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 refinements were made with the introduction of cuff anastomosis^3,4, and Schleimer et al.⁵ determined the optimal stent diameter for regulating blood flow to obtain physiological portal flow and avoid hyper- or hypo-perfusion of the graft. Other investigators developed significant alterations to the method by using the splenic⁶ or common iliac⁷ artery for graft blood supply, while some developed models that used only venous blood⁸ or only arterial blood via the hepatic artery⁹ to supply the auxiliary liver graft.

The present study hypothesized that functional competition from the native liver would not interfere with allograft rejection, so we developed a protocol based on the flow-regulated Schleimer model¹⁰ that did not include any size reduction of the native or auxiliary liver. The left side of the recipient was selected to locate the graft because it provided optimal orientation between the recipient's renal and donor liver vessels. Initially, we attempted biliary reconstruction via hepaticoduodenostomy but these trials simply confirmed Schleimer's assertion that "biliary drainage is the Achilles heel of liver transplantation"¹⁰. This prompted the development of a new technique where the bile duct is anastomosed end-to-end using a stent with the recipients' ureter, permitting the discharge of bile via the bladder. A noteworthy benefit of using a hepaticoureterostomy is that graft liver functionality can be monitored daily by observing the urine; a bile-producing liver graft colors the urine a bright yellow. Figure 1 represents the schematic overview of the HALT-PVA method.

An important advantage of heterotopic over orthotopic rat LTx relates to the absence of any anhepatic or total lower body ischemia time, which permits quicker and easier recoveries for heterotopic recipients. Additionally, LTx immunological studies utilizing orthotopic methods often rely on severe rejection or death of the recipient as an experimental endpoint, which is not the case with heterotopic transplants, where the animal remains healthy even if the allograft stops functioning due to rejection. Both of these features of the heterotopic method support principles of the international 3R's initiative (Replacement, Reduction, and Refinement)¹¹, which promotes a framework for minimizing the pain, suffering, and distress experienced by research animals and improving their welfare.

The HALT-PVA model reported here is a practical and reliable method for investigating the mechanisms of hepatic allograft rejection in preclinical studies. This useful experimental technique helps overcome the considerable surgical demands and animal stress of orthotopic LTx in rats. In the future, we intend to use this method to investigate the mechanisms of acute immune rejection while exploring novel targets and therapeutic strategies to suppress hepatic allograft rejection.

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 Use adult Lewis female rats weighing 205-235 g and Lewis …

Representative Results

Presently, 29 pairs of rats have been used to establish the HALT-PVA protocol, 17 syngeneic transplants, and 12 allogeneic transplants. The syngeneic transplanted livers survived to their designated 8 or 30-day experimental endpoint with a 70% success rate, while allogeneic transplanted livers survived to their designated 3 or 8-day endpoints with a 50% success rate. Failures include rats that died due to surgical complications and auxiliary livers that failed even when the recipient survived. <p class="jove_content"…

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

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