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 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.
Protocol
Representative Results
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 UC17,18,19. Since Parks et al.20 introduced total colectomy with an ileal pouch-anal anastomosis (IPAA) procedure in 1978 to remove altere…
Divulgations
The authors have nothing to disclose.
Acknowledgements
None
Materials
| 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 |
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