A Comparison between splenic fossa and subhepatic fossa auxiliary partial heterotopic liver transplantation in a porcine model

Animals

All animal experiments were performed according to the guidelines set by the US National Institutes of Health (1985). The Animal Care and Use Committee of Zhejiang Chinese Medical University approved this experimental protocol. Eighteen female Guangxi Bama miniature pigs weighing between 16 and 22 kg were used in the subhepatic fossa APHLT model (group A). Twenty female Guangxi Bama miniature pigs weighing between 14 and 22 kg were used in the splenic fossa APHLT model (group B). An additional animal was used as a whole blood donor for each donor and recipient pair. Animals were followed for 2 weeks, at which time they were killed.

Animals were fed with a standard diet and fasted for 24 hours before surgery. Ketamine hydrochloride (20 mg/kg) and atropine sulfate (0.1 mg/kg) were administered intramuscularly, followed by endotracheal intubation and administration of an anesthetic gas composed of isoflurane (1%-1.5%), oxygen (2 L/minute), and room air (2 L/minute) under mechanical ventilation. Anesthesia was supplemented by the addition of pentobarbital sodium (30 mg/kg). Catheters were introduced through an ear lateral marginal vein for intravenous infusions, drug administration, and blood sampling. Ringer's solution was given for hemodynamic support as needed. Depending on the amount of blood loss, 1-3 IU of whole blood (200 mL each) was given in the operation.

Donor Operation

After laparotomy, the donor liver was skeletonized from all surrounding ligaments. After hilar dissection at the base of the hepatoduodenal ligament, the left hepatic artery (LHA) and the left PV branch supplying the left lateral and left medial lobes were identified and isolated. The left bile duct branch was transected, and a polyethylene tube was inserted for external bile drainage (Fig. 1A). The LHA and the left PV were temporarily occluded with vascular clamps in order to reveal the demarcation line between the left and right hemiliver. The demarcation line is an improved Taira line,12 deviated to the left of the middle HV. Parenchymal transaction was initiated with Peng's multifunctional operative dissector and a harmonic scalpel (Generator 300, Ethicon Endo-Surgery LLC, Johnson-Johnson Co., New Brunswick, NJ) by scoring the liver surface just to the left of the gallbladder fossa at the demarcation line (Fig. 1B and Fig3A). The small penetrating vessels and biliary radicles were sutured and ligated as required. The left HV was encircled at the junction to the inferior vena cava (IVC). During these procedures the vessels remained unclamped. After an intravenous injection of 2 mg/kg of heparin sodium, the left hemihepatic graft without the middle HV was removed and preserved in cold hypertonic citrate adenine solution (HCA), which is an improved Ross solution. Intravenous injection of 1 mg/kg of protamine sulfate was used to reverse the previous heparinization. After thorough hemostasis, abdominal closure was performed.

Back-table Operation

The harvested liver was immersed in a sterile bag containing 4°C HCA solution. At the back table, 1 L of 4°C HCA solution was flushed via the left PV. The biliary system was washed with 4°C HCA solution. The liver graft comprised approximately 40% of the whole liver weight.

In group A, an 8-mm, internal diameter, thin-walled polytetrafluoroethylene (PTFE) graft (GORE-TEX, W.L. Gore & Associates, Inc., Newark, DE) approximately 2.5 cm long was connected to the left HV. Then, another 8-mm diameter PTFE graft approximately 2.5 cm long was connected to the left PV (Fig. 1C). In group B, an 8-mm diameter PTFE graft approximately 2.5 cm long was connected to the left HV (Fig. 1D).

Recipient Operation

Two consecutive series of transplantations were performed. In group A (n = 9), animals received an auxiliary partial liver transplant in the subhepatic fossa. All animals in group B (n = 10) received a splenic fossa heterotopically placed partial liver graft.

Postoperative Care

Intravenous therapy consisted of 1500 mL of 5% dextrose and saline containing 4 million units of penicillin G and 100 mg of ranitidine daily until postoperative day (POD) 3. No immunosuppressant was administered. The animals were fed with a standard diet from POD 1. The recipients were given 500 mL of dextran-40 during the operation and daily until POD 3. Then warfarin (1.25 mg) was mixed with oral feeding daily thereafter.

Animal Survival

Surviving pigs were killed on POD 15, and all pigs that died before POD 15 were further studied for the cause of death. At death or autopsy, both the NL and the graft were carefully dissected, and all vessels and anastomoses were examined for patency.

Biochemistry

Blood samples were taken before the operation and at 1, 3, 7, and 14 days after operation. Serum aspartate aminotransferase (AST), total bilirubin (TBil), blood urea nitrogen (BUN), and creatinine (Cr) levels were determined with an autoanalyzer (type 7020, Hitachi Co., Ltd., Tokyo, Japan).

Histological Examination

All animals were autopsied, and hepatic specimens for light microscopy were fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin-eosin (H & E) for histological examination.

Statistical Analysis

All values were expressed as the mean ± standard deviation (SD). Statistical analysis of the values in both groups was made by 1-way analysis of variance and Student t test. Survival curves were constructed with the Kaplan-Meier method. The log-rank test for trends was used when survival curves were compared across groups. P value < 0.05 was considered statistically significant. All statistical procedures were performed on a commercially available computer program (SPSS for Windows 23.0, SPSS Inc., Chicago, IL).

Survival and Clinical Course

Table 1 depicts the average donor, recipient, and transplant characteristics. The miniature pigs were usually awake in 3 or 4 hours following the operation and began food intake the next morning. Kaplan-Meier survival curves for group A and group B can be seen in Fig.4.

In group A, 7 of the 9 donors survived more than 14 days. The remaining 2 donors died from intraperitoneal hemorrhage within 24-48 hours postoperatively. Four of the 9 recipients survived less than 14 days. Table 2 depicts the survival times and causes of death in the group A recipients. In 5 recipients that survived for 2 weeks, neither the size of the liver graft nor the size of the host liver had changed at autopsy. The liver grafts of 2 recipients were heavily mottled with patches of necrosis and abscess formation. Hepatic artery thrombosis was present in those 2 animals. The remaining 3 liver grafts appeared to be normal macroscopically; all vascular channels were found to be patent and free of thrombosis at autopsy.

In group B, 7 of the 10 donors survived more than 2 weeks. One donor died on POD 5 of an abdominal infection. The remaining 2 donors died from intraperitoneal hemorrhage on PODs 1 and 2. Table 2 depicts the survival times and causes of death in the group B recipients. In 6 recipients that survived for 2 weeks, neither the size of the liver graft nor the size of the host liver had changed at autopsy. The liver grafts of 3 recipients were heavily mottled with patches of necrosis and abscess formation. Thrombosis of vascular anastomoses occurred in those 3 animals, thrice in the hepatic artery (HA) and once in the PV. The remaining 3 liver grafts appeared to be normal macroscopically; all vascular channels were found to be patent and free of thrombosis at autopsy.

Patency Rate (PR) of the PTFE Graft

Overall, the 2-week PR of the PTFE graft was 88% (14/16), but the 2-week mural thrombus formation rate of the PTFE graft was as high as 44% (7/16). In group A, 2 liver grafts were dysfunctional. Anastomotic and luminal stenoses were found in 2 PV PTFE grafts and 1 HV PTFE graft; more than half of each PTFE graft lumen was filled with thrombus. Another HV PTFE graft was thrombotic. In group B, 3 liver grafts were dysfunctional. Anastomotic and luminal stenoses were found in 2 HV PTFE grafts, and more than half of each PTFE graft lumen was filled with thrombus. Another HV PTFE graft was thrombotic.

Biochemistry

Five animals in group A and 6 animals in group B survived longer than 14 days after transplantation; the data of these animals were used for analysis. The preoperative and serial postoperative measurements of serum TBil, serum AST, BUN, and Cr are shown in Table 3.

Quantification of hepatocellular injury using the serum level of TBil and AST did not demonstrate any differences between group A and group B at any time point. The TBil serum level peaked at POD 3, gradually declined afterward, and nearly normalized by POD 14. Compared with the preoperative TBil (1.3 ± 0.6 μmol/L), TBil on POD 1 (4.0 ± 2.8 μmol/L), TBil on POD 3 (4.6 ± 4.0 µmol/L), TBil on POD 7 (3.3 ± 2.0 μmol/L), and TBil on POD 14 (1.7 ± 0.6 μmol/L) did not demonstrate any statistically significant differences in group A. However, in group B, TBil on POD 1 (3.6 ± 1.6 µmol/L) and TBil on POD 3 (3.6 ± 2.1 µmol/L) were significantly higher, which compared to preoperative TBil (1.4 ± 0.6 µmol/L). The AST serum level sharply peaked at POD 1, gradually declined afterward, and nearly normalized by POD 7. Compared with preoperative serum AST, AST on POD 1 and AST on POD 14 were significantly higher in both groups.

Serum level of BUN and Cr peaked at POD 1, gradually declined afterward, and nearly normalized by POD 7. Compared with preoperative BUN, BUN on POD 1 and BUN on POD 3 were significantly higher in both groups. Cr serum level evolution exhibited a similar pattern. Still, at each time point, there was no statistically significant difference between the 2 groups in terms of BUN and Cr serum level.

Pathology

The autopsy demonstrated 10 normal NLs, with mild sinusoidal congestion and slight edema of the liver cells (Fig. 5A,C,G). Only 1 shows a dilated hepatic central vein with the normal liver architecture (Fig. 5E).

The autopsy demonstrated that the 5 deceased liver grafts had thrombosis of the vascular anastomoses, and all patients showed necrotic coagulation of the liver cells (Fig. 5B). The autopsy demonstrated that the 6 vital liver grafts had patency of all vascular anastomoses and that all but 1 patient in group B showed severe hemorrhage necrosis of the liver cells (Fig. 5D). None showed evidence of changes due to rejection. Only slight amounts of lymphocyte, plasma cell, and other mononuclear cell infiltration were present in the portal tracts and interlobular septa (Fig. 5H).