Orthotopic liver transplantation of pigs using porto/caval-jugular shunt for research work or off- site training of young surgeon

Masashi Kadohisa Department of Transplantation and Pediatric Surgery, Kumamoto University, Kumamoto, Japan https://orcid.org/0000-0002-5552-5539 Yukihiro Inomata (  yino@kuh.kumamoto-u.ac.jp ) Kumamoto Rosai Hospital, Kumamoto, Japan Hiroo Kasamatsu SCREEN Holdings Co, Ltd, Kyoto, Japan Shinji Torai SCREEN Holdings Co, Ltd, Kyoto, Japan Syuhei Yoshimoto SCREEN Holdings Co, Ltd, Kyoto, Japan Eiji Kobayashi Department of Organ Fabrication, Keio University School of Medicine, Tokyo, Japan


Introduction
Liver transplantation is a long and complicated procedure. For residents or fellows, the technical skill is taught on site, usually part by part. In countries or areas where the clinical cases are limited in number, like in Japan, even senior surgeons have to operate by him-herself for maintain of the skill, and there is quite limited space for young surgeons to improve their clinical skill on site. Simulation of the operation using a big animal can be an alternative for the practical training, if burdens related to the cost and the facility are supported. An interinstitutional training program was launched in 2014 in Japan assisted by the Ministry of Education, Culture, Sports, Science and Technology (MEXT). The system was operated by six National Universities, with supervision of additional one University (Kyoto University) and a National Center (National Center for Child Health and Development), each with high volume of cases. The program was called "Six National University Consortium in Liver Transplant Professionals Training Program" or SNUC-LT). The trainee was selected among the young HBP surgeons in the six Universities. The curriculum for each trainee was completed in 3 years. Two to six trainees joined to this program each year. It consisted of practical training, lectures, seminars, and web conferences. In the practical training, on job learning is most interesting and highly educational. However, the chance was limited. In Japan, living donor liver transplantation is still dominant, and the deceased donor liver transplantation including the organ harvest is much smaller in number. For supplement of the practical training in this aspect, operative simulation using a pig was induced. It was done twice every year, with gathering of the trainees in a wet-lab. This could offer very valuable experience of real operation to each trainee who could not touch enough in the clinical setting. In the simulation, enough learning should be secured while keeping the animal alive until the end of the procedure. The trainees should have room to breathe. For this purpose, to guarantee stable circulatory dynamics during the anhepatic phase of pigs, operative procedure using a porto-caval-jugular shunt was applied. This met the intended purpose by addressing liver dysfunction, portal hypertension and intestinal congestion 1 . Here, we describe the precise practical procedure. We believe that our procedure enables young transplant surgeons to gain surgical skills e ciently and effectively. In addition, this procedure may assist the researchers to use in the in-vivo study of liver transplantation using a big animal kept alive after the surgery. The simple and active shunt system is composed of 2 motors (BMU series, Oriental Motor Inc., Tokyo, Japan) and 2 pump heads (custom-made item, SCREEN Inc., Kyoto, Japan) of roller pump for blood perfusion and blood aspiration, a manometer (ISE80 series, SMC Inc., Tokyo, Japan) that measures in ux pressure, a blood container, and a circulation circuit material (silicone tube, φ5×8) (Fig1). The circulation circuit consists of 2 blood aspiration lines (Infraheptic inferior vena cava (IHIVC) and splenic vein (SV) lines) connected to the roller pump, and a blood container and blood infusion line (left jugular vein (LJV) line). In an active shunt system, Multi-pore suction connector (MPSC) is developed to prevent the occlusion of the vascular wall due to the suction pressure and enable stable blood aspiration (Fig2).

Reagents
Ringer's lactate mixed with heparin at a concentration of 1000 IU/L was connected to the infusion line and drip-infused to prime the entire circuit. The solution volume in the circuit is reduced (within 400 mL).
As a result of this arrangement, the effect of blood loss ex vivo could be minimized. A blood container (150 mL) with vent port was incorporated in the circuit, and the air was removed by opening this port. This container enables us to lower the risk of infusing air to the vein of a recipient from the blood aspiration line.

Sedation and anesthesia induction
Sedate the pigs by cervical intramuscular injection of a mixture of midazolam (0.2 mg/kg) and medetomidine (0.04 mg/kg). Insert a catheter in an ear vein, followed by slow intravenous administration of Thiamylal Sodium (3-5mg/kg).

Tracheal intubation
In supine position, con rm loss of swallowing re ex. Intubate the trachea with visual con rmation of airway. Con rm the expiration from tracheal tube, followed by securing tracheal tube with tape.

Hair shaving
Shave the operative eld from inferior border of thorax to groin using electrical hair clipper.

Maintenance of anesthesia and intraoperative monitoring
Place in the supine position on the operating table. Maintain anesthesia with 1.5-3.5% inhaled iso urane and set the arti cial ventilator with 40-100% concentration of oxygen. Keep the pigs warm and continuously monitor the electrocardiogram, heart rate, non-invasive blood pressure, oxygen saturation by pulse oximetry at the pig's tail, end-tidal carbon dioxide, body temperature and depth of anesthesia during anesthesia management. 10. Cross clamp the SCA using a DeBakey aortic clamp. Then, euthanize the donor pig by blood removal from IRIVC cannulation tube.
11. After con rming the death, perfuse amount Ringer's lactate into the IRA and venting from IRIVC and, if necessary, SVC to the right thoracic cavity.
Excision of the Liver and vascular graft 1. Place the crushed ice in the abdominal cavity, for example around the surface of liver, in the bursa omentalis and around small intestine, for maintain a low temperature.
2. Dissect the hepatoduodenal ligament and identify each vascular channel such as the common bile duct, the hepatic artery, the gastroduodenal artery, and the portal vein (PV). If necessary, ligate the lymph vessels within the hepatoduodenal ligament to prevent lymphatic leakage. Troubleshooting: If the active shunt system cause blood pressure reduction, administer adrenaline solution to maintain blood pressure as required.
5. Cross-clamp the SHIVC including a diaphragmatic rim using a Satinsky clamp while retracting the liver caudally. Subsequently, cut it at its border to the liver tissue. Subsequently, excise the liver.
Troubleshooting: If hypoglycemia occurs during the anhepatic phase, infuse 5% dextrose solution until glucose levels in the blood rise adequately.
Liver transplantation 1. Place the donor liver into the abdominal cavity.
2. For an end-to-end anastomosis of SHIVC, trim the donor SHIVC appropriately. Stitch on each side corner of the recipient SHIVC with 5-0 prolene sutures. Using the inside needles of its stitches, perform an inside-outside stitches on each side corner of the donor SHIVC.
3. Shoot both ends of the right suture. Approximate recipient and donor SHIVC, then tie both ends of the left suture.
4. Using the longer end of left suture, perform an outside-inside stitch of the recipient SHIVC back wall next to the tie. Subsequently, perform the running suture of the back wall to the right corner. 5. After reaching the right corner, tie the other right suture ends at the right corner and tie both sutures. Then add 2-3 front wall stitches using the running suture.
6. Add front wall stitch using the new suture near the left corner tie. Then perform the running suture of the front wall to right corner. Tie together both sutures of the back and front walls. 7. After trimming the donor portal vein appropriately, perform an end-to-end portal vein anastomosis in the same manner with 6-0 prolene, leaving approximately 0.5 cm of growth factor. 8. After performing these anastomoses, block the blood aspiration lines of the circulation circuit and stop the centrifugal pump. Open the SHIVC clamp, subsequently, re-perfuse the liver by opening the portal vein clamp.
Troubleshooting: If blood pressure reduction occurs after liver re-perfusion, administer more intravenous uid and adrenaline solution.
9. Return the remaining blood in the circuit to the jugular vein as e ciently as possible and withdraw the circulation circuit completely. Schematic image of the active shunt system. The system is composed of 2 motors and 2 pump heads of roller pump for blood perfusion and blood aspiration, a manometer that measures in ux pressure, a blood container, and a circulation circuit material.

Figure 2
Multi-pore suction connector (MPSC). The connector has 10 pores in the tip to prevent the occlusion of the vascular wall due to the suction pressure and enable stable blood aspiration.

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