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.
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.
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 inflammation4,5,6,7.
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 guidelines9,10. 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 DSS13,14.
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.
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
2. Preoperative preparation
3. Establishment of the rat ileal pouch model
4. Establishment of rat ileal pouchitis model with DSS
5. Histological analysis
6. Immunohistochemical assay
7. ELISA test
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 31st 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 9th to 12th day after surgery.
Compensatory increase in ileal pouch
On the 35th 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 35th 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 31st 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 35th 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 35th 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 35th 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).
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 UC17,18,19. 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.
The authors have nothing to disclose.
None
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 |