Heterotopic Auxiliary Whole Liver Rat Transplant Model Utilizing a Hepaticoureterostomy for Allograft Rejection Studies

Summary

The rat heterotopic auxiliary liver transplant protocol described here offers a practical investigational tool for exploring mechanisms of hepatic allograft rejection. This model helps to alleviate the surgical hurdles and animal stress of orthotopic liver transplantation in rats.

Abstract

Small animal transplant models are indispensable for organ tolerance studies investigating feasible therapeutic interventions in preclinical studies. Rat liver transplantation (LTx) protocols typically use an orthotopic model where the recipients' native liver is removed and replaced with a donor liver. This technically demanding surgical procedure requires advanced micro-surgical skills and is further complicated by lengthy anhepatic and lower body ischemia times. This prompted the development of a less complicated heterotopic method that can be performed faster with no anhepatic or lower body ischemia time, reducing post-surgery stress for the recipient animal.

This heterotopic LTx protocol includes two main steps: excising the liver from the donor rat and transplanting the whole liver into the recipient rat. During the excision of the donor liver, the surgeon ligates the supra-hepatic vena cava (SHVC) and hepatic artery (HA). On the recipient side, the surgeon removes the left kidney and positions the donor liver with the portal vein (PV), infra-hepatic vena cava (IHVC), and bile duct facing the renal vessels. Further, the surgeon anastomoses the recipient's renal vein end to end with the IHVC of the liver and arterializes the PV with the renal artery using a stent. A hepaticoureterostomy is utilized for biliary drainage by anastomosing the bile duct to the recipient's ureter, permitting the discharge of bile via the bladder.

The average duration of the transplantation was 130 min, cold ischemia duration was around 35 min, and warm ischemia duration was less than 25 min. Hematoxylin and eosin histology of the auxiliary liver from syngeneic transplants showed normal hepatocyte structure with no significant parenchymal alterations 30 days post-transplant. In contrast, 8-day post-transplant allogeneic graft specimens demonstrated extensive lymphocytic infiltration with a Banff Schema rejection activity index score of 9. Therefore, this LTx method facilitates a low morbidity rejection model alternative to orthotopic LTx.

Introduction

Small animal LTx is an invaluable model for investigating mechanisms of liver rejection. Heterotopic auxiliary liver transplantation with portal vein arterialization (HALT-PVA) in rats was introduced in 1968 by Lee and Edgington¹when they reported using a recipient's renal vein and artery to re-vascularize a grafted auxiliary liver. Subsequently, Hess et al.² enhanced the protocol with the mitigation of functional competition between the native and auxiliary livers by reducing the native and donor liver size along with reconstructing the donor bile duct connection, resulting in long-term graft survival. Further refin....

Protocol

Animals were bred and housed in specific pathogen-free conditions in the animal care facilities at the University of Wisconsin (UW)-Madison Institute for Medical Research in accordance with institutional guidelines. The study protocol (No. M006022) was approved by the Institutional Animal Care and Use Committee at the UW School of Medicine and Public Health, and all animals were treated ethically.

1. Animals

1. Use adult Lewis female rats weighing 205-235 g and Lewis .......

Discussion

Liver transplantation is the only treatment option for patients with end-stage liver disease, with almost 9,000 LTxs performed yearly in the US¹³. Unfortunately, immunological rejection is seen in up to 25% of LTx recipients, and this rejection is detrimental to the transplanted organ and patient^14,15. To improve outcomes after LTx, the development of innovative models to study organ rejection and implement strategies to decrease rejection.......

Materials

Name	Company	Catalog Number	Comments
3-0 Silk Suture	Ethicon	C013D
5-0 Silk ties	Fine Science Tools	18020-50
6-0 Silk ties	Fine Science Tools	18020-60
7-0 Silk ties	Teleflex	103-s
9-0 Polyamide Suture	AROSurgical	T05A09N10-13	Black
Bipolar Cautery	Codman & Shurtleff Inc.	P.H. 234
Buprenorphine HCL	Hospira	409201232
Forceps, Adson-Brown	Fine Science Tools	11627-12	12.5 cm
Forceps, Angled Dumont	Fine Science Tools	11253-25	Medical #5/45 11 cm
Forceps, Suture Tying	Fine Science Tools	18025-10	10 cm
Heparin Sodium Injection, USB	Fresenius Kabi	504015	10,000 USP units per 10 mL
Hydrodissection Cannula	Ambler Surgical	1021E	27 G
Isoflurane	Dechra Vet. Products	17033-091-25
I.V. Catheter	Kendall	2619PUR	26 G x 3/4"
Magnetic Retraction System	Fine Science Tools	18200-50
Micro Clamps	Fine Science Tools	18055-05	6 mm
Micro Clamps	Fine Science Tools	18055-06	4 mm
Micro Clamp Applicator	Fine Science Tools	18057-14	14 cm
Micro Needle Holder	S&T	C-14	14 cm
Microscope	Zeiss	Universal S3	Dual head
Ophthalmic Ointment	Puralube	14590500
Polyimidi Tubing	Cole Parmer	95820-04	OD 0.0215", ID 0.0195", wall 0.0010"
Saline	Baxter	281324	0.9% Sodium Chloride
Surgical Spring Scissors	S&T	SDC-15	Blunt 14 cm
Surgical Spring Scissors	Fine Science Tools	15021-15	Vannas 14 cm