Development of a new auxiliary heterotopic partial liver transplantation technique using a liver cirrhosis model in minipigs: Preliminary report of eight transplants

Introduction

Auxiliary heterotopic partial liver transplantation (AHPLT), which is an important branch of liver transplantation, has been considered as the best choice for the treatment of acute liver failure and liver metabolic diseases (1–4). However, its clinical application is unsatisfactory for the treatment of chronic end-stage liver failure, for which further investigation is required to solve issues, including the lack of ideal operative technique, the liver function competition between the transplanted and host livers, and other post-operative complications (5–8). Established AHPLT in the past mainly used small or normal large animals, and was unable to simulate the characteristics of auxiliary liver transplantation of end-stage liver failure (9,10). Thereby motivated to resolve these issues, our study herein aimed to establish a new operative technique for AHPLT in minipigs using a model of liver cirrhosis.

Materials and methods

Experimental animals

Twenty-two Chinese experimental minipigs (either gender) were purchased from the laboratory animal farm of the Inner Mongolia Medical College. Fourteen minipigs (weighing 21–25 kg) were used for the establishment of the cirrhosis model, and 8 minipigs (weighting 19–23 kg) were used as donors for the improved AHPLT. The experimental protocol was approved by the Laboratory Animal Ethics Committee of the Inner Mongolia Medical College and conformed to the NIH Guidelines for the Care and Use of Laboratory Animals.

Establishment of the cirrhosis model in minipigs

Our previous study successfully produced liver cirrhosis in minipigs (11). Liver cirrhosis was induced by the intraperitoneal injection of carbon tetrachloride (CCl4) twice a week for 9 weeks. Maize flour was the only food and a 5% alcohol-water mixture was provided for the animals. A piece of liver tissue was obtained in the 9th week and was stained with H&E and Van Gieson’s (VG) stain. Meanwhile, portal vein pressure (PVP) and hepatic venous pressure (HVP) were measured by direct puncture with a 27G needle and pressure tubing attached to the normal central venous pressure monitoring transducer, and then the portal vein pressure gradient (PVPG) was directly calculated by PVP and HVP.

Establishment of the donor procedure model

Under general anaesthesia, the donor abdominal cavity was exposed via a midline incision. The donor liver was perfused with lactated Ringer’s solution at 4°C via an abdominal aorta and portal vein. Once the perfusion was finished, the whole donor liver was resected. The right lobe was resected along the line connecting the front side of the median fissure of the liver with the right edge of the suprahepatic vena cava. The collateral aorta, porta and cava vessels were ligated. The left lobe was used for the transplantation. The 2.5-cm abdominal aorta of the donor was reserved.

Recipient procedure and testing index

The venous channel was established through the internal jugular vein, and then general anesthesia and assisted respiration were carried out. After opening the abdomen, the condition of the spleen was observed and splenectomy was performed. The reserved abdominal aorta was anastomosed end-to-side with the suprahepatic vena cava of the recipient. Latex drainage tube matching the diameter of the abdominal aorta was adopted. One end of the common latex drainage tube was inserted into the anastomosed abdominal aorta and sutured. The donor liver was placed on the splenic bed, then the portal vein of the transplanted liver was anastomosed end-to-end with the splenic vein of the recipient, while the other end of the latex drainage tube was secured with the suprahepatic vena cava of the transplanted liver. Blood flow to the portal vein was thus reestablished. The common hepatic artery of the transplanted liver was end-to-end anastomosed with the splenic artery of the recipient. The donor’s common bile duct was intubated and bile was collected with an extracorporeal bag (Fig. 1). After the operation, fluid infusion was given for the symptomatic treatment, and anti-rejection drugs were not administered. Throughout the transplantation process, vital signs [including the heart rate (HR), mean arterial pressure (MAP) and central venous pressure (CVP)] of the recipient pigs were measured by a central venous catheter in the right internal jugular vein and a catheter in the right internal carotid artery that was connected to a cardiac output monitor (NPB4000, USA). The PVP, HVP and PVPG of the recipients were measured at different time intervals. Additionally, the PVP, HVP and PVPG of the donors were also measured after the operation. On the 3rd day after the operation, the blood flow of every anastomosis was examined with Doppler vascular ultrasound. After being anesthetized, 2 cases were randomly selected to perform laparotomy.

Figure 1. Diagram of the new auxiliary heterotopic partial liver transplantation. (A) The recipient spleen before transplantation (single arrow); (B) the transplanted liver after transplantation (double arrow).

Statistical analysis

SPSS13.0 software was used for data analysis. Results are expressed as the means ± SEM, unless otherwise noted. All variables were analyzed by two-way ANOVA. P≤0.05 denoted statistical significance.

Results

Fourteen minipigs were utilized to establish the cirrhosis model through administration of CCl4 by intraperitoneal injection. After administration for 9 weeks, cirrhosis developed in 8 pigs, and 4 cases of ascites were found. An increasing spleen volume was discerned by visual inspection. Before the establishment of the cirrhosis model, the PVP, HVP and PVPG of the experimental animals were 13.66±1.15, 6.73±1.00 and 6.92±1.42 cmH2O, respectively. After 9 weeks, the PVP, HVP and PVPG of the cirrhotic pigs were 24.52±2.84, 6.81±1.05 and 17.70±2.71 cmH2O, respectively. The normal liver and the cirrhotic liver stained by H&E and VG are shown in Fig. 2.

Figure 2. Normal liver and cirrhotic liver stained with H&E and VG. (A) Normal liver (H&E stain). (B) Normal liver (VG stain). (C) Formation of liver cirrhosis (H&E stain). (D) Formation of liver cirrhosis (VG stain). Magnification, x40.

Eight cases were used to establish the novel AHPLT model, 7 of which survived on the 3rd day after the operation, whereas 1 case died of bleeding of the transplanted liver section and shock at 5 h after the operation; the surgical success rate was thus 87.5% (7/8). Changes in the vital signs of the surviving 7 recipients at different time periods during the operation are shown in Table I, and the PVP, HVP and PVPG of the recipients are shown in Table II. After the blood flow to the transplanted liver was regained for 15 min, the PVP, HVP and PVPG of the donor were 21.59±1.26, 6.08±0.66 and 15.51±1.35 cmH2O, respectively. After the blood flow of the portal vein was regained for 0.5–3 h, all experimental pigs had bile excretion. On the 3rd day after the operation, 1 case of abnormal blood flow between the suprahepatic vena cava of the transplanted liver and the latex drainage tube was discovered employing Doppler vascular ultrasound examination. Moreover, the laparotomy showed a distortion at the anastomosis, but no obvious abnormality in the blood flow of each anastomosis of the other animals was observed. Two pigs were vivisected, in which a small amount of thrombosis was found at the anastomosis between the vena cava of the transplanted liver and the latex drainage tube.

Table I. Changes in the vital signs of the recipients during liver transplantation (χ̄ ± s, n=8).

Table I.

Changes in the vital signs of the recipients during liver transplantation (χ̄ ± s, n=8).

Time	HR (/min)	MAP (mmHg)	CVP (cmH2O)
A	130.75±6.11	120.88±4.91	5.33±0.37
B	127.87±4.29	119.13±6.10	5.31±0.43
C	136.87±5.89a	120.50±4.66b	5.20±0.40c

{ label (or @symbol) needed for fn[@id='tfn1-etm-03-05-0865'] } A, 5 min after opening of the abdomen of the recipient pigs. B, 10 min after the splenectomy. C, 15 min after regaining blood flow to the transplanted liver.

a Compared to the HR of Group A and Group B, P<0.05.

b Compared to the MAP of Group A and Group B, P>0.05.

c Compared to the CVP of Group A and Group B, P>0.05. HR, heart rate; MAP, mean arterial pressure; CVP, central venous pressure.

Table II. Changes in PVP, HVP and PVPG of the recipients (χ̄ ± s; n=8).

Table II.

Changes in PVP, HVP and PVPG of the recipients (χ̄ ± s; n=8).

Time	PVP (cmH2O)	HVP (cmH2O)	PVPG (cmH2O)
A	24.01±2.57	6.41±0.97	17.60±2.52
B	23.10±1.78	6.18±0.93	16.93±1.68
C	21.59±1.26a	6.08±0.66b	15.51±1.35c

{ label (or @symbol) needed for fn[@id='tfn5-etm-03-05-0865'] } A, 5 min after opening of the abdomen of the recipient pigs. B, 10 min after the splenectomy. C, 15 min after regaining blood flow to the transplanted liver.

a Compared to the PVP of Group A, P<0.05, and compared to the PVP of Group B, P>0.05.

b Compared to the HVP of Group A and Group B, P>0.05.

c Compared to the PVPG of Group A, P <0.05, and compared to the PVPG of Group B, P>0.05. PVP, portal vein pressure; HVP, hepatic venous pressure; PVPG, portal vein pressure gradient.

Discussion

Since Welch introduced the first case of auxiliary whole liver transplantation to dogs in 1955 (12), many improvements have been made in the urgical techniques. However, no satisfactory auxiliary heterotopic liver transplantation method has been reported hitherto. In the previous study concerning the auxiliary heterotopic liver transplantation, donor livers were mostly transplanted into the abdominal cavity, which led to an increase in abdominal pressure and circulatory disturbance. Recently, a liver was transplanted to the splenic bed of a normal animal to solve the shortage of space in the animal model (13). However, the method suffered from the limited availability of splenic vessels in the normal condition. In the present study, the auxiliary heterotopic liver transplantation model was established using a model of liver cirrhosis; the spleen of the recipient increased to further expand the transplantation space. Theoretically, cirrhosis and portal hypertension leads to hemangiectasis of the spleen, which favors the anastomosis of the blood vessels of the recipient spleen in the donor. However, no changes in the blood vessel diameter of the spleen were observed prior to and after establishment of the liver cirrhosis model, which is in need of further investigation in the future.

Moreover, in auxiliary heterotopic liver transplantation, the function competition between the recipient liver and the transplanted liver is a long-term complication, which is the most common problem requiring an urgent solution. Nevertheless, the underlying mechanism has not been completely clarified (6,10,14). Portal vein blood flow of the transplanted liver is inadequate, which is considered as an important manifestation that results from the function competition. Once the portal vein blood flowing to the transplanted liver was restored, the function competition was found to vanish (15). Portal vein blood flow is driven by the PVPG, i.e., by the pressure difference between the portal vein and the hepatic vein. Previous studies were carried out mostly using rat models of transplantation without arterial blood supply or the established normal large animal model of AHPLT, which differs greatly from the pathophysiological characteristics of clinical auxiliary heterotopic liver transplantation. The results of the present study (Table II) showed that the established minipig model of AHPLT on the basis of liver cirrhosis better simulated the characteristics of liver transplantation for human end-stage liver failure, which is therefore suitable for further exploration of the function competition mechanism. Due to the pressure gradient in the vena system, lower venous pressure occurs at the position closer to the heart. In our study, the end-to-side anastomosis of the donor vena cava and the recipient suprahepatic vena cava replaced the previous method of the donor vena cava and the recipient renal vein or the infrahepatic inferior vena cava above its level, which aimed to reduce the outflow tract pressure of the donor hepatic vein. The interaction of higher PVP and lower HVP results in a further increase in PVPG of the transplanted liver. However, in this study, 1 case of suprahepatic vena cava anastomotic distortion of the transplanted liver and the drainage tube was noted on the 3rd day after the operation. This may have resulted from the long outflow tract and the abnormal location of the liver transplantation, which is a shortcoming of this operation. Two pigs were vivisected, revealing a small amount of thrombosis at the anastomosis between the vena cava of the transplanted liver and the latex drainage tube, which may be avoided by adopting a man-made blood vessel instead of the common latex drainage tube in the living donor liver transplantation or donor conduit (donor aorta, donor vena cava and iliacs) in the cadaver liver transplantation.

Early studies have proven that AHPLT outweighs other techniques due to advantages, such as slight injury of the operation, short time of the operation, no anhepatic phase and stable hemodynamics (16,17). The results of this study also supported the above findings; the surgery success rate in the group was 87.5%, and changes in the vital signs of the recipients at different time periods were more stable.

In this study, although the observation time was short (only 3 days), a novel AHPLT technique was successfully established using a model of liver cirrhosis. In a future study, we will assess the comparison of the difference between a suprahepatic and infrahepatic venae cavae drainage model. In addition, post-transplant aspects will also be observed, including liver function of the transplanted liver compared to that of the cirrhotic liver on the long term, evolution of the blood flow and pressure over time, and the histologic analysis of both the transplanted and cirrhotic livers over time.

Abbreviations:

Acknowledgements

The authors are grateful to Jian-liang Qiao and Rui-Fang Zhang for the technical assistance. This study was supported by a research fund of the Inner Mongolia Medical College (no. Y2003ZD001), and the Natural Science Foundation of Inner Mongolia of China (no. 2010MS1126).

References