Sharing Experience on Long-Term Survival in A Non-Arterialized Rat Liver Transplantation Model

Summary

The goal of this presented protocol is to share optimized surgical experience for establishing a non-arterialized rat liver transplantation model, specifically focusing on technical refinements that ensure long-term animal survival beyond 100 days.

Abstract

In experimental liver transplantation, the rat model is the gold standard for investigating transplant immunology and metabolic processes; however, reconstructing the exceedingly fine hepatic artery (~0.5 mm) presents a formidable technical challenge. To circumvent this, the simplified non-arterialized rat liver transplantation (NArLT) model is widely utilized. Unfortunately, severe biliary complications—such as bile leakage and ischemic cholangitis—arising from arterial deprivation frequently restrict the long-term survival of recipients. This obstacle significantly limits research on key topics, including long-term host-graft immunocompatibility and chronic rejection. Herein, we present an optimized protocol for the NArLT model that incorporates critical technical refinements to successfully overcome these ischemic biliary injuries. Key improvements include a significantly shortened anhepatic phase, a “no-touch” biliary dissection technique, thorough biliary tract flushing, and an optimized internal biliary stenting method. After implementing this improved procedure, the recipient survival rate exceeded 94% during the 100-day observation period. Ultimately, this optimized protocol provides a highly reproducible and robust platform to investigate the mechanisms underlying long-term transplant tolerance, thereby supporting the development of novel therapeutic strategies in transplantation medicine.

Introduction

The rat liver transplantation model serves as the most widely utilized experimental platform for basic transplant research. However, its broader application is frequently hindered by a notoriously steep learning curve, which can significantly impede the progress of downstream experimental studies^1,2. Furthermore, conventional rat liver transplantation models often suffer from suboptimal surgical success and low long-term survival rates. By critically streamlining the surgical procedures, meticulously refining operative details, and performing vascular and biliary reconstructions under direct visualization, the....

Protocol

All animal experiments and surgeries in this study were approved by the Experimental Animal Ethics Committee of Qingdao University (No. 20260301SD5020260401065). Make all efforts to minimize animal suffering.

1. Preoperative Preparation

1. Experimental animals
   1. Use adult male Sprague–Dawley (SD) rats, aged 8–10 weeks and weighing 220–250 g, as donors. Ensure that the recipient’s weight is slightly greater than the donor’s, with the difference within 50 g.
   2. House rats under specific pathogen-free conditions at a controlled temperature of 22°C ± 2°C and relative humidity of 50% ± 10%.
   3. ....

Results

To ensure consistent surgical quality and reproducibility across the NArLT models, all procedures were performed by the same experienced microsurgeon. A total of 50 rat liver transplantations were completed during the study period. The mean durations for the surgical procedures were as follows: donor surgery averaged 25.3 ± 2.6 min, ex vivo graft preparation averaged 13.1 ± 2.1 min, recipient surgery averaged 43.0 ± 1.6 min, and the anhepatic phase averaged 15.7 ± 1.2 min.

Recipients undergoin.......

Discussion

The NArLT model is a cornerstone for experimental transplant research due to its avoidance of complex microvascular arterial anastomosis¹¹. However, conventional NArLT methods are historically plagued by high rates of biliary complications—up to 30%—which severely limit long-term survival and hinder chronic immunological studies². The significance of the proposed method lies in its ability to circumvent these ischemic biliary injuries. In this study, the 100-day.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4-0 Absorbable Suture	Ethicon	W9109H	Used for closure of musculofascial layer (linea alba)
4-0 Silk Suture	Ethicon	VCP304H	Used for skin closure
7-0 or 8-0 Suture / Silk Tie	Ethicon	Z1711E	Used for ligation of vessels (e.g., left phrenic vein)
22G Intravenous Catheter	B. Braun	4252098B	Trimmed to 5–7 mm with inclined ends to prepare biliary stent
23G Scalp Vein Needle	Shandong Qinkai Medical Industry Co., Ltd.	MDLSSVS	Used for insertion into abdominal aorta for liver perfusion
Buprenorphine	Tianjin Institute of Pharmaceutical Research Pharmaceutical Co., Ltd.	H12020275	Administered as subcutaneous analgesia post-surgery
Cotton Swab	Aoocn	216-236	Used to gently elevate liver during dissection
Crushed Ice	N/A	N/A	Used to maintain graft temperature during ex vivo preparation
Elastic Retractors	N/A	N/A	Used to retract abdominal wall during laparotomy
Epidural Catheter Sheaths	Jiangsu Maichuang Medical Device Co., Ltd.	20173084015	Sizes 5F and 6F; used as cuffs for PV and IHIVC
Gauze	Central Medical Technology Co., Ltd.	CH-1001	Saline-soaked; used to cover and protect intestines
Glucose Solution (5%)	Sichuan Kelun Pharmaceutical Co., Ltd.	H20044307	Used to flush intrahepatic blood during recipient hepatectomy
Heparin Saline	Shenzhen Life Biotechnology Co., Ltd.	H2012003	100 IU/mL; used to soak biliary stent
Indwelling Needle	B. Braun	4254074B	Used to flush SHIVC to remove air before completing anastomosis
Isoflurane	Jinan Guangshengyuan Biotechnology Co., Ltd.	153107015	5% concentration; used for anesthesia induction
Microvascular Clamps	Shanghai Medical Devices Co., Ltd.	JCZ200	Used for vessel occlusion during surgery
Mosquito Forceps	Shanghai Medical Devices Co., Ltd.	J31010	Used for fine manipulation and cuff preparation
Normal Saline (0.9%)	Sichuan Kelun Pharmaceutical Co., Ltd.	H20044304	Used for irrigation and gauze soaking (37°C)
Pentobarbital (1%)	Shanghai Yuansi Technology Co., Ltd.	P0500000	40–45 mg/kg; used for surgical anesthesia
Peripherally Inserted Venous Catheter (PIVC)	Covidien	8888145015	Used to flush cuffed PV and confirm patency
Povidone-Iodine	Shanxi Trinda Biotechnology Co., Ltd.	41836054	Used for surgical site disinfection
Rubber Band (from Surgical Glove)	Ansell	N/A	Used for SHIVC traction during recipient surgery
Sprague–Dawley Rats	Huafukang (Beijing) Biotechnology Co., Ltd.	22001A	Adult male rats, 8–10 weeks, 220–250 g
Sterile Petri Dish (10 cm)	Shandong Enyou Biotechnology Co., Ltd.	H905001	Used to hold graft in UW solution during ex vivo preparation
Syringe (20 mL)	Shandong Weigao Group Medical Polymer Products Co., Ltd.	20163141593	Used for controlled liver perfusion (3–3.5 mL/min)
UW Preservation Solution	Bridge to Life	BTL-UW-1L	Maintained at 4°C; used for donor liver perfusion and graft preservation