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
<ol>
	<li>Select a healthy animal (body weight of ~220-240 g).</li>
	<li>Ensure that t...

Experimental Rat Model of Pouchitis for Studying Ulcerative Colitis Pathogenesis and Treatment

<ol>
	<li>Yang, M. L., Brar, M. S., Kennedy, E. D., De Buck Van Overstraeten, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laparoscopic+versus+transanal+IPAA+for+ulcerative+colitis:+A+patient-centered+treatment+trade-off+study.">Laparoscopic versus transanal IPAA for ulcerative colitis: A patient-centered treatment trade-off study.</a> Dis Colon Rectum. 67 (1), 107-113 (2024).</li><li>Aktas, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+and+surgical+technique+for+restorative+proctocolectomy+with+ileal+pouch-anal+anastomosis.">Current status and surgical technique for restorative proctocolectomy with ileal pouch-anal anastomosis.</a> Balkan Med J. 40 (4), 236-243 (2023).</li><li>Zhao, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiota+DNA+translocation+into+mesentery+lymph+nodes+is+associated+with+early+development+of+pouchitis+after+ipaa+for+ulcerative+colitis.">Microbiota DNA translocation into mesentery lymph nodes is associated with early development of pouchitis after ipaa for ulcerative colitis.</a> Diseases of the Colon &amp; Rectum. 66 (11), e1107-e1118 (2022).</li><li>Ng, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Worldwide+incidence+and+prevalence+of+inflammatory+bowel+disease+in+the+21st+century:+A+systematic+review+of+population-based+studies.">Worldwide incidence and prevalence of inflammatory bowel disease in the 21st century: A systematic review of population-based studies.</a> Lancet. 390 (10114), 2769-2778 (2017).</li><li>Pardi, D. S., D'haens, G., Shen, B., Campbell, S., Gionchetti, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+guidelines+for+the+management+of+pouchitis.">Clinical guidelines for the management of pouchitis.</a> Inflamm Bowel Dis. 15 (9), 1424-1431 (2009).</li><li>Shen, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+pouchitis,+Crohn's+disease,+cuffitis,+and+other+inflammatory+disorders+of+the+pouch:+Consensus+guidelines+from+the+international+ileal+pouch+consortium.">Treatment of pouchitis, Crohn's disease, cuffitis, and other inflammatory disorders of the pouch: Consensus guidelines from the international ileal pouch consortium.</a> Lancet Gastroenterol Hepatol. 7 (1), 69-95 (2022).</li><li>Dalal, R. L., Shen, B., Schwartz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+pouchitis+and+other+common+complications+of+the+pouch.">Management of pouchitis and other common complications of the pouch.</a> Inflamm Bowel Dis. 24 (5), 989-996 (2018).</li><li>Li, K. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+rat+model+of+pouchitis+after+proctocolectomy+and+ileal+pouch-anal+anastomosis+using+2,4,6-trinitrobenzene+sulfonic+acid.">A new rat model of pouchitis after proctocolectomy and ileal pouch-anal anastomosis using 2,4,6-trinitrobenzene sulfonic acid.</a> J Gastrointest Surg. 25 (6), 1524-1533 (2021).</li><li>Drzymala-Czyz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discrepancy+between+clinical+and+histological+effects+of+dha+supplementation+in+a+rat+model+of+pouchitis.">Discrepancy between clinical and histological effects of dha supplementation in a rat model of pouchitis.</a> Folia Histochem Cytobiol. 50 (1), 125-129 (2012).</li><li>Santiago, P., Barnes, E. L., Raffals, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classification+and+management+of+disorders+of+the+J+pouch.">Classification and management of disorders of the J pouch.</a> Am J Gastroenterol. 118 (11), 1931-1939 (2023).</li><li>Lichtman, S. N., Wang, J., Hummel, B., Lacey, S., Sartor, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rat+model+of+ileal+pouch-rectal+anastomosis.">A rat model of ileal pouch-rectal anastomosis.</a> Inflamm Bowel Dis. 4 (3), 187-195 (1998).</li><li>Guarner, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inulin+and+oligofructose:+Impact+on+intestinal+diseases+and+disorders.">Inulin and oligofructose: Impact on intestinal diseases and disorders.</a> Br J Nutr. 93, S61-S65 (2005).</li><li>Kim, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-tryptophan+exhibits+therapeutic+function+in+a+porcine+model+of+dextran+sodium+sulfate+(DSS)-induced+colitis.">L-tryptophan exhibits therapeutic function in a porcine model of dextran sodium sulfate (DSS)-induced colitis.</a> J Nutr Biochem. 21 (6), 468-475 (2010).</li><li>Valatas, V., Bamias, G., Kolios, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+colitis+models:+Insights+into+the+pathogenesis+of+inflammatory+bowel+disease+and+translational+issues.">Experimental colitis models: Insights into the pathogenesis of inflammatory bowel disease and translational issues.</a> Eur J Pharmacol. 759, 253-264 (2015).</li><li>Letson, H. L., Morris, J., Biros, E., Dobson, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conventional+and+specific-pathogen+free+rats+respond+differently+to+anesthesia+and+surgical+trauma.">Conventional and specific-pathogen free rats respond differently to anesthesia and surgical trauma.</a> Sci Rep. 9 (1), 9399 (2019).</li><li>Gu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saccharomyces+boulardii,+a+yeast+probiotic,+inhibits+gut+motility+through+upregulating+intestinal+serotonin+transporter+and+modulating+gut+microbiota.">Saccharomyces boulardii, a yeast probiotic, inhibits gut motility through upregulating intestinal serotonin transporter and modulating gut microbiota.</a> Pharmacol Res. 181, 106291 (2022).</li><li>Akiyama, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+phenotype+of+the+j+pouch+in+patients+with+inflammatory+bowel+disease:+A+new+classification+for+pouch+outcomes.">Endoscopic phenotype of the j pouch in patients with inflammatory bowel disease: A new classification for pouch outcomes.</a> Clin Gastroenterol Hepatol. 20 (2), 293-302 (2022).</li><li>Hata, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pouchitis+after+ileal+pouch-anal+anastomosis+in+ulcerative+colitis:+Diagnosis,+management,+risk+factors,+and+incidence.">Pouchitis after ileal pouch-anal anastomosis in ulcerative colitis: Diagnosis, management, risk factors, and incidence.</a> Dig Endosc. 29 (1), 26-34 (2017).</li><li>Gallo, G., Kotze, P. G., Spinelli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgery+in+ulcerative+colitis:+When?+How.">Surgery in ulcerative colitis: When? How.</a> Best Pract Res Clin Gastroenterol. 32-33, 71-78 (2018).</li><li>Parks, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proctocolectomy+without+ileostomy+for+ulcerative+colitis.">Proctocolectomy without ileostomy for ulcerative colitis.</a> BMJ. 2, 85-88 (1978).</li><li>Shebani, K. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pouchitis+in+a+rat+model+of+ileal+J+pouch-anal+anastomosis.">Pouchitis in a rat model of ileal J pouch-anal anastomosis.</a> Inflamm Bowel Dis. 8 (1), 23-34 (2002).</li></ol>

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...

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&#39;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anhydrous ethanol</td>
			<td>Tianjin Fengchuan Chemical Reagent Technology Co., Ltd</td>
			<td>China</td>
			<td>Hematoxin-eosin Staining</td>
		</tr>
		<tr>
			<td>Dextran Sulfate Sodium&#160;</td>
			<td>&#160;Yeasen&#160;</td>
			<td>60316ES76</td>
			<td>Used to induce pouch inflammation</td>
		</tr>
		<tr>
			<td>Formaldehyde solution</td>
			<td>Tianjin Zhiyuan Reagent Company</td>
			<td>China</td>
			<td>Hematoxin-eosin Staining</td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Jiangxi Zhonggan Medical Equipment Company</td>
			<td>China</td>
			<td>Used for animal microsurgery</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Beijing Zhongshan Jinqiao Company</td>
			<td>China</td>
			<td>Hematoxin-eosin Staining</td>
		</tr>
		<tr>
			<td>Interferon &#947;&#160; Detection reagent kit</td>
			<td>Cloud-clone</td>
			<td>SEA049Ra</td>
			<td>Detecting inflammatory factors</td>
		</tr>
		<tr>
			<td>Interleukin-10 detection kit</td>
			<td>Cloud-clone</td>
			<td>SEA056Ra</td>
			<td>Detecting inflammatory factors</td>
		</tr>
		<tr>
			<td>Interleukin-17 detection kit</td>
			<td>Cloud-clone</td>
			<td>SEA063Ra</td>
			<td>Detecting inflammatory factors</td>
		</tr>
		<tr>
			<td>Interleukin-6 detection kit</td>
			<td>Cloud-clone</td>
			<td>SEA079Ra</td>
			<td>Detecting inflammatory factors</td>
		</tr>
		<tr>
			<td>Iodophor</td>
			<td>Tangpai Medical Equipment Co., Ltd</td>
			<td>China</td>
			<td>Used for animal microsurgery</td>
		</tr>
		<tr>
			<td>Microscopic manipulation instruments</td>
			<td>Aesculap</td>
			<td>Germany</td>
			<td>Used for animal microsurgery</td>
		</tr>
		<tr>
			<td>Occludin</td>
			<td>abcam</td>
			<td>ab216327</td>
			<td>Immunohistochemical testing</td>
		</tr>
		<tr>
			<td>Sewing needle</td>
			<td>Yangzhou Fuda Medical Equipment Co., Ltd</td>
			<td>China</td>
			<td>Used for animal microsurgery</td>
		</tr>
		<tr>
			<td>tumor necrosis factor &#945; Detection reagent kit</td>
			<td>Cloud-clone</td>
			<td>SEA133Ra</td>
			<td>Detecting inflammatory factors</td>
		</tr>
		<tr>
			<td>Two person binocular surgical microscope</td>
			<td>OPTON</td>
			<td>Germany</td>
			<td>Used for animal microsurgery</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Tianjin Yingda Rare and Precious Reagent Factory</td>
			<td>China</td>
			<td>Hematoxin-eosin Staining</td>
		</tr>
	</tbody>
</table>

a rat model of pouchitis following proctocolectomy and ileal pouch-anal anastomosis using dextran sulfate sodium

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.

Author Spotlight: Developing a Rat Model for Pouchitis Research and Treatment

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

Due to the challenges associated with conducting clinical trials on pouchitis, animal models can serve as valuable tools for studying pouchitis drugs and their mechanisms. This protocol describes the method for establishing a rat model of pouchitis. This experimental animal model of pouchitis provides a foundation for studying the pathogenesis and the treatment of the condition.With previous experience in mouse feeding and IPAA surgery modeling, compressing the learning curve down to around 10 subjects is feasible. A critical step in this process is construction and anastomosis of the J-pouch. Postoperative management also play a pivotal role in successful modeling.The successful establishment of the rat pouchitis model provide a solid platform for subsequent drug and mechanism research. Our team will always be committed to studying the pathogenesis of pouchitis and the mechanism of therapeutic drugs.

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 &#177; 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...

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 ...

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Research

JoVE Journal

Medicine

Establishment of the Rat Ileal Pouch Model Using Microsurgical Techniques

To begin, position the rat on its back in dorsal recumbency on an anatomical bench. Cover the surgical site with sterile gauze soaked in bacteriostatic saline. After creating a six-centimeter midline incision, reveal the peritoneal surface using a retractor.Then, cut the terminal jejunum. Position the cecal stump one to two centimeters from the ileocecal valve and then cut it. Isolate the blood supply to the terminal jejunum and tie it off with an 8-0 suture thread.Now, begin the resection of the right hemicolon, carefully tying off the right hemicolon and middle colic vein. Next, separate the inferior mesenteric artery and isolate the inferior rectal artery. Proceed to isolate down the rectum until reaching two centimeters from the anal verge.Then, cut the distal rectum at an angle of approximately 45 degrees to prevent postoperative narrowing. Then, fold the terminal ileum into a J shape to create a pouch for the ileum. Using an interlocking stitch, perform posterior wall anastomosis and enhance the anterior wall with a modified Connell stitch.Ensure there is no torsion in the mesentery. At intervals, suture the anterior side walls of the pouch's opening and the lateral wall of the rectal stump using an 8-0 suturing thread for traction. Then, apply a full layer continuous lock suture to both the anterior and posterior walls.Sequentially close the abdominal muscle fascia and skin using 4-0 sutures. To treat the rat ileal pouchitis model with DSS, dissolve four grams of DSS in 100 milliliters of pure water, freshly prepared daily. Administer pure water to the IPAA group and 4%DSS to the pouchitis group from postoperative day 31 to day 35.Initially, both the IPAA and ileal pouchitis groups had equal body weights and were generally in good condition. By the 35th day after surgery, the weight of the ileal pouchitis group was significantly lower than that of the IPAA group.

167 Views.  Tianjin Medical University General Hospital.  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.

Experimental Rat Model of Pouchitis for Studying Ulcerative Colitis Pathogenesis and Treatment

Summary