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 control of signaling cues, success has been achieved in generating multiple tissues at the scale of organoids. For full-scale organ generation, although significant progress has recently been made in specific organs such as the eyes^3,4, allo-transplantation from human donors remains the mainstream approach for most vital organs.

Among internal organs, intestinal transplantation continues to show unsatisfactory prognostic outcomes and requires further surgical refinement or drug intervention⁵. Novel interventions are seldom tested directly in human subjects and are often evaluated first in animal models of intestinal transplantation. The current article details a rat model of intestinal graft rejection to serve as an assessment platform for these potential therapeutic advancements.

To mimic the subtle human leukocyte antigen (HLA) mismatch observed in humans, a sex-matched (male) ileum segment isolated from a Fischer 344 (F344) rat was transplanted into a Lewis (LEW) rat, followed by administration of suboptimal doses of the immunosuppressant tacrolimus during the acute phase to induce chronic-phase graft rejection. This donor-recipient pair shares the same major histocompatibility complex loci but differs in minor histocompatibility antigens⁶. In this method, a portion of the grafted mucosa was exteriorized as an ileostomy to enable visual time-course monitoring of transplantation prognosis and rejection status. In addition, a complementary ileostomy image-based method for quantification of the rejection state, validated by corresponding histological changes in the transplanted graft, is presented.

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.

1. Donor surgery

1. Fast the donor rat for 12 h prior to surgery.
2. Anesthetize the donor rat with inhaled isoflurane (following institutionally approved protocols). Position the rat perpendicular to the operator. Set the stereomicroscope to 5x magnification during the procedure. Use 5-0 silk for ligation.
   NOTE: The isoflurane flow rate was 1.5 L/min. Isoflurane was delivered at 2.5% during the initial abdominal incision and maintained at 1.5% during surgery. Adequate anesthesia was confirmed by the absence of toe reflex upon toe pinch. If needed, apply vet ointment to the eyes to prevent dryness after confirming anesthesia.
3. After aseptically preparing the rat, confirm a surgical plane of anesthesia, and then perform an 8 cm midline incision to open the abdominal cavity. Extend the upper incision to the xiphoid process to ensure clear visualization of the portal vein (PV).
4. Dissect the Treitz ligament and rotate the small intestine 180 degrees clockwise to reverse intestinal rotation.
5. Observe the mesenteric vascular loops and select a well-vascularized graft site, approximately 10 cm in length from the ileum (Figure 1A1).
6. Dissect the distal mesentery and ligate the superior mesenteric artery (SMA) and superior mesenteric vein (SMV) of the distal ileum. Ensure that SMA/SMV are cut away from the proximal ligation. Improper ligation may cause bleeding and compromise graft perfusion.
7. Displace the stomach and spleen cranially to clearly expose the PV.
8. Dissect and ligate the middle colonic artery/vein and right colonic artery/vein, located on the left side of the SMA/SMV.
9. Dissect the mesentery on the right side of the SMA/SMV and ligate the jejunal branch. Avoid ligating too close to the SMV to prevent kinking (Figure 1A2).
10. Gently separate the SMA base using a cotton swab and ligate the mesentery between the aorta above the SMA and the PV. This area contains the superior mesenteric lymph nodes and numerous lymphatic vessels.
11. Identify and ligate the right renal artery, located on the right side of the aorta (Figure 1A3).
12. Inject heparinized saline (200 U / 2 mL) into the penile vein.
13. Determine the final graft length and ligate excess mesenteric branches. Ligate straight arteries at both the proximal and distal ends of the graft.
14. Ligate the aorta below the SMA, then ligate the aorta above the SMA and excise the SMA base along with a circular patch of the aorta. Cut the portal vein at the hepatic hilum, ensuring an oblique cut to create a large anastomotic opening.
    1. Proceed to cut the proximal and distal vascular loops and the intestine. Place the extracted graft in cold saline (4 °C) (Figure 1A4).
15. Remove the proximal aortic ligation to induce exsanguination and euthanize the donor rat (following institutionally approved protocols).

2. Graft processing

1. Identify the superior mesenteric artery (SMA) and portal vein (PV).
2. Gently perfuse the graft with 15 mL of cold saline via the SMA (Figure 1B1). Confirm that all blood is replaced and the graft color turns pale or white.
3. Wash the lumen of the intestinal graft and remove fecal content.
4. Keep the graft immersed in fresh cold saline until transplantation.

3. Recipient surgery

1. Anesthetize the recipient rat with inhaled isoflurane (following institutionally approved protocols). Disinfect the operative field with iodine/chlorhexidine based scrub and 70% alcohol. After aseptically preparing the rat, place the rat perpendicularly to the operator.
   1. Set the stereomicroscope to 5× for general procedures and to 12.5× during vascular anastomosis. Use 5-0 silk for ligation and 8-0 synthetic polypropylene suture for vascular anastomosis. Place the rat on a heating pad to prevent hypothermia during the operation.
      NOTE: The isoflurane flow rate was 1.5 L/min. Isoflurane was delivered at 2.5% during the initial abdominal incision and reduced to 1.5% during surgery. Anesthesia was confirmed by the absence of toe reflex following a toe pinch. Apply ophthalmic ointment to the eyes to prevent dryness after confirming anesthetic plane.
2. Before surgery, inject 0.5 mg/kg tacrolimus intramuscularly. Make a midline incision (~5 cm) and open the abdominal cavity.
3. Move the intestine and colon cranially and dissect the Treitz ligament. Wrap the intestine and colon in damp gauze and place them on the rat's body.
4. Dissect the peritoneum over the inferior vena cava (IVC) and aorta (Figure 1B2). Identify and ligate the right gonadal artery and vein. Locate the right ureter.
5. Rotate the rat with the anesthetic nose cone 90° counterclockwise so the head faces the operator's left. Clamp the IVC above the right renal vein using a curved Bulldog vascular clamp.
6. Incise the IVC wall with scissors to create an oval opening. Flush with saline and place a stay suture on the caudal edge of the IVC (Figure 1B3-1). Ensure the venous anastomotic opening is large (~1 cm), and stretch the PV wall adequately at the anastomosis.
7. Place the graft on the rat's left side, positioning the PV and SMA near the IVC and aorta. Orient the distal ileum toward the head. Wrap the graft in sterile, cold, and damp gauze and apply sterile ice to maintain a low temperature.
8. Pass the stay suture needle (8-0 synthetic polypropylene suture) through the caudal edge of the PV. Use a mosquito clamp to control suture tension.
9. Suture and tie the cranial edge of the PV to the IVC (PV to IVC). Anastomose the posterior PV wall (IVC to PV) (Figure 1B3-2). Tie the stay suture at the right edge of the PV. Next, anastomose the anterior PV wall (PV to IVC).
   1. Before completing the anastomosis, clamp the graft PV and inject heparinized saline. Tie the suture and release the IVC clamp (Figure 1B3-3).
      ​NOTE: Initial bleeding may occur at the anastomosis but is typically controlled spontaneously.
10. Clamp the aorta above the iliac artery bifurcation with a curved Bulldog clamp.
11. Incise the aortic wall. Place a stay suture (8-0 synthetic polypropylene suture) on the cranial edge of the aorta and the SMA (~4-5 mm) (Figure 1B4-1). Hold the stay suture with a mosquito clamp to control tension.
12. Pass another 8-0 needle through the caudal edge of the SMA and the aorta. Tie and begin anastomosis on the rat's right side (Figure 1B4-2). Tie the stay suture.
13. Rotate the rat with the anesthetic nose cone 180° so the head faces the operator's right. Complete the SMA-to-aorta anastomosis on the left side. Before the final suture, clamp the graft PV and inject heparinized saline. Tie the suture (Figure 1B4-3).
14. Release the aortic and PV clamps for reperfusion (Figure 1B5). Initial bleeding may occur, but can be controlled with gentle pressure using a cotton swab. The graft mucosa should turn pink within minutes (Figure 1B6).
15. Rotate the rat with the anesthetic nose cone 90° so the head faces the opposite side. Make two 8 mm skin incisions on the right abdomen and exteriorize the proximal and distal ends of the ileal graft.
    1. Suture the skin and graft serosa using 5-0 polydioxanone (PDS) (Figure 1B7). Close the midline incision by suturing linea alba and skin separately with 5-0 PDS to complete the surgery.
       NOTE: Post-operative rats must be carefully monitored until full recovery of consciousness. Do not return to the cage until sternal recumbency is achieved.

4. Post-operative management

1. To maintain the graft during the acute phase, inject tacrolimus at 0.5 mg/kg intramuscularly on post-operative days (POD) 1-13, 20, and 27⁷.
   NOTE: Administer consecutive injections on alternating legs. Alternatively, tacrolimus may be given subcutaneously.
2. Inject meloxicam at 0.4 mg/kg subcutaneously on POD 1-3.
   NOTE: Recipient rats typically lose weight until POD 7, with recovery by POD 14. Subcutaneous saline injections may be administered to mitigate dehydration due to fluid loss from the ileostomy. Unexpected mortality occurs in approximately 20% of cases before POD 7. Survival and general health are typically stable thereafter.

5. Image assessment of the health state of the intestinal graft

1. Open an image of the ileostomy in ImageJ. Convert the image to an RGB stack by selecting Image > Type > RGB Stack.
   NOTE: If the image is not already in RGB format, first convert it to RGB Color using Image > Type > RGB Color.
2. Select a region of interest (ROI) that includes only the visible mucosal area of the ileostomy.
3. Measure the intensity of each channel (Red, Green, and Blue) within the selected ROI.
4. Calculate the health score of the graft using the following formula:
   Score =
   NOTE: Normalize the final score such that: If Score > 10, assign a value of 10; If Score < 0, assign a value of 0.

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