A Rat Graft Rejection Model of Intestinal Transplantation with Exteriorized Ileostomy for Longitudinal Prognosis Assessment

Summary

This protocol describes a rat-based intestinal transplantation model with ileostomy for time-course assessment of drug intervention on graft rejection.

Abstract

To date, organ transplantation remains the mainstream treatment for organ failure. Among various solid organs, patient prognosis following intestinal transplantation remains unsatisfactory, primarily due to graft rejection. This challenge necessitates further improvement in surgical techniques or the development of novel post-surgical pharmacological interventions. This article presents a rat model of intestinal transplantation graft rejection to serve as a platform for such investigations. A segment of the ileum isolated from the Fischer 344 (F344) strain was allografted to a Lewis (LEW) rat, followed by administration of tacrolimus at a defined dose and schedule. An exteriorized ileostomy was constructed to allow non-invasive, longitudinal monitoring of the tissue status of the allograft. A trichromatic linear regression model was trained using ileostomy images and used to construct an image-based assessment scheme capable of evaluating ileostomy status on a scale from 0 to 10. Evaluation indicated allograft health deterioration beginning in the third week after transplantation. Histological analyses of the allograft confirmed signs of chronic rejection from post-operative day (POD) 28 onward, similar to those observed in human patients, and validated the results of the non-invasive ileostomy image assessment. The intestinal allograft model and associated assessment methods presented here are expected to facilitate treatment development for intestinal transplantation.

Introduction

Organ failure is a condition in which one or more vital organs function suboptimally and cannot sustain health. Delayed medical attention is often fatal or associated with secondary complications such as infections¹. For most organs, replacement of the malfunctioning organ is the only viable option for prolonged recovery. Since the discovery of re-acquisition of stemness by somatic cells², it has been postulated that these induced pluripotent stem cells (iPSCs) may enable full organogenesis and provide a virtually unlimited source of organs for replacement. By combining knowledge of embryonic development with careful con....

Protocol

All animal procedures were approved by the Institutional Animal Care and Use Committee of the Graduate School of Medicine, The University of Osaka, under reference number 04-018-003 and were performed in accordance with the guidelines of The University of Osaka. Recipient male Lewis (LEW) rats (weighing 200-250 g, 7-9 weeks old) and donor male Fischer 344 (F344) rats (weighing 200-250 g, 10-12 weeks old) were used. Prior to surgery, inject meloxicam at 0.4 mg/kg subcutaneously, shave and disinfect the abdominal area with 70% ethanol. Details of the reagents and equipment used in this study are listed in the Table of Materials.

Results

Representative images of key surgical checkpoints are shown in Figure 1. The reperfusion success rate was nearly 100%, and the post-operative survival rate was 80.0% (20 out of 25 surgeries). When the procedures were correctly performed and tacrolimus was administered at the specified dosage and schedule, the exteriorized ileostomy typically turned whitish -- an indication of graft rejection -- beginning in the third week post-surgery (Figure 2A,B

Discussion

While animal studies are essential to the development of improved surgical and pharmacological interventions for better short- and long-term outcomes of intestinal transplantation, it has been empirically difficult to establish the technique of intestinal transplantation surgery in rats. Here, surgical procedures of heterotopic intestinal transplantation are described in detail. In this method, donor and recipient operation times were approximately 35 min and 90 min, respectively. Warm ischemia time, which directly corre.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5-0 silk braided 40cm black	alfresa pharma	62-9965-34
5-0 silk braided 40cm white	alfresa pharma	62-9964-99
8-0 PROLENE BV130-5 45cm	ETHICON	EP8730H
Alexa Fluor 594 goat anti-mouse IgG(H+L)	Invitrogen	A11005	working conc.: 2ug/ml
Alexa Fluor 647 goat anti-rabbit IgG(H+L)	Invitrogen	A21245	working conc.: 2ug/ml
Barraquer Micro Needle Holder, 8mm CVD Jaw, 6"
Bulldog vascular clamp 35mm	Natsume	C-42-S-2
Fischer 344 (F344)	Japan SLC (Shizuoka, Japan)		donor, male, 10 to 12 weeks old
Lewis (LEW) rats	Japan SLC (Shizuoka, Japan)		recipients, male, 7 to 9 weeks old
Microscissors	YDM	CC06C
Microsurgery Suture Forceps - Curved, Platforms, Round Handles: 15 cm/6 in
Ms mAb to Collagen I	abcam	AB90395	working conc.: 1000x dilution
NORMAL SALINE 500 mL	OTSUKA	3311401A8024
Plastic vascular double clamp	Microsurgery training ltd
Prograf injection 5 mg	Astellas Pharma	3999416A1028
Rb pAb to Laminin	abcam	AB11575	working conc.: 2ug/ml
Small animal anesthetizer	Muromachi kikai	MK-A100
Surgical microscope OPMI-1FR	Carl Zeiss	OPMI-1FR
SUTURE, 5/0 18 PDS II CLR MONO P, VA	ETHICON	Z493G