Modeling experiments on animal models of liver transplantation

Summary

Liver transplantation is currently one of the most effective ways to treat end-stage liver disease. The increasing shortage of liver supplies has led to the development of diverse clinical liver transplantation techniques. Measures to expand the donor pool for liver transplantation, such as split liver transplantation (SLT) and living donor liver transplantation (LDLT), have significantly reduced the morbidity and mortality of patients waiting for liver transplantation. Further studies on the regenerative mechanism of transplanted liver and its influencing factors are needed to improve the clinical outcome after partial liver transplantation. The establishment of animal models of liver transplantation is a prerequisite for organ transplantation and a platform for basic research. Among them, rat partial liver transplantation is the ideal model for its study. Currently, there is one main method for modeling animal models of liver transplantation: the rat in situ liver transplantation model by the two-cuff method.

Principle

The basic principle of the rat in situ liver transplantation model by the two-cuff method is to establish the model by using the principle of reconstructing the subhepatic inferior vena cava and portal vein by the cannula, reconstructing the bile duct by the internal stent tube, and anastomosing the upper and lower hepatic vena cava directly.

Operation method

Rat in situ liver transplantation model by the two-cuff method

Principle

The basic principle of the rat in situ liver transplantation model by the two-cuff method is to establish the model by using the principle of reconstructing the subhepatic inferior vena cava and portal vein by the cannula, reconstructing the bile duct by the internal stent tube, and anastomosing the upper and lower hepatic vena cava directly.

Materials and Instruments

Equipment:
A 5/7 F cardiac catheter sheath, a 22 GA intravenous retention needle sheath, 9-0 non-invasive sutures, a two-person binocular surgical microscope, microsurgical instruments, a fully automated biochemical analyzer, an optical microscope, and male SD or Wistar rats were used.
Reagents:
① Lactated Ringer's solution.
② Ketamine; ③ Saline.
Saline.

Move

The basic procedure of the rat in situ liver transplantation model by the two-cuff method can be divided into the following steps:

(a) Clean-grade male SD or Wistar rats, weighing 220-240 g, were sheathed with 5/7F cardiac catheters to make portal vein (PV) and infrahepatic vena cava (IVC) cuffs; biliary stents were sheathed with 22 GA venous retention needles; 9-0 nondestructive sutures, double binoculars, surgical microscopes, microsurgical instruments, fully automatic biochemical analyzers, and optical microscopes. operating microscope, microsurgical instruments, automatic biochemical analyzer, optical microscope, etc.

(II) Donor surgery

A After successful anesthesia, the abdomen was entered through a transverse incision, and the liver was exposed by a homemade back pad. After systemic heparinization, the abdominal aorta was isolated above the celiac artery behind the first hepatic hilar and ligated with a wire.

B 4 ℃ lactated Ringer's solution 20 mL was infused via the abdominal aorta with an infusion pump at a rate of 50-150 mL/h. The IVC was quickly cut at the upper edge of the left renal vein, the thoracic vena cava was disconnected by cutting the diaphragm, and iced saline was poured onto the surface of the donor liver.

C While perfusing the donor liver, the perihepatic ligament was freed in clockwise order, and the left diaphragmatic vein, esophagus into the left hepatic vascular branch, the right adrenal gland, the right kidney, and the inferior vena cava were severed in turn, and the upper and lower vena cava of the donor liver was cut off immediately against the diaphragmatic ring (suprahepatic vena cava (SVC)). The anterior wall of the common bile duct was wedge incised 0.5 cm distal to the confluence of the left and right hepatic ducts, and a biliary stent was inserted into the proximal hepatic segment and secured with a silk ligature.

D The hepatic artery was isolated, and the pyloric vein and splenic vein were dissected near the PV.

(C) Donor liver trimming

A The operations were performed in a 4 ℃ ice bath. Use microvascular forceps to pass through the lumen of the homemade cuff, clip the broken end of the IVC and drag it out to turn it outward on the wall of the cuff, and ligate it with silk threads in the notch; treat the PV cuff in the same way to ensure that there is no twisting of the cuff. After slowly injecting 5 mL of 4 ℃ lactated Ringer's solution through the PV cuff, the donor liver was stored in 4 ℃ lactated Ringer's solution.

(iv) Recipient surgery

A After successful induction of ether inhalation anesthesia, ketamine 100 mg/kg was injected intraperitoneally.

B The abdomen was entered through a median incision, and a homemade pulling hook was used to retract the rib arches and pull the raphe cephalad; the intestinal tube was pushed to the lower left and wrapped with warm wet gauze. Free the perihepatic ligament in a clockwise direction, ligate the left phrenic vein, the esophageal branch into the left hepatic vessel, free the IVC in the plane of the superior border of the right renal vein, disconnect the right superior adrenal vein, and fully free the SVC.

C Free the common bile duct and cut it at the confluence; free and cut the hepatic innominate artery; free the PV above the level of the pyloric vein; block the PV and the IVC with vascular clips close to the liver in turn; puncture the bifurcation of the PV and inject 2-3 mL of warm physiological saline; use Satinsky clamp to block the SVC; cut off the SVC, the IVC, and the PV close to the liver; remove the original liver.

D Place the donor liver into the original position of the liver, clamp the PV trocar handle with microscopic forceps, and push 2-3 mL of warm saline from the PV to drive out the intrahepatic preservative.

E A 9-0 nylon thread was used to enter the needle on the lateral side of the vena cava wall at the diaphragmatic vein of the donor liver, and the posterior and anterior walls were anastomosed consecutively in the order of "inside-outside-outside-inside" after locking the edges, and the intracavitary air bubbles were expelled with lactated Ringer's solution containing heparin at 125 U/mL before tightening, and then tied with a knot with a pre-determined thread after tightening.

F Move the vascular clamp blocking the recipient PV down to the level of the pyloric vein to drain blood and clots from the PV, insert the PV cuff quickly after rinsing, and remove the vascular clamp and Satinsky clamp after ligation to restore blood flow to the liver and end the hepatic-free period.

G The recipient IVC vascular clamp is moved down to the upper edge of the right renal vein, the blood clot in the IVC is removed, and the IVC cuff is inserted immediately after flushing and secured with a silk ligature.

H A biliary stent was inserted into the lumen of the recipient's common bile duct, ligated and covered with a large omentum, and 2 mL of warm saline containing 200,000 U of penicillin was injected into the abdominal cavity and the abdomen was closed. Postoperatively, the patient was rewarmed with a lamp until awakening, and was given 10% glucose and water ad libitum, and was fed ad libitum on the first postoperative day.

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