**2. Materials and methods**

#### **2.1 Materials**

Healthy rhesus monkeys provided by the Laboratory Animal Center of Kunming Medical University were used as donor and recipient. Recipients were male rhesus monkeys weighing 7.2–11.5 kg, and donors were of either gender weighing 5.3–8.1 kg. The animals were housed in the Laboratory Animal Center of Kunming Medical University, and were allowed free access to food and water. Food access for donors was restricted, and recipients were starved of food for 12 hours and of water for 6 hours preoperatively. Recipients were given cefazolin sodium (0.1 g/kg) before transplantation. Experimental procedures were performed in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals formulated by the Ministry of Science and Technology of the People's Republic of China.

#### **2.2 Surgery for donor and recipient animals**

We operated on 9 rhesus monkeys using our original surgical model, and then modified our model for the remaining 16 monkeys. In the original model, the hepatic vein, portal vein and hepatic artery were directly anastomosed, and a supporting tube was placed in the biliary tract. The modified model is described below.

Causes of Death of Rhesus Monkeys Undergoing Liver Transplantation 539

We successfully performed liver transplantation in 25 pairs of rhesus monkeys. In the early postoperative period (within 6 hours after portal vein opening), seven animals (25%) died; five (20%) due to abdominal hemorrhage, one (4%) due to primary nonfunction and 1 (4%) due to pneumothorax-induced respiratory failure. In the short-term postoperative period (12–72 hours after portal vein opening), seven animals (28%) died; one due to hyperacute rejection within 12 hours, one due to hyperacute rejection and arterial thrombosis within 12 hours, one due to pulmonary infection and one due to accidental death at 72 hours. In the long-term postoperative period (> 72 hours after portal vein opening), eleven animals (44%) died; six due to acute rejection, three due to arterial thrombosis and two due to pulmonary infection. Abdominal hemorrhage occurred mainly in the early and short-term postoperative periods, and acute rejection occurred mainly in the long-term postoperative

Liver transplantation in the rhesus monkey frequently uses the classical model because the inferior vena cava is embedded in the parenchyma of the posterior segment of the liver, making the anatomy unsuitable for piggyback liver transplantation. The rhesus monkey is fragile and often dies early following experimental transplantation. In this study, seven animals (66.7%) died within 6 hours after portal vein opening, seven died at 12–72 hours and eleven died at > 72 hours. The double-cuff method has been used extensively in established liver transplantation models. This model significantly shortens the duration of the anhepatic phase, decreases the incidence of portal vein bleeding and stenosis, and

**4.1 Abdominal hemorrhage in rhesus monkeys following liver transplantation** 

In this study, the main cause of death was abdominal hemorrhage in the early postoperative period (within 6 hours after portal vein opening). In small animals such as rats, abdominal hemorrhage is also the major cause of death after reduced-size liver transplantation. This bleeding is often from the inferior vena cava anastomosis above the liver but has also been observed from the ligation points, liver capsule, right adrenal vein, lumbar veins, portal vein and inferior vena cava below the liver. In this study, abdominal hemorrhage was most often from the anastomoses of the portal vein and the inferior vena cava below the liver, and was also observed from the anastomosis of the inferior vena cava above the liver, liver bed, liver capsule, right adrenal vein and lumbar veins, similar to the bleeding points observed in rats. Abdominal hemorrhage sometimes involved multiple sites in one animal. In this study, the animals did not tolerate bleeding well and showed signs of decreased peripheral circulation after a blood loss of 100 mL. Animals with abdominal hemorrhage commonly died within 6 hours after portal vein opening. Rhesus monkeys may also have a preoperative hypercoagulable state and a postoperative hypocoagulable state, greatly influencing the stability of this model. Hemostasis and fluid balance are therefore very important for successful liver transplantation in the rhesus monkey. As a variety of factors contribute to the development of abdominal hemorrhage, surgeons should be familiar with the surgical procedures used including microsurgical techniques. Our original model prior to

**3. Conclusion** 

period.

**4. Discussion** 

decreases the incidence of early death.

#### **2.3 Donor surgery and liver perfusion**

Donor animals were anesthetized by intravenous injection of 3% pentobarbital sodium dissolved in normal saline (0.5 mL/kg). Under sterile conditions, a large, crucial incision was made in the abdominal wall and the liver was harvested. One cannula was placed in the portal vein and another was placed in the inferior vena cava below the liver. The splenic and renal veins were ligated. The liver was perfused with HTK solution at 4 °C, and bleeding tissues were ligated. A 2 mm diameter supporting tube was placed in the bile duct.

#### **2.4 Recipient surgery**

Recipient animals were anesthetized by intravenous injection of 3% pentobarbital sodium dissolved in normal saline (0.5 mL/kg), followed by subcutaneous injection of atropine (0.03–0.04 mg/kg). Recipient surgery was undertaken while the donor liver was undergoing Histidine- Tryptophan- Ketoglutarate (HTK) perfusion. Briefly, a large, cross-shaped incision was made in the abdominal wall. The perihepatic ligaments and inferior vena cava above and below the liver were separated from adjacent structures. The hepatic artery, portal vein and biliary tract were separated at the porta hepatis. Tissues surrounding the inferior vena cava below the liver between the right renal and right adrenal veins were separated over 0.5–1.0 cm. The right suprarenal and lumbar veins were ligated adjacent to the inferior vena cava using a 4-0 suture. Blood was collected from the liver according to the autotransfusion method described for rat liver transplantation. The inferior vena cava below the liver and the portal vein were clamped, and 60–100 mL of sterile balanced salt solution was slowly injected into the portal vein until the liver became khaki in color. The inferior vena cava above the liver was then immediately clamped. The inferior vena cava above the liver was cut adjacent to the liver, and was trimmed into a bellmouth shape at the bifurcation of the portal vein. The inferior vena cava was cut below the liver with some liver tissue included. The donor liver was transplanted using standard orthotopic liver transplantation techniques (double-cuff and one support tube). The inferior vena cava above the liver was anastomosed using 5-0 prolene, the cuff of the portal vein was anastomosed and the portal vein was declamped. When blood was observed flowing from the inferior vena cava below the liver, the cuff was anastomosed. The inferior vena cava above and below the liver were declamped to terminate the anhepatic phase. The liver and gastrointestinal tract were perfused with 0.9% sodium chloride injection at 40–50 °C for rewarming until the color of the liver was restored. The common hepatic artery was anastomosed and a supporting tube was placed in the common bile duct. The abdominal cavity was washed with warm saline. If no hemorrhage or bile leakage was detected, the abdominal wall was closed.

#### **2.5 Postoperative observation and treatment**

Animal activities, facial expressions, food and water intake and reactions to stimulation were observed. Animals who died were immediately dissected to obtain samples and to analyze the cause of death. Each monkey was caged separately at 22–25 °C and was allowed access to water after 24 hours and food after 48 hours. Intramuscular cefazolin sodium (0.1 g/kg) was administered twice a day for 2 days. Colloid and sugar water (500–1 000 mL per day) was administered postoperatively to maintain electrolyte and acid-base balance.
