Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Summary

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

Abstract

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

Introduction

Ischemia-reperfusion injury (IRI) is a common complication after liver transplantation. Hepatic IRI is a pathological process involving ischemia-mediated cell damage and abnormal deterioration of liver reperfusion. Hepatic IRI and the local innate immune response can be divided into hot and cold IRI, according to differences in the clinical environment¹. Hot IRI is induced by stem cell injury, usually as a result of liver transplantation, shock, and trauma². Cold IRI is a complication of liver transplantation caused by endothelial cells and peripheral circulation³. Clinical reports have shown that hepatic IRI is associated with 10% of early organ failures and may increase the incidence of acute and chronic rejection^4,5. In addition, hepatic IRI may also induce multiple organ dysfunction syndromes or systemic inflammatory response syndrome, with high mortality⁶. Patients with extrahepatic organ involvement tend to stay longer in the hospital, spend more money, and have a worse prognosis⁷. The development of complications is closely related to the length of the anhepatic phase of liver transplantation⁸.

Orthotopic liver transplantation (OLT) in rats was first reported by the American professor Lee in 1973. The experimental operation simulated the steps of clinical liver transplantation and the anastomosis of blood vessels and the common bile duct (CBD) using the suture method. The procedure is difficult and time-consuming with a low rate of success⁹. In 1979, Kamada et al. made a significant improvement to OLT in rats by creatively using the 'two-cuff method' for anastomosis of the portal vein to control the anhepatic phase within 26 minutes¹⁰. In the same year, Zimmermann proposed the 'single biliary stent method.' On the basis of Lee's work, Zimmermann used polyethylene tubes to directly anastomose the CBD of the donor and recipient, simplified the reconstruction of CBD, and preserved the function of the sphincter, and this method became the standard for biliary reconstruction of OLT models¹¹. In 1980, Miyata et al. proposed the 'three-cuff method' where the portal vein (PV), suprahepatic vena cava (SVC), and intrahepatic vena cava (IVC) were anastomosed by the cuff method. However, there is a risk of distortion of the cannula with this method, which can lead to the obstruction of inferior vena cava reflux¹². In 1983, the 'two-cuff method' was proposed using the cuff method for anastomosis of the PV and IVC, but adopting the suture method for the SVC¹³. This method was adopted by scholars globally to establish OLT models. Since then, the cuff anastomosis steps have been improved to shorten the anhepatic phase and improve the survival rate of the rats¹⁴. Similarly, improved methods are used in clinical practice to shorten the anhepatic phase¹⁵. However, basic research into IRI after liver transplantation has shown that the survival rate is inversely related to the degree of injury to extrahepatic organs. Therefore, further research is required, and a simple and reproducible animal model is needed to simulate IRI after liver transplantation.

Based on the definition of the anhepatic phase, we simulated the hemodynamic changes in liver transplantation resulting in IRI of extrahepatic organs in rats. Herein, we provide a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into IRI after liver transplantation.

Protocol

The Animal Ethics Committee approved the experiment of Guangxi Medical University (No20190920). All animals were supplied by the Animal Experiment Center of Guangxi Medical University. We used SPF male Sprague Dawley rats (200-250 g, 10-12 weeks), kept under the room temperature of 25 ± 2°C and humidity of 50 ± 10%. Feeding was stopped 24 hours before operation; however, water was provided.

NOTE: One operator can perform all operations without a microsurgery basis or surgical microscope.

1. Operation

1. After weighing, anesthetize the rats with isoflurane (5%) using an animal anesthesia machine.
2. After 1-2 minutes, gently clamp the toes of the rat with tweezers. If the rat does not respond after pinching, it has entered a state of anesthesia. Use vet ointment on the eyes to prevent dryness. Use animal heating lamps to keep the rats' body temperature at 37-38 °C.
3. Following abdominal disinfection (povidone iodine solution), fix the rat on the animal dissection table. Make a median incision of 3 cm below the xiphoid process using forceps and scissors.
4. Open the abdominal cavity, expose the liver using a retractor, and mobilize the hepatogastric ligament. Use cotton swabs to flip the middle lobe of the liver gently and turn it upward to expose the porta hepatis. Identify the CBD, PV, and HA.
5. Push the small intestine toward the left lower abdominal cavity using cotton swabs, cover it with wet gauze, and move the intrahepatic vena cava to the right renal vein.
6. Isolate the portal vein, hepatic artery, and the inferior vena cava above the right renal vein with an intraocular lens and forceps marked with 3-0 silk thread, each with a slip knot.
7. Cut open the left and right lower extremity skin and expose the femoral vein using ophthalmic forceps. Slowly inject low molecular weight heparin 625 IU/kg through the femoral vein to heparinize the whole body.
8. Ligate the portal vein, hepatic artery, and inferior vena cava above the right renal vein with No. 3-0 sutures, lasting 45 minutes (Figure 1). Replace the small intestine in the abdominal cavity and cover it with gauze. Reduce inhalation anesthesia during these periods.
9. After 45 minutes, release the portal vein, hepatic artery, and the inferior vena cava above the right renal vein.
10. Suture the muscle and skin, layer by layer, and terminate the inhalational anesthesia. Provide postoperative analgesia using subcutaneous morphine of 5 mg/kg every 4 hours.
11. Observe the rat until it is awake and feed under a temperature of 25 ± 2 °C and humidity of 50 ± 10%. Animal heating lamps are necessary.

Results

Rats' tolerance to liver ischemia
In this animal model, the sites at which blood vessels were ligated during operating are shown in Figure 1. The rats were randomly divided into 5 groups for ischemia for 15 minutes (I15 group), 30 minutes (I30 group), 45 minutes (I45 group), 60 minutes (I60), and sham group, with 10 rats in each group. The survival rate of each group was observed 14 days after the operation. All rats survived in the I15 group, I30 group, and sham g...

Discussion

OLT in rats is an ideal model for studying organ preservation in liver transplantation, IRI, transplant rejection, immune tolerance, transplantation pathology and pharmacology, homotransplantation, and xenotransplantation. At present, it is widely used in the experimental research of liver transplantation.

During pilot studies we first administered pentobarbital sodium intraperitoneal anesthesia and found that this led to high postoperative mortality and short tolerance to hemodynamic changes....

Materials

Name	Company	Catalog Number	Comments
4% paraformaldehyde solution	Shanghai Macklin Biochemical Co.,Ltd	P804536
air drying oven	Shanghai Binglin Electronic Technology Co., Ltd.	BPG
Alanine aminotransferase (ALT)Kit	Elabscience Biotechnology Co.,Ltd	E-BC-K235-S
ammonia	Sinopharm Chemical Reagents Co. Ltd	10002118
amylase Kit	Elabscience Biotechnology Co.,Ltd	E-BC-K005-M
anhydrous ethanol	Sinopharm Chemical Reagents Co. Ltd	100092183
Animal anesthesia machine	Shenzhen Ruiwode Life Technology Co. Ltd	R640
aspartate aminotransferase (AST)kit	Rayto Life and Analytical Sciences Co., Ltd.	S03040
automatic biochemical analyzer.	SIEMENS AG FWB:SIE, NYSE:SI Co., Ltd.	2400
Biosystems (when nessary)	Chengdu Taimeng Electronics Co., Ltd.	BL-420F
Centrifuge	Baiyang Medical Instrument Co., Ltd.	BY-600A
cover glass	Jiangsu Shitai Experimental Equipment Co. Ltd	10212432C
creatinine Kit	Rayto Life and Analytical Sciences Co., Ltd.	S03076
dewatering machine	Hungary 3DHISTECH Co.,Ltd	Donatello Series 2
embedding machine	Hubei Xiaogan Kuohai Medical Technology Co., Ltd.	KH-BL1
frozen machine	Wuhan Junjie Electronics Co., Ltd	JB-L5
hematoxylin-eosin dye solution	Wuhan Saiwell Biotechnology Co., Ltd	G1005
high-efficiency paraffin wax	Shanghai huayong paraffin wax co., Ltd	Q/YSQN40-91
hydrochloric acid	Sinopharm Chemical Reagents Co. Ltd	10011018
intraocular lens (IOL)forceps	Guangzhou Guangmei Medical Equipment Co., Ltd.	JTZRN
Isoflurane	Shenzhen Ruiwode Life Technology Co. Ltd	—
micro Scissors(when nessary)	Shanghai Surgical Instrument Factory	WA1010
needle holders	Shanghai Surgical Instrument Factory	J32010
neutral gum	Shanghai Huashen Healing Equipment Co.,Ltd.	—
normal optical microscope	Nikon Instrument Shanghai Co., Ltd	Nikon Eclipse CI
ophthalmic forceps	Shanghai Surgical Instrument Factory	J3CO30	straight
ophthalmic forceps	Shanghai Surgical Instrument Factory	JD1060	bending
ophthalmic Scissors	Shanghai Surgical Instrument Factory	J1E0
pathological slicer	Shanghai Leica Instrument Co., Ltd	RM2016
pipettes	Dragon Laboratory Instruments Co., Ltd.	7010101008
retractors	Beijing Jinuotai Technology Development Co.,Ltd.	JNT-KXQ
scanner	Hungary 3DHISTECH Co.,Ltd	Pannoramic 250
slide	Wuhan Saiwell Biotechnology Co., Ltd	G6004
xylene	Sinopharm Chemical Reagents Co. Ltd	1330-20-7