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 there were no obvious secretions from the eyes and nose. Weight loss before and after surgery was 21.17 g ± 1.59 g. The initial postoperative weight showed a decreasing trend, with a maximum reduction of 69.58 g ± 33.19 g. Stable growth began on the 8.25 day ± 2.53 day after surgery, and by the 31^st day after surgery, the weight exceeded 9.35% ± 4.7% of the weight on the day after surgery. Rats were able to defecate within 24 h after surgery, with loose stools but no bloody stools. The formation of soft stools occurred on the 9^th to 12^th day after surgery.

Compensatory increase in ileal pouch
On the 35^th day after surgery, the abdominal cavity of the rat was opened under anesthesia, revealing severe adhesion between the ileal pouch and the pelvic cavity. After carefully separating the adhesion between the pouch and surrounding tissues, compensatory enlargement of the ileal pouch, thickening of the intestinal wall, and mild dilation of the distal small intestine were observed (Figure 1). The length of the ileal pouch was 2.89 cm ± 0.28 cm at the time of surgery, while on the 35^th day after surgery, it measured 3.86 cm ± 0.87 cm, showing a significant statistical difference of P = 0.000). The mucosal area (cm²) of the ileal pouch was also significantly increased compared to the surgical measurement (6.46 ± 0.85 vs. 17.02 ± 4.61, P = 0.000). There was no significant difference between the IPAA group and the ileal pouchitis group (P > 0.05).

General condition evaluation and fecal score of rats with ileal pouchitis
On the 31^st day after surgery, DSS was administered to the ileal pouchitis group. At the beginning of the administration, both the IPAA group and the ileal pouchitis group had equal body weight (352.00 g ± 30.03 g vs. 352.00 g ± 25.92 g, P = 1) and were generally in good condition. However, the amount of food and water consumed in the ileal pouchitis group decreased, resulting in mental fatigue, lackluster hair, secretion in the eyes and nose, decreased mobility, and no significant changes were observed in the IPAA group. On the 35^th day after surgery, the weight of the ileal pouchitis group was significantly lower than that of the IPAA group (322.83 g ± 29.24 g vs. 364.83 g ± 30.13 g, P = 0.028) (Figure 2).

The ileal pouchitis group experienced significant diarrhea on the first day after DSS administration, and on the second day after DSS administration, they had mucus, pus, and bloody stools (Figure 3). On the 35^th day after surgery, the stool scores¹¹ of the IPAA group and the ileal pouchitis group were (4.33 ± 0.82 vs. 2.17 ± 0.75, P = 0.001), respectively. The IPAA group rats had a clean and pollution-free anus, while the ileal pouchitis rats had swelling of the anus accompanied by mucus, pus, and bloody stool attachment.

Histopathological changes of ileal pouch

Macroscopic observation of ileal pouch specimens
In the IPAA group, the intestinal wall of the ileal pouch thickened, and no erosion, ulcer, or bleeding point was observed. In the ileal pouchitis group, the blood vessels in the storage bag mesentery become thicker, and the mucosa of the ileal pouch is extensively or locally inflamed, with visible erosion, ulcers, and bleeding points, some of which are accompanied by significant distal small intestinal dilation (Figure 4).

Microscopic observation of ileal pouch tissue
In the IPAA group of rats, the tip of some intestinal villi in the pouch tissue became blunt, accompanied by a small amount of neutrophil infiltration, and occasionally, a small amount of exudation was observed on the mucosal surface.

In contrast, the arrangement of intestinal villi in the ileal pouch tissue of rats in the ileal pouchitis group was extremely disordered. Some intestinal villi were missing, and extensive erosion and multiple patchy ulcers were visible without involving the muscle layer. This was accompanied by a large number of neutrophils and lymphocyte infiltration, excessive exudation, and visible crypt inflammation. The pathological score of the ileal pouch tissue in the ileal pouchitis group was significantly higher than that in the IPAA group, with a significant statistical difference observed (8.50 ± 1.76 vs. 1.33 ± 0.52, P = 0.000) (Figure 5).

Expression level of intestinal barrier functional protein occludin
Occludin, an important functional protein of the intestinal barrier, is expressed on the cell membrane of the ileal pouch tissue in both the IPAA and ileal pouchitis groups of rats¹¹. However, the expression level of occludin protein in the ileal pouchitis group was significantly lower than that in the IPAA group (0.25 ± 0.03 vs. 0.15 ± 0.02, P = 0.000) (Figure 6).

Detection of inflammatory factors in ileal pouch
The expression levels of IL-6, IL-17, TNF-α, and INF-γ in the ileal pouch tissue of rats in the ileal pouchitis group were significantly higher than those in the IPAA group (as determined by the ELISA test¹⁶). Conversely, IL-10 showed the opposite results, with statistical differences observed (P = 0.000) (Table 1).

Figure 1: The state of the ileal pouch. The arrow indicates the ileal pouch (A) at the time of surgery and (B) the compensatory enlargement of the ileal pouch on the 35^th day after surgery (during specimen collection). Please click here to view a larger version of this figure.

Figure 2: Trend of body weight changes in rats after IPAA surgery. The error bar is related to the standard deviation, n = 6 in each group. Please click here to view a larger version of this figure.

Figure 3: Photograph of rat feces. (A) IPAA group. (B) Ileal pouchitis group. Please click here to view a larger version of this figure.

Figure 4: Macroscopic observation of ileal pouch specimens. (A) IPAA group. (B) Ileal pouchitis group. Please click here to view a larger version of this figure.

Figure 5: Pathological changes of rat ileal pouch tissue. (A) IPAA group. (B) Ileal pouchitis group. Scale bars: 60 µm. Please click here to view a larger version of this figure.

Figure 6: Immunohistochemical detection of occludin protein expression in rat ileal pouch tissue. (A) IPAA group. (B) Ileal pouchitis group. Scale bars: 60 µm. Please click here to view a larger version of this figure.

Group	n	IL-6	IL-10	IL-17	TNF-α	INF-γ
IPAA group	6	2.60 ± 0.36	5.81 ± 0.66	17.48 ± 4.81	86.94 ± 24.06	4.08 ± 0.56
Pouchitis group	6	6.94 ± 1.18	2.77 ± 0.60	34.82 ± 2.41	213.00 ± 26.11	9.67 ± 1.70
P	0	0.000	0.000	0.000	0.000	0.000

Table 1: Expression level of inflammatory factors in rat ileal pouch tissue (pg/mL).

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 altered tissue and restore bowel continuity, this operation has become the international standard for surgical treatment of UC. Inflammatory disorders of the pouch are the most common complications following IPAA surgery.

Due to the challenges faced in conducting clinical trials on pouchitis, animal models have been used as complementary experiments for studying drugs and mechanisms related to pouchitis. In 1998, Lichtman et al.¹¹ developed a rat model involving the complete removal of the large intestine and reconnection of the pouch to the rectum. This model showed signs of inflammation, such as mononuclear cell infiltration, luminal exudation, mucosal ulceration, and serosal inflammation within four weeks post-surgery. The level of inflammation was correlated with an increased bacterial count in the pouch, and distinct host genetic susceptibility could be observed. In 2002, Shebani et al.²¹ successfully constructed an IPAA surgical rat model via SDD, which effectively induced pouchitis. This model confirmed the efficacy of using animals to study the pathogenesis of pouchitis and provided a robust experimental platform for basic research on pouchitis. They also identified similar patterns of abundant bacterial growth in both human and rodent reservoirs, validating its usefulness in investigating intestinal bacterial imbalance and identifying potential pathogens related to pouchitis.

With previous experience in mouse feeding and IPAA surgery modeling, compressing the learning curve down to around ten subjects is feasible. A critical step in this process is the construction and anastomosis of the J pouch. Given that the intestinal tract of mice is relatively slender, 8-0 suture was used for the sutures. Continuous locking sutures and a Connel stitch were separately employed for the sutures of the posterior wall and anterior wall of the pouch. During the pouch and anal canal anastomosis, creating an inclined surface can effectively reduce stenosis at the anastomotic site by taking the rectal stump as an example. Initially, securing two stitches on each side of the anastomotic site, then proceeding with continuous locking sutures to suture the posterior and anterior walls individually ensures the integrity of the anastomosis and shortens surgical time.

Postoperative management also plays a pivotal role in successful modeling. A 72 h fasting period postoperatively can effectively reduce the occurrence of intestinal obstruction. On the other hand, an early moderate liquid diet can ensure the nutritional and fluid requirements of the mice. After mastering the learning curve, surgeons can achieve a more efficient and consistent success rate in model establishment.

This study has built on this foundation and, through preliminary exploration and refinement, successfully established a rat ileal pouch model using microsurgery. This model was then utilized to examine the status of pouch inflammation and intestinal barrier indicators. The results demonstrated that rats exhibited notable symptoms such as bloody stool, diarrhea, and weight loss after being exposed to DSS. This led to pouch mucosa edema and erosion, inflammation as indicated by histopathological scoring, and increased levels of proinflammatory factors, including interleukin-6 (IL-6), IL-17, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (INF-γ), along with diminished levels of the anti-inflammatory factor interleukin-10 (IL-10). Furthermore, the protein expression of the gut barrier indicator occludin decreased. These findings align with previous studies¹¹ and affirm the successful establishment of the rat pouchitis model, providing a solid platform for subsequent drug and mechanism research.

Materials

List of materials used in this article

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