and food with a constant light-dark cycle. The donors were fasted for 12 h before the operation, whereas the recipients were not. Operations were performed under aseptic conditions. The studies met the guidelines for the Care of Laboratory Animals (The Ministry of Science and Technology of China, 2006).

Experimental procedure

The experiment was conducted on three groups of rats with 40 pairs in each group. In group I, the median lobe of the liver was used as grafts; in group II, the right half of the median lobe and the right lobe were used as grafts; in group III, the median and the right lobes were used as grafts. In groups I and II, the bodyweights of donors were similar to those of recipients. In group III, the bodyweights of donors were 100-120 g less than those of the recipients. The bodyweights and graft sizes among the three groups are shown in Table 1. All operations were performed under ether anesthesia. The donor livers were perfused via the abdominal aorta with 4°C lactated Ringer’s solution containing heparin sodium (50 U/mL). Thereafter, liver sizes were reduced in situ using the lobe ligation technique [5]. The harvested grafts were preserved in a bath of lactated Ringer’s solution at 4°C. The sleeve fixture of the portal vein, the infrahepatic inferior vena cava (IVC), and the bile duct, as well as the methods used for the operation on the recipients and liver transplantation are similar to that of whole orthotopic liver transplantation. The suprahepatic IVC was reconstructed using continuous 7-0 polypropylene sutures. Furthermore, the portal vein and infrahepatic IVC were anastomosed using the two-cuff method modified from that described previously [6-9]. The graft was reperfused before anastomosing with the infrahepatic IVC. The bile duct was connected by telescoping a tube in the bile duct of the donor into that of the recipient. After operation, the rats were allowed to recover spontaneously with no further treatment given. Up to 6 rats were sacrificed at 1 and 7 days after operation to estimate serum alanine transferase (ALT), total bilirubin (TB), and blood ammonia (AMON), as well as for histological examination of the livers under light microscope. The duration of the operation, the 7-day survival rate, and the technical complication rates were compared between the three groups.

Statistical analysis

Data analysis and statistical procedures were performed using SPSS software version 16.0 (SPSS, Inc). The measurements are expressed as mean±SD, which were compared using analysis of variance (ANOVA). The enumeration data were compared using chi-square test. Survival rates were assessed using the Kaplan–Meier method. A value of P<0.05 was considered significant.

Results

The duration of the hepatectomy was 8.8±0.7 min in group III, which was significantly shorter than 11.5±1.1 min in group I (P = 0.009) and 10.1±1.0 min in group II (P = 0.001). The cold ischemia time for the graft was 61.5±6.7 min in group I, 62.3±6.5 min in group II, and 57.2±5.3 min in group III. The anhepatic time was 11.3±2.2 min in group I, 10.8±2.3 min in group II, and 11.2±2.1 min in group III. The operating time for the recipients was 40.2±3.5 min in group I, 41.5±2.4 min in group II, and 39.5±4.0 min in group III. The above parameters were not significantly different among the three groups (P>0.05). After the operation, 7 cases of bleeding from the cut surfaces of the liver occurred in group I, 5 cases in group II, but none in group III. Furthermore, 5 cases of bile leakage were observed in group I, 6 cases in group II, but none in group III. There were 7 cases of IVC stricture and 4 cases of vascular thrombosis in group I, but none in the other two groups. The above parameters were significantly different among the three groups (P<0.05). There were 2, 1, and 2 cases of graft malreperfusion among groups I, II, and III, respectively. There were 3, 3, and 5 cases of suprahepatic inferior vena caval bleeding among groups I, II, and III, respectively. The above parameters were not significantly different among the three groups (P>0.05). Serum ALT, TB, and AMON levels increased on day 1 post-operation and declined subsequently over the following days in all three groups. There were no significant differences in the serum levels of ALT, TB, and AMON between the three groups at 1 day and 7 days post-operation (Table 2). At 7 days post-operation, the histopathological examination of the liver using hematoxylin and eosin staining under light microscope revealed small amounts of lymphocytic infiltrates around the portal area, normal hepatic lobular architecture, and mild acute rejection in all three groups (Fig. 1). Group III had more survivors at 7 days post-operation and longer median survival time than the other two groups, but the differences were not statistically significant (Fig. 2).

Discussion

In situations where the bodyweight of the donor and the recipient are similar, majority of current studies choose to use the median lobe (as with group I) or the right half of the median lobe with the right lobe (as with group II) as grafts for the 30% small-for-size liver transplantation in rats [4,10, 11]. This means that the left lobe, caudal lobe, disciform lobe, and the right lobe or the left half of the median lobe will have to be excised from the six lobes of the rat liver (Fig. 3A). Among these lobes, the left, caudal, and disciform lobes can be more easily removed from its root. However, the cut surfaces of the right lobe and the left half of the median lobe would leave a broader cross section, increasing the risk of bleeding and leakage of bile after reperfusion of the graft. Moreover, only two thirds of the right lobe of the liver could be cut off and the suprahepatic IVC stricture can easily occur when it is tied close to its root. In our experiment, seven cases of IVC stricture and 4 cases of vascular thrombosis occurred in the group that used the median lobe of the liver as grafts, but they did not occur among the other two groups. To avoid the complication caused by the resection of liver lobes, and obtain a small-for-size graft, Xu et al. [12] used rats whose bodyweights were much larger than those of the donors, as the recipients, so that the weight of the whole donor liver was much less than that of the recipient liver in the liver transplantation of rats. However, to establish such a 30% difference in livers between the donor and the recipient in small-for-size liver transplantation of rats, the bodyweight difference between the donor and the recipient must be above three times. Such a significant difference of bodyweight between the donor and the recipient would result in a larger difference in vessel diameter, which will affect surgical operation when the vein is being anatomosed, and the bile duct of a rat weighing less than 200 g is too thin to indwell the stent. In addition, the rats weighing too much were elder or even in the aging period, leading to poor tolerance for surgery. The success rate of liver transplantation was low in their report. But in our study, we explored the possibility of using a method that does not involve the division of the right and median lobes of the rat liver. The liver makes up 3% of the bodyweight of rats, whereas the median lobe and the right lobe of the liver make up 50% of the liver weight. With that in mind, the study design limited the donor bodyweight to 100-120 g less than that of the recipients, thereby achieving the desired aim of a 30% small-for-size liver transplantation for utilizing the median and right lobes of the liver as grafts (Fig. 3B). Of the 40 pairs constituting group III, there was no apparent difference in blood vessel size nor difficulty with the anastomosis of the IVC and the veins even though the bodyweights of the donors and recipients differed by 100-120 g (Figs. 3C-3D). Our findings differ from those of a previous report by Xu et al. [12]. In conclusion, the use of the median and right lobes of the liver in small-for-size liver transplantation reveals a trend toward the shorter duration of hepatectomy, less bleeding and bile leakage from the cut surfaces of the liver, less stricture formation in the IVC after graft reperfusion, and longer median survival time.

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