Experimental animals
54 healthy adult BAMA pigs (age: 10–12 months, weight: 30–40 kg; Shenyang Agricultural University, Shenyang, Liaoning, China) of either gender were enrolled for model establishment. The animal experiments were approved by the Animal Care and Use Committee of the First Affiliated Hospital of China Medical University (No. 20190512) and were performed in accordance with the guidelines of the Provincial Laboratory Animal Science Association of Liaoning Province.
Experimental design
The pigs were numbered 1–54, weighed, and renumbered based on their body weight. The pigs were divided into 5 groups: in (i) the sham group (n = 6), the were anesthetized, the abdomen was incised and perihepatic ligament mobilized, followed by extubation for 2 h after abdominal closure, (ii) the WIT 30 min + CS 4 h DCD group (n = 12), in which the pigs were subjected to warm ischemia for 30 min and the excised livers cold preserved for 4 h, followed by liver transplantation, (iii) the WIT 30 min + CS 4 h + SP600125 DCD group (n = 12), which were subjected to warm ischemia for 30 min and cold preserved for 4 h, with addition of 20 µM SP600125 into the preservation solution, followed by liver transplantation), (iv) the WIT 30 min + CS 2 h + HMP 2 h DCD group (n = 12), which were subjected to warm ischemia for 30 min and cold preserved for 2 h, followed by HMP for 2 h and liver transplantation, and (v) the WIT 30 min + CS 2 h + HMP 2 h + SP600125 group (n = 12), in which the DCD modeled pigs were subjected to warm ischemia for 30 min and cold preserved for 2 h, after which 20 µM SP600125 was added to the preservation/perfusion solution, followed by 2 h HMP and liver transplantation. No immunosuppressants were used in the liver transplantation procedure. In the sham group, the pigs were numbered as 1, 2, 3, 4, 5, and 6, and in the WIT 30 min + CS 4 h DCD group, the pigs were numbered as 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, etc. We randomly specified a point in a random number table to record pig numbers in horizontal order. The random number of each block was divided by 5, leaving a remainder of 0, 1, 2, 3, or 4. The pigs with remainder 0 were assigned to the sham group; the remainder 1 was assigned to the WIT 30 min + CS 4 h DCD group, the remainder 2 was assigned to the WIT 30 min + CS 4 h + SP600125 DCD group, the remainder 3 was assigned to the WIT 30 min + CS 2 h + HMP 2 h DCD group, and the remainder 4 was assigned to the WIT 30 min + CS 2 h + HMP 2 h + SP600125 group. If the remainders of two consecutive numbers were the same, the allocation of the latter number would be one group less. When a group was completed, the remaining pig was assigned to the last group.
The DCD model was established as previously described [
9,
20]. All pigs were sent to the laboratory 1 week before the experiment for acclimation, thus minimizing confounds due to physiological stress reactions. The pigs were subjected to 12-h fast and 6-h water deprivation prior to operation. They were the anesthetized through the pump-controlled intravenous injection of propofol and cisatracurium, with the simultaneous inhalation of isoflurane, along with mechanical ventilation through an endotracheal tube. The right internal jugular vein and the right common carotid artery were exposed through a transverse incision on the neck, and a central venous catheter and arterial catheter were inserted, to be used for blood collection, drug administration, and monitoring. After this surgical procedure, the ventilator was removed and warm ischemia was initiated when mean arterial pressure (MAP) was < 60 mmHg. The presence of cardiac arrest or MAP < 25 mmHg and pulse pressure < 20 mmHg indicated cardiac death. The liver was collected from each subject when warm ischemia had lasted for the designated period.
Collection and preparation of donor livers
Abdominal organs were collected using the rapid en bloc technique for liver and pancreas procurement [
21]. Following entry to the abdomen, the liver quality was detected by visual inspection. Once liver function had been confirmed to be sufficient for liver transplantation, the abdominal aorta was isolated and superior mesenteric vein was ligated. Then, 30 min after onset of warm ischemia, LPS-1 solution (based on the formula for KPS-1, 1000 mL, 4 °C) was perfused through the abdominal aorta and superior mesenteric vein via gravity perfusion at a height of 0.8 m. The composition of KPS-1 (Organ Recovery Systems Inc., Itasca, IL, USA) and the additional agents added to produce LPS-1 are provided in Table
1. Ice chips were placed around the liver to decrease it temperature quickly. The gall bladder was cut open to remove the bile. The gastrointestinal tract and corresponding mesenterium were freed. The biliary tract was washed with the use of 50 mL LPS-1, which was poured via the lower segment of the bile duct. Subsequently, the liver, kidneys, pancreas, and spleen were harvested and stored in a sterile container containing LPS-1 at 4 °C. The suprahepatic inferior vena cava, infrahepatic vena cava, portal vein, hepatic artery, and common bile duct were adjusted to the appropriate lengths for machine perfusion attachment. Next, kidneys, pancreas, spleen, and excess muscle, along with adipose tissues were removed. For the HMP treatment, donor livers were placed in the portal vein and hepatic artery casing prior to beginning the perfusion.
Table 1
Composition of KPS-1 and agents added to produce LPS-1
KPS-1 | 1 L |
6N HCl (as a pH preadjustment additive) | 1.6 mL |
α-Ketoglutaric acid | 2 mg |
N-Acetylcysteine | 2 mg |
l-Arginine | 2 g |
Nitroglycerin | 50 mg |
Alprostadil | 2 vials (concentration, 500 μg/mL) |
Recipient liver transplantation model
Classic orthotopic liver transplantation without venovenous bypass was performed on pigs in the experimental groups [
22]. After Mercedes incision laparotomy, the liver was freed from its attachments. The hepatoduodenal ligament was freed and resected to isolate the common bile duct, hepatic artery, and portal vein. The common bile duct and hepatic artery were ligated and cut near the liver. Subsequently, the suprahepatic inferior vena cava and infrahepatic vena cava were isolated, followed by casing. Three phrenic veins were sutured and ligatured close to suprahepatic inferior vena cava. The infrahepatic vena cava and portal vein were experimentally pre-blocked three times under ordinary circumstances through colloid supply and norepinephrine application to increase blood pressure as required. When blood pressure and pulse rate were stable, the portal vein, suprahepatic inferior vena cava, and infrahepatic vena cava were blocked, followed by the removal of recipient livers, whereupon the anhepatic phase began. At this point, methylprednisolone (500 mg) was intravenously injected to alleviate inflammatory reaction in the recipient pigs, whereupon the colloid was quickly supplied and the amount of norepinephrine was adjusted according to present blood pressure. The restoration of blood flow to the liver following the anastomosis of suprahepatic inferior vena cava and portal vein indicated the end of the anhepatic phase. Subsequently, calcium gluconate (20 mL, 10%), sodium bicarbonate (100 mL), and methylprednisolone (500 mg) were injected. The transplanted liver was washed in saline water for rapid rewarming, and the infrahepatic vena cava, hepatic artery, and common bile duct were anastomosed, with end-to-end anastomosis for the common bile duct. Then, a silicon tube was placed in the bile duct to serve as a stent for fixation. Once substantial abdominal bleeding had stopped, the abdominal cavity was closed, and the pigs were allowed to recover.
Tissue sampling and analysis
The animals were followed up to 5 days following transplant. Venous and arterial blood samples were collected from the catheters after opening of the abdomen of recipient livers in the sham and transplantation groups, at 1 h after blood restoration of hepatic artery, and at 1, 2, 3, 4, and 5 days after the operation for further serological examination. Blood was immediately centrifuged, and the plasma was frozen and stored at − 20 °C until analysis.
Appropriate portions of liver tissues were collected through relaparotomy from recipient livers before warm ischemia, 30 min after warm ischemia, 2 h after cold storage, 4 h after cold storage, 2 h after HMP, and 24 h after transplantation. The biopsy samples were either stored at − 80 °C or preserved in 10% formaldehyde for further analysis. At the end of the study, the animals were tranquilized and then euthanized by injection of an overdose of pentobarbital sodium (> 150 mg/kg) after establishment of an intravenous passage.
Biochemical assay of blood sample
The dry chemistry method was applied to determine glutamic pyruvic transaminase (ALT), glutamic oxaloacetic transaminase (AST), and total bilirubin (TBIL) using the Vitros 5600 Automatic Biochemical Immunoanalyzer (Johnson & Johnson, New Brunswick, NJ, USA). The international normalized ratio (INR) was measured by immunomagnetic beads using an automatic coagulometer (Stago, France). The lactate was analyzed by GEM 3500 blood electrolyte analyzer.
Western blot analysis
Proteins obtained from liver tissues were analyzed by Western blot analysis. Blots underwent incubation with antibodies against JNK (Anti-JNK1/2/3 antibody bs-2592R; Bioss, Beijing, China), phosphorylated-JNK (P-JNK) (Anti-phospho-JNK1/2/3 antibody bs-1640R; Bioss), B-cell lymphoma-2 (Bcl-2) (Anti-Bcl-2 antibody ab117115; Abcam Inc., Cambridge, MA, UK), Bcl-2 associated protein X (Bax) (Anti-Bax antibody bs-0127R; Bioss), Cleaved Caspase-3 (Anti-Cleaved Caspase-3 ab49822; Abcam), Cytochrome C (Anti-Cytochrome C antibody ab90529; Abcam), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Anti-GAPDH antibody ab8245; Abcam). The blots were scanned with ChemiDoc™ XRS (Bio-Rad, USA). Image J 7.0 software was used for the analysis of the gray area density.