A Rat Model of Pouchitis Following Proctocolectomy and Ileal Pouch-Anal Anastomosis Using Dextran Sulfate Sodium

Summary

This protocol describes the method for establishing a rat model of pouchitis. The ileal pouch model was created by performing ileal pouch-anal anastomosis (IPAA) surgery using microsurgical techniques. After the surgery, the rat was treated with 4% dextran sulfate sodium (DSS) for 4 days.

Abstract

Ulcerative colitis (UC) is a chronic immune-mediated disease that affects the entire colon and rectum with a relapsing and remitting course, causing lifelong morbidity. When medical treatment is ineffective, especially in cases of massive gastrointestinal bleeding, perforation, toxic megacolon, or carcinogenesis, surgery becomes the last line of defense to cure UC. Total colorectal resection and ileal pouch-anal anastomosis (IPAA) offer the best chance for long-term treatment. Pouchitis is the most common and troublesome postoperative complication. In this investigation, microsurgery is employed to create an ileal pouch model in experimental rats via IPAA surgery. Subsequently, a sustained rat model of pouchitis is established by inducing inflammation of the ileal pouch with dextran sulfate sodium (DSS). The successful establishment of rat pouchitis is validated through analysis of postoperative general status, weight, food and water intake, fecal data, as well as pouch tissue pathology, immunohistochemistry, and inflammatory factor analysis. This experimental animal model of pouchitis provides a foundation for studying the pathogenesis and treatment of the condition.

Introduction

Pouchitis is a non-specific inflammation that affects the ileal pouch and is a prevalent complication following total proctocolectomy and ileal pouch-anal anastomosis (IPAA) in individuals with ulcerative colitis (UC)^1,2,3. This condition has a relatively high occurrence rate of up to 50% and can cause various clinical manifestations, including diarrhea, abdominal pain, fecal blood loss, and fever. The exact cause of pouchitis remains elusive, although some researchers believe that a shift in the pouch flora may trigger immune activation and subsequent inflammation^4,5,6,7.

Due to the challenges associated with conducting clinical trials on pouchitis, animal models can serve as valuable tools for studying pouchitis drugs and mechanisms. There are growing concerns regarding the creation of rat ileal pouches, with reports indicating possible inflammation⁸. However, research in this field remains sparse due to the intricate nature of the manufacturing process, which lacks clear guidelines^9,10. In 1998, Lichtman was the first to establish an ileal pouch model in Lewis rats and Sprague-Dawley (SD) rats by performing total colectomy¹¹. They observed macrophage infiltration, mucosal ulceration, and an increase in anaerobic bacterial flora within the intestines of these rats, providing a solid foundation for further research on ileal pouch inflammation. This experimental model of rat pouchitis closely mimics the physical signs and underlying mechanisms observed in human pouchitis.

Commonly applied preclinical ulcerative colitis models include the DSS and TNBS models. The inducing chemical 2,4,6-trinitrobenzene sulfonic acid (TNBS) typically simulates Crohn's disease¹². The DSS model, respected for its efficacy, safety profile, and affordability, is often used as a reliable tool for UC induction due to the evident symptoms observed. Given the colonization of the pouch tissue, we successfully induced a pouchitis model using DSS^13,14.

In the present study, microsurgery was used to successfully create an ileal pouch model in experimental rats via IPAA surgery. Subsequently, a sustained rat pouchitis model was established by inducing inflammation of the ileal pouch with DSS. Accuracy during surgery is essential for successful model formation, and postoperative care is crucial as well. This model can be used to investigate the pathogenesis of pouchitis, evaluate potential therapeutic agents, and further our understanding of this complex condition. The study streamlines the ileal pouch manufacturing procedure, reducing operation duration and boosting efficiency, thereby establishing a robust foundation for fundamental research into postsurgical pouch disorders.

Protocol

All animal experiments were performed in accordance with the policies of the Tianjin Medical University General Hospital ethical committees. Male Sprague-Dawley rats aged between 9 and 12 weeks, weighing approximately 320-360 g, were used for this study. The details of the reagents and equipment used are listed in the Table of Materials.

1. Animal selection and maintenance

1. Select a healthy animal (body weight of ~220-240 g).
2. Ensure that they meet the criteria of specific pathogen-free (SPF)¹⁵ level and are adaptively reared for a minimum of 2 weeks in an environment with adequate ventilation (8 to 12 air changes per hour), a comfortable temperature (20-25 °C), appropriate humidity (40%-70%), minimal noise (below 70 decibels), and a natural light cycle (12 h of light and 12 h of darkness).
3. During this period, house them in standard clean cages at a density of three to five per cage, and change the bedding twice weekly. Provide ad libitum access to food and water, as well as standard maintenance feed for laboratory rats.

2. Preoperative preparation

1. Select a healthy rat with an approximate body weight of 320 g to 360 g. Fast the rat for 8-12 h before the operation and provide access to a solution of physiological saline and 5% glucose in a 1:1 ratio for voluntary consumption.
2. Prepare the autoclaved microsurgical instruments (preoperative soaking in alcohol for 1 h), a microscope, a rat dissecting table, 8-0 non-absorbable high molecular suture, 4-0 non-absorbable suture, sterile gauze, sterile cotton swabs, etc.

3. Establishment of the rat ileal pouch model

1. Intraperitoneally inject a 1% pentobarbital sodium solution at a dose of 6 mL/kg and incisional infiltration of local anesthesia with 0.5% ropivacaine to anesthetize the rat. Maintain the rat at a comfortable temperature during anesthesia with an electric lamp.
2. Perform total colorectal resection following the steps below:
   1. Secure the rat in dorsal recumbency on an anatomical bench following a satisfactory anesthetic protocol. Confirm the depth of anesthesia via a toe-pinch. Use shaving clippers to eliminate hair debris, and apply iodophor solution twice for surgical field sterilization.
   2. Make a midline incision, approximately 6 cm long, to dissect through the skin, white line fascia, and peritoneum, providing entry into the abdominal cavity. Use a retractor to expose the peritoneal surface and cover it with bacteriostatic saline-drenched sterile gauze.
   3. Isolate the vascular flow of the terminal jejunum, ligate its origin using an 8-0 suture thread, and subsequently sever it. Set the cecal stump, located 1-2 cm from the ileocecal valve, at rest, and then sever it.
   4. Commence right hemicolon resection, ligating the right hemicolon and middle colic vein conservatively.
   5. Further segregate the inferior mesenteric artery and isolate the inferior rectal artery. Continue isolation down the rectum until an interval equating to two centimeters from the anal verge. Obliquely resect the distal rectum at around 45 degrees to avoid postoperative stenosis.
3. Perform J pouch construction.
   1. Use a microtome scalpel to separate the transverse section of the terminal ileum from the mesentery intestine, which should measure about 6-7 cm.
   2. Fold the terminal ileum in a J-shaped shape to create a pouch for the ileum. Perform posterior wall anastomosis via an interlocking stitch. Augment the anterior wall using a modified Connell stitch and retain an appropriate intestine to match the cross-sectional size of the distal rectal stump.
   3. Reinforce the J-shaped pouch with 8-0 stitching if needed. The length of the J pouch should fall within the range of 2.5 cm to 3.5 cm.
4. Perform ileal pouch-anal anastomosis, IPAA.
   1. Confirm the absence of torsion in the mesentery and suture the ant side walls of the pouch's opening and the lateral wall of the rectal stump at intervals with an 8-0 suturing thread for traction.
   2. Ultimately, apply a full-layer continuous lock suture to both the anterior and posterior walls.
   3. Verify the absence of active bleeding and physiologically cleanse the abdominal cavity with normal saline. Sequentially close the abdominal muscle fascia and skin with 4-0 sutures.
5. Perform postoperative management.
   1. Administer ketoprofen as postoperative analgesia (40 mg/kg, subcutaneously) to all the rats after abdominal closure. Ensure that the rat is warmed using an electric lamp until restoration occurs.
   2. Restrict the animal from food consumption for a minimum period of 72 h postoperatively to reduce the risk of intestinal obstruction. Allow access to a diet of 5% glucose ad libitum to quickly replenish energy and body fluids, and adjust the feeding frequency according to defecation patterns.
   3. A week after the surgery, begin with limited nourishment and gradually increase the quantity consumed. Then, provide the rats with regular rodent feed to consume freely, while keeping a daily record of their dietary intake, water intake, and body weight.

4. Establishment of rat ileal pouchitis model with DSS

1. Perform the experimental grouping.
   1. Randomly assign 12 rats who have undergone total colorectal resection and IPAA surgery into the IPAA group (Group A, n = 6) and the pouchitis group (Group B, n = 6).
   2. Prepare a 4% DSS solution by adding 4 g of DSS to 100 mL of pure water, freshly prepared daily.
   3. Administer the IPAA group and the pouchitis group with pure water or 4% DSS on postoperative day 31 to day 35, respectively. Allow rats to freely drink and eat rat food during the four consecutive days of administration.
2. Perform the pouch sampling.
   1. On the morning of day 35, after the operation, anesthetize the rats with an intraperitoneal injection of 1% pentobarbital sodium solution, administering a dose of 6 mL/kg (following institutionally approved protocols).
   2. Sever the pouch perpendicularly from the stoma of the pouch to the junction of the input and output pouches in each group. Obtain the pouch specimen.
   3. Open the pouch along the anterior wall suture line and wash the intestinal canal with saline.
   4. Place a fragment of pouch tissue in a microcentrifuge tube and promptly refrigerate it at -80 °C. Use this part for ELISA detection of inflammatory indicators. Fix the remaining pouch section with 10% formaldehyde for histopathological scoring and immunohistochemical staining.
   5. Finally, euthanize the rat by air embolism under deep anesthesia (following institutionally approved protocols).

5. Histological analysis

1. After acquiring pouch tissue from the rat, immerse it in 4% paraformaldehyde for 24 h. Subsequently, proceed with dehydration and embedding protocols. Section the processed tissue for histological examination¹⁶.
2. Apply hematoxylin and eosin staining to identify histopathological differences across groups. Examine the samples microscopically and capture photographs for documentation.

6. Immunohistochemical assay

1. Dewax and dehydrate the tissue sections carefully. Then, conduct antigen retrieval by cooling the sections in sodium citrate solution and blocking them with blocking serum for 20 min.
2. Subsequently, expose the slices to the primary anti-occludin antibody and incubate them overnight at 4 °C. Afterward, treat them with secondary antibodies for 30 min before hematoxylin counterstaining¹⁶.
3. Once the slices have dried, observe and photograph them under a light microscope.

7. ELISA test

1. 7.1 Mince and homogenize the tissue fragments in lysis buffer using sonication to ensure thorough homogenization. Utilize the resulting supernatant for detection.
2. Allocate wells for blanks, samples, and replicates per group. Determine protein concentration by reading the absorbance at 450 nm (OD value) and conducting linear regression analysis¹⁶.

Results

General condition evaluation of ileal pouch model rats after establishment
After the operator passed the IPAA surgical learning curve, the rats tolerated the surgery well, with a surgical duration of 192.94 min ± 27.15 min, and fewer postoperative complications occurred. During the early postoperative period, rats experienced a decrease in dietary intake, but their preoperative appetite was restored within 10 days to 14 days after surgery. Early postoperative activity slightly decreased, and t...

Discussion

Ulcerative colitis (UC) is a chronic intestinal inflammation characterized by recurrent epigastric pain, diarrhea, and mucus bloody stool. It primarily affects the rectum and may involve the progressing colon to varying degrees. Surgery plays a crucial role in managing UC^17,18,19. Since Parks et al.²⁰ introduced total colectomy with an ileal pouch-anal anastomosis (IPAA) procedure in 1978 to remove altere...

Materials

Name	Company	Catalog Number	Comments
Anhydrous ethanol	Tianjin Fengchuan Chemical Reagent Technology Co., Ltd	China	Hematoxin-eosin Staining
Dextran Sulfate Sodium	Yeasen	60316ES76	Used to induce pouch inflammation
Formaldehyde solution	Tianjin Zhiyuan Reagent Company	China	Hematoxin-eosin Staining
Gauze	Jiangxi Zhonggan Medical Equipment Company	China	Used for animal microsurgery
Hematoxylin	Beijing Zhongshan Jinqiao Company	China	Hematoxin-eosin Staining
Interferon γ  Detection reagent kit	Cloud-clone	SEA049Ra	Detecting inflammatory factors
Interleukin-10 detection kit	Cloud-clone	SEA056Ra	Detecting inflammatory factors
Interleukin-17 detection kit	Cloud-clone	SEA063Ra	Detecting inflammatory factors
Interleukin-6 detection kit	Cloud-clone	SEA079Ra	Detecting inflammatory factors
Iodophor	Tangpai Medical Equipment Co., Ltd	China	Used for animal microsurgery
Microscopic manipulation instruments	Aesculap	Germany	Used for animal microsurgery
Occludin	abcam	ab216327	Immunohistochemical testing
Sewing needle	Yangzhou Fuda Medical Equipment Co., Ltd	China	Used for animal microsurgery
tumor necrosis factor α Detection reagent kit	Cloud-clone	SEA133Ra	Detecting inflammatory factors
Two person binocular surgical microscope	OPTON	Germany	Used for animal microsurgery
Xylene	Tianjin Yingda Rare and Precious Reagent Factory	China	Hematoxin-eosin Staining