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 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 &#176;C), appropriate humidity (40%-70%), minimal noise (below 70 decibels), and a natural light cycle (12 h of light and 12 h of darkness).</li>
	<li>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.</li>
</ol>
2. Preoperative preparation
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Establishment of the rat ileal pouch model
<ol>
	<li>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.</li>
	<li>Perform total colorectal resection following the steps below:
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Commence right hemicolon resection, ligating the right hemicolon and middle colic vein conservatively.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Perform J pouch construction.
	<ol>
		<li>Use a microtome scalpel to separate the transverse section of the terminal ileum from the mesentery intestine, which should measure about 6-7 cm.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Perform ileal pouch-anal anastomosis, IPAA.
	<ol>
		<li>Confirm the absence of torsion in the mesentery and suture the ant side walls of the pouch&#39;s opening and the lateral wall of the rectal stump at intervals with an 8-0 suturing thread for traction.</li>
		<li>Ultimately, apply a full-layer continuous lock suture to both the anterior and posterior walls.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Perform postoperative management.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
4. Establishment of rat ileal pouchitis model with DSS
<ol>
	<li>Perform the experimental grouping.
	<ol>
		<li>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).</li>
		<li>Prepare a 4% DSS solution by adding 4 g of DSS to 100 mL of pure water, freshly prepared daily.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Perform the pouch sampling.
	<ol>
		<li>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).</li>
		<li>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.</li>
		<li>Open the pouch along the anterior wall suture line and wash the intestinal canal with saline.</li>
		<li>Place a fragment of pouch tissue in a microcentrifuge tube and promptly refrigerate it at -80 &#176;C. Use this part for ELISA detection of inflammatory indicators. Fix the remaining pouch section with 10% formaldehyde for histopathological scoring and immunohistochemical staining.</li>
		<li>Finally, euthanize the rat by air embolism under deep anesthesia (following institutionally approved protocols).</li>
	</ol>
	</li>
</ol>
5. Histological analysis
<ol>
	<li>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.</li>
	<li>Apply hematoxylin and eosin staining to identify histopathological differences across groups. Examine the samples microscopically and capture photographs for documentation.</li>
</ol>
6. Immunohistochemical assay
<ol>
	<li>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.</li>
	<li>Subsequently, expose the slices to the primary anti-occludin antibody and incubate them overnight at 4 &#176;C. Afterward, treat them with secondary antibodies for 30 min before hematoxylin counterstaining16.</li>
	<li>Once the slices have dried, observe and photograph them under a light microscope.</li>
</ol>
7. ELISA test
<ol>
	<li>7.1 Mince and homogenize the tissue fragments in lysis buffer using sonication to ensure thorough homogenization. Utilize the resulting supernatant for detection.</li>
	<li>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.</li>
</ol>

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.

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...

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

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.

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 ...

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263 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.

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

Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal cancer (CRC) over healthy individuals. This risk is proportional to the duration and extent of disease, with a cumulative incidence as high as 30% in individuals with longstanding UC with widespread colonic involvement.1 Colonic dysplasia in IBD and colitis associated cancer (CAC) are believed to develop as a result of repeated cycles of epithelial cell injury and repair while these cells are bathed in a chronic inflammatory cytokine milieu.2 While spontaneous and colitis-associated cancers share the quality of being adenocarcinomas, the sequence of underlying molecular events is believed to be different.3 This distinction argues the need for specific animal models of CAC.
Several mouse models currently exist for the study of CAC. Dextran sulfate sodium (DSS), an agent with direct toxic effects on the colonic epithelium, can be administered in drinking water to mice in multiple cycles to create a chronic inflammatory state. With sufficient duration, some of these mice will develop tumors.4 Tumor development is hastened in this model if administered in a pro-carcinogenic setting. These include mice with genetic mutations in tumorigenesis pathways (APC, p53, Msh2), as well as mice pre-treated with genotoxic agents (azoxymethane [AOM], 1,2-dimethylhydrazine [DMH]).5 
The combination of DSS with AOM as a model for colitis associated cancer has gained popularity for its reproducibility, potency, low price, and ease of use. Though they have a shared mechanism, AOM has been found to be more potent and stable in solution than DMH. While tumor development in other models generally requires several months, mice injected with AOM and subsequently treated with DSS develop adequate tumors in as little as 7-10 weeks.6, 7 Finally, AOM and DSS can be administered to mice of any genetic background (knock out, transgenic, etc.) without cross-breeding to a specific tumorigenic strain. Here, we demonstrate a protocol for inflammation-driven colonic tumorigenesis in mice utilizing a single injection of AOM followed by three seven-day cycles of DSS over a 10 week period. This model induces tumors with histological and molecular changes closely resembling those occurring in human CAC and provides a highly valuable model for the study of oncogenesis and chemoprevention in this disease.8

Modeling Colitis-Associated Cancer with Azoxymethane AOM and Dextran Sulfate Sodium DSS

We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

Reproducible Motor Deficit Following Aortic Occlusion in a Rat Model Of Spinal Cord Ischemia

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing and treating this complication using experimental models of spinal cord ischemia. The model described here demonstrates varying degrees of paraplegia that relate to the length of occlusion following thoracic aortic occlusion in a rat spinal cord ischemia model.
A 2-Fr. balloon-tipped catheter was advanced through the femoral artery into the descending thoracic aorta until the catheter tip was placed at the left subclavian artery in anesthetized male Sprague-Dawley rats. Spinal cord ischemia was induced by inflating the catheter balloon. After a set period of occlusion (9, 10, or 11 min), the balloon was deflated. Neurologic assessment was performed using the motor deficit index at 24 h after surgery, and the spinal cord was harvested for histopathological examination.
Rats that underwent 9 min of aortic occlusion showed mild and reversible motor impairment in the hind limb. Rats subjected to 10 min of aortic occlusion presented with moderate but reversible motor impairment. Rats subjected to 11 min of aortic occlusion displayed complete and persistent paralysis. The motor neurons in the spinal cord sections were more preserved in rats subjected to shorter duration of aortic occlusion.
Researchers can achieve a reproducible hind limb motor deficit following thoracic aortic occlusion using this spinal cord ischemia model.

This study demonstrates the technique to make a minimally invasive and easily reproducible model of spinal cord ischemia in rats. Various degrees of hind limb motor deficit can be produced by controlling the aortic occlusion time.

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing ...

A Rat Model of Orthotopic Liver Transplantation Using a Novel Magnetic Anastomosis Technique for Suprahepatic Vena Cava Reconstruction

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep learning curve. The introduction of the cuff technique for anastomosis of the portal vein (PV) and infrahepatic vena cava (IHVC) has significantly simplified the transplant procedure in rats. However, due to the short anterior wall of the recipients' suprahepatic vena cava (SHVC), the cuff technique is very difficult to use for the reconstruction of the SHVC. Most researchers in this field still use the hand-suture technique for SHVC reconstruction, which makes it the bottleneck step in rat orthotopic liver transplantation. The magnetic anastomosis technique (i.e., magnamosis) is a method of connecting two vessels using the attractive force between two magnets. Our recent study has shown that the magnetic anastomosis technique is superior to the hand-suture technique for SHVC reconstruction in rats. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using the novel magnetic anastomosis technique. In this model, the reconstruction of the PV and IHVC was performed by the standard cuff technique, while the reconstruction of the bile duct (BD) was performed by a stent technique. The hepatic re-arterialization was not performed. The magnetic anastomosis technique made SHVC reconstruction much easier and significantly shortened the anphepatic phase. After a reasonable learning curve, even researchers without advanced microsurgical skills can produce reliable and reproducible results using this rat model of OLT.

The reconstruction of the suprahepatic vena cava (SHVC) remains a difficult step in rat orthotopic liver transplantation. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using a novel magnetic anastomosis technique.

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Vessel-sparing Excision and Primary Anastomosis

Urethroplasty is considered to be the standard treatment for urethral strictures since it provides excellent long-term success rates. For isolated short bulbar or posterior urethral strictures, urethroplasty by excision and primary anastomosis (EPA) is recommended. As EPA only requires the excision of the narrowed segment and the surrounding spongiofibrosis, a full-thickness transection of the corpus spongiosum, as performed in the traditional transecting EPA (tEPA), is usually unnecessary. Jordan et al. introduced the idea of a vessel-sparing approach in 2007, aiming to reduce surgical trauma, especially to the dual arterial blood supply of the urethra, and, thus, potentially reducing the risk of postoperative erectile dysfunction or glans ischemia. This approach could also be beneficial for subsequent urethral interventions such as redo urethroplasty using a free graft, in which a well-vascularized graft bed is imperative. Nevertheless, these potential benefits are only assumptions as prospective studies comparing the functional outcome of both techniques with validated questionnaires are currently lacking. Moreover, vessel-sparing EPA (vsEPA) should at least be able to provide similar surgical outcomes as tEPA. The aim of this paper is to give an elaborate, step-by-step overview of how to manage patients with isolated short bulbar or posterior urethral strictures with vsEPA. The main objective of this manuscript is to outline the surgical technique and to report the representative surgical outcome. A total of 117 patients were managed according to the described protocol. The analysis was performed on the entire patient cohort and on the bulbar (n = 91) and posterior (n = 26) vsEPA group separately. Success rates were 93.4% and 88.5% for the bulbar and posterior vsEPA, respectively. To conclude, vsEPA, as outlined in the protocol, provides excellent success rates with low complication rates for isolated short bulbar and posterior urethral strictures.

Here, we present an elaborate and efficient protocol to treat isolated short bulbar or posterior urethral strictures with vessel-sparing excision and primary anastomosis.

Urethroplasty is considered to be the standard treatment for urethral strictures since it provides excellent long-term success rates. For isolated short ...

Creation of Colonic Anastomosis in Mice

Intestinal anastomoses are commonly performed in both elective and emergent operations. Even so, anastomotic leaks are a highly feared complications of colonic surgeries and can occur in up to 26% of surgical anastomoses, with mortality being up to 39% for patients with such a leak. Currently, there remains a paucity of data detailing the cellular mechanisms of anastomotic healing. Devising preventative strategies and treatment modalities for anastomotic leak could be greatly potentiated by a better understanding of appropriate anastomotic healing.
A murine model is ideal as previous studies have shown that the murine anastomosis is the most clinically similar to the human case as compared with other animal models. We offer an easily reproducible murine model of colonic anastomosis in mice that will allow for further illustration of anastomotic healing.

Anastomotic leak or breakdown after surgery is a major cause of postoperative morbidity and mortality. Our surgery for creating a colonic anastomosis is a reliable and reproducible method for studying the healing mechanisms of anastomoses.

Intestinal anastomoses are commonly performed in both elective and emergent operations. Even so, anastomotic leaks are a highly feared complications of ...

One-anastomosis Gastric Bypass (OAGB) in Rats

The goal of this protocol is to set up a preclinical model of bariatric surgery and, more specifically, OAGB in obese rats. Based on this preclinical model, longitudinal studies can be carried out to provide an improved understanding of the mechanisms underlying the outcomes seen after bariatric surgery in humans. For this purpose, rats are operated on through a laparotomy under general anesthesia with isoflurane. First, the surgeon creates a long and tubular gastric pouch: after greater curve and hiatal dissection, the nonglandular stomach is stapled and removed. Then, the remaining stomach is also stapled in order to create a gastric tube and exclude the antrum of the stomach. After that, the surgeon performs a single end-to-side gastrojejunostomy 35 cm from the duodenojejunal angle. This limb length has been chosen in order to reproduce the same ratio between the biliopancreatic limb (BPL) and common limb (CL) length as in human bariatric surgery. The operation ends by aponeurotic and cutaneous closure. The early postoperative management consists of subcutaneous hydration, an intramuscular prophylactic antibiotic injection, a parietal injection of xylocaine, the administration of painkillers, and a progressive reintroduction of diet.

One-anastomosis Gastric Bypass OAGB in Rats

This protocol is for the performance of one-anastomosis gastric bypass (OAGB) on rat. The operator performs a long and tubular-stapled gastric pouch followed by hand-sewn anastomosis. For this model, the operator reproduces the same ratio between biliopancreatic and common limb in humans; therefore, the biliopancreatic limb measures 35 cm.

The goal of this protocol is to set up a preclinical model of bariatric surgery and, more specifically, OAGB in obese rats. Based on this preclinical ...

Acupuncture in a Rat Model of Asthma

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes methods for the fabrication of the high platform, establishes a rat model of asthma and measures changes in respiratory function using a noninvasive and real-time whole-body plethysmography (WBP) system.

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes ...