All male rats like inbred SD, Lewis and BN (weighing 180-380g) were used as donors and recipients purchased from Beijing Vital River Laboratory Animal Technology Corporation. SD→SD OLT was performed for microsurgery skills (control group, n=9); for the project of tolerance induction through stem cells, chronic rejection model with SD→Lewis (chronic rejection, n=11) and acute rejection with Lewis→ BN (acute rejection, n=63) were used, the acute rejection group was sub-grouped into the transverse incision group (n=26) and midline group (n=37). The rats were housed in a temperature and light-controlled environment, the standard food and tap water was freely accessible; they were fasted 12 hours before the operation. All experimental protocols were approved by Committee of Henan Provincial People’s Hospital and all experiments were carried out in compliance with the ARRIVE guideline and the standards for animal use and care set by the Institutional Animal Care Committee of Henan Provincial People’s Hospital.
Under inhalation anesthesia of isoflurane with a few modifications, a transverse incision was made to enter the abdominal cavity of the all donor rats. The liver was flushed first through the aorta with lactated Ringer's solution and re-flushed through the portal vein (PV). Cold storage time was less than 3 hours in all cases. In the recipient, after the abdominal cavity was opened using the transverse incision (Figure 1), the midline incision was made only for Lewis→BN subgroup(Figure 2). The native liver was removed and the graft was placed orthotopically, the suprahepatic vena cava was anastomosed with 8-0 polypropylene running suture, PV and the inferior vena cava were reconnected with the cuff, the anhepatic time was generally less than 20 minutes, and the hepatic artery was reconstructed with a stent , the bile duct was reconnected with a stent too. After the muscle layer was closed and coated with lidocaine cream, the skin was interruptedly sutured (Figure 3). The rat recipient was kept in a cage under an infrared light for warm. Both tap water and 10% glucose solution were supplied for first 3 days, and later regular food and tap water were available. Naloxone was additionally used to distract the recipients for the acute rejection group of the transverse incision. Antibiotic (ceftriaxone) was injected subcutaneously once a day, 3 days in total. For the rejection models, the recipient stem cells were mobilized, cyclosporine A was subcutaneously injected at 2 mg/kg once daily for 14 days and ceased afterwards. The detailed protocol of tolerance induction was out of scope here. All recipient rats were closely monitored, the severity of self-biting was rated simply on the scale adapted from the clinical setting: 0= no evidence of biting, 1=mild injury (erythema, edema), 2=moderate injury (minor tissue loss, such as single digit, fur removal, or involvement of multiple sites), 3=severe injury (amputation of several digits, significant abdominal or thoracic lesions, animal requires euthanasia due to self-biting injuries) . Only the recipient rat alive for more than 3 days was included in this study.
The comparisons of incision-related self biting morbidity or mortality between groups were made with the chi-squared test or Fisher exact test; the cumulative survival rates of different groups was evaluated with the Kaplan-Meier Curve, the analysis was made with SPSS 22.0 software (IBM Corp, Armonk, NY, USA), and P<0.05 was considered significant