Name: a rat model of pouchitis following proctocolectomy and ileal pouch-anal anastomosis using dextran sulfate sodium
Uploaded: 2024-05-31T13:30:00.000+00:00
Duration: 4 min 5 s
Description: Ulcerative colitis (UC) is a chronic immune-mediated disease that affects the entire colon and rectum with a relapsing and remitting course, causing ...

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

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	<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><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&amp;nbsp;</td><td>&amp;nbsp;Yeasen&amp;nbsp;</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 &amp;gamma;&amp;nbsp; 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 &amp;alpha; 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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Research

JoVE Journal

Medicine

286 Views.  Tianjin Medical University General Hospital. Vanwege de uitdagingen die gepaard gaan met het uitvoeren van klinische onderzoeken naar pouchitis, kunnen diermodellen dienen als waardevolle hulpmiddelen voor het bestuderen van medicijnen tegen pouchitis en hun mechanismen. Dit protocol beschrijft de methode voor het opstellen van een rattenmodel van pouchitis. Dit experimentele diermodel van pouchitis biedt een basis voor het bestuderen van de pathogenese en de behandeling van de aandoening.Met eer...

Video: Auteur in de schijnwerpers: ontwikkeling van een rattenmodel voor onderzoek en behandeling van pouchitis

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 ...

Murine Colitis Modeling using Dextran Sulfate Sodium (DSS)

Colitis can occur from viral or bacterial infections, ischemic insult, or autoimmune disorders; most notably Ulcerative Colitis and the colonic variant of Crohn&rsquo;s Disease - Crohn&rsquo;s Colitis. Acute colitis may present with abdominal pain and distention, malabsorption, diarrhea, hematochezia and mucus in the stool. We are beginning to understand the complex interactions between the environment, genetics, and epithelial barrier dysfunction in Inflammatory Bowel Disease and animal models of colitis have been essential in advancing our understanding of this disease. One popular model involves supplementing the drinking water of mice with low-molecular weight Dextran Sodium Sulfate (DSS), resulting in epithelial damage and a robust inflammatory response in the colon lasting several days 1.Variations of this approach can be used to model acute injury, acute injury followed by repair, and repeated cycles of DSS interspersed with recovery modeling chronic inflammatory diseases 2. After a single four-day treatment of 3% DSS in drinking water, mice show signs of acute colitis including weight loss, bloody stools, and diarrhea. Mice are euthanized at the conclusion of the treatment course and at necropsy dissected colons are processed and can be 'Swiss rolled" 3 to allow microscopic analysis of the entire colon or infused with formalin as "sausages" to allow macroscopic analysis. Tissue is then embedded in paraffin, sectioned, and stained for histologic review.

Murine Colitis Modeling using Dextran Sulfate Sodium DSS

Dextran sulfate sodium (DSS) administered in the drinking water is an established murine inflammatory injury model of acute colitis. This protocol outlines the method for DSS treatment and the preparation of tissues.

Colitis can occur from viral or bacterial infections, ischemic insult, or autoimmune disorders; most notably Ulcerative Colitis and the colonic variant of ...

Biology

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 ...

Investigating Intestinal Inflammation in DSS-induced Model of IBD

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease (CD). Both UC and CD, when present in the colon, generate a similar symptom profile which can include diarrhea, rectal bleeding, abdominal pain, and weight loss.1 Although the pathogenesis of IBD remains unknown, it is described as a multifactorial disease that involves both genetic and environmental components.2
There are numerous and variable animal models of colonic inflammation that resemble several features of IBD. Animal models of colitis range from those arising spontaneously in susceptible strains of certain species to those requiring administration of specific concentrations of colitis-inducing chemicals, such as dextran sulphate sodium (DSS). Chemical-induced models of gut inflammation are the most commonly used and best described models of IBD. Administration of DSS in drinking water produces acute or chronic colitis depending on the administration protocol.3 Animals given DSS exhibit weight loss and signs of loose stool or diarrhea, sometimes with evidence of rectal bleeding.4,5 Here, we describe the methods by which colitis development and the resulting inflammatory response can be characterized following administration of DSS. These methods include histological analysis of hematoxylin/eosin stained colon sections, measurement of pro-inflammatory cytokines, and determination of myeloperoxidase (MPO) activity, which can be used as a surrogate marker of inflammation.6
The extent of the inflammatory response in disease state can be assessed by the presence of clinical symptoms or by alteration in histology in mucosal tissue. Colonic histological damage is assessed by using a scoring system that considers loss of crypt architecture, inflammatory cell infiltration, muscle thickening, goblet cell depletion, and crypt abscess.7 Quantitatively, levels of pro-inflammatory cytokines with acute inflammatory properties, such as interleukin (IL)-1&beta;, IL-6 and tumour necrosis factor (TNF)-&alpha;,can be determined using conventional ELISA methods. In addition, MPO activity can be measured using a colorimetric assay and used as an index of inflammation.8
In experimental colitis, disease severity is often correlated with an increase in MPO activity and higher levels of pro-inflammatory cytokines. Colitis severity and inflammation-associated damage can be assessed by examining stool consistency and bleeding, in addition to assessing the histopathological state of the intestine using hematoxylin/eosin stained colonic tissue sections. Colonic tissue fragments can be used to determine MPO activity and cytokine production. Taken together, these measures can be used to evaluate the intestinal inflammatory response in animal models of experimental colitis.

Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis. Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease.

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease ...

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a diverting enterostomy. The etiology of this disease remains ill-defined but appears to differ from that of classical inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Research aimed to decipher the pathophysiological mechanisms leading to the development of this disease has been severely hampered by the lack of an appropriate murine model. This protocol generates a murine model of DC that facilitates the study of the immune system's role and its interaction with the microbiome in the development of DC. In this model using C57BL/6 animals, distal parts of the colon are excluded from the fecal stream by creating a distal colostomy, triggering the development of mild to moderate inflammation in the excluded bowel segments and reproducing the hallmark lesions of human DC with a moderate systemic inflammatory response. In contrast to the rat model, a large number of genetically-modified murine models on the C57BL/6 background are available. The combination of these animals with our model allows the potential roles of individual cytokines, chemokines, or receptors of bioactive molecules (e.g., interleukin (IL)-17; IL-10, chemokine CXCL13, chemokine receptors CXCR5 and CCR7, and the sphingosine-1-phosphate receptor 4) to be assessed in the pathogenesis of DC. The availability of congenic mouse strains on the C57BL/6 background largely facilitates transfer experiments to establish the roles of distinct cell types involved in the etiology of DC. Finally, the model offers the opportunity to assess the influences of local interventions (e.g., modification of the local microbiome or local anti-inflammatory therapy) on mucosal immunity in affected and non-affected bowel segments and the on systemic immune homeostasis.

Murine distal colostomy provides a murine model for human diversion colitis, a predominantly lymphocytic colitis in colon segment excluded from the fecal stream.

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a ...

Systematic Scoring Analysis for Intestinal Inflammation in a Murine Dextran Sodium Sulfate-Induced Colitis Model

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. However, robust standards for objective and reproducible quantification of disease severity remain to be defined. Most colitis analysis methods rely on limited histological scoring of small segments of intestine, leading to partial or biased analyses. Here, we combine high-resolution image acquisition and longitudinal analysis of the entire colon to quantify intestinal injury and ulceration in the dextran sodium sulfate (DSS) induced model of murine colitis. This protocol allows for the generation of objective and reproducible results without extensive user training. Here, we provide comprehensive details on sample preparation and image analysis using examples of data from DSS induced colitis. This method can be easily adapted to other models of murine colitis that have significant inflammation associated with mucosal injury. We demonstrate that the fraction of inflamed/injured and eroded/ulcerated mucosa relative to the complete length of the colon closely parallels clinical findings such as weight loss amid DSS-induced disease progression. This histological protocol provides a reliable time and cost-effective aid to standardize analyses of disease activity in an unbiased way in DSS colitis experiments.

Systematic scoring of intestinal inflammation using a free computer-assisted system is a powerful tool to quantitatively compare histopathological changes in colitis models characterized by the presence of ulcers and inflammatory changes. Histological colitis score evaluation strengthens clinical observations and facilitates data interpretation.

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. ...

Immunology and Infection

Murine Model for Studying Postoperative Ileus through Intestinal Manipulation

Postoperative Ileus Murine Model

Most patients experience postoperative ileus (POI) after surgery, which is associated with increased morbidity, mortality, and hospitalization time. POI is a consequence of mechanical damage during surgery, resulting in disruption of motility in the gastrointestinal tract. The mechanisms of POI are related to aberrant neuronal sensitivity, impaired epithelial barrier function, and increased local inflammation. However, the details remain enigmatic. Therefore, experimental murine models are crucial for elucidating the pathophysiology and mechanism of POI injury and for the development of novel therapies.
Here, we introduce a murine model of POI generated via intestinal manipulation (IM) that is similar to clinical surgery; this is achieved by mechanical damage to the small intestine by massaging the abdomen 1-3 times with a cotton swab. IM delayed gastrointestinal transit 24 h after surgery, as assessed by FITC-dextran gavage and fluorescence detection of the segmental digestive tract. Moreover, tissue swelling of the submucosa and immune cell infiltration were investigated by hematoxylin and eosin staining and flow cytometry. Proper pressure of the IM and a hyperemic effect on the intestine are critical for the procedure. This murine model of POI can be utilized to study the mechanisms of intestinal damage and recovery after abdominal surgery.

We describe a murine model of postoperative ileus generated via intestinal manipulation. Gastrointestinal transit function, pathologic changes, and immune cell activation were assessed 24 h after surgery.

Most patients experience postoperative ileus (POI) after surgery, which is associated with increased morbidity, mortality, and hospitalization time. POI ...

Inflammatory Bowel Disease  V: Surgical Management

Surgical interventions for inflammatory bowel disease (IBD), which includes ulcerative colitis and Crohn&#39;s disease, are essential in managing symptoms and addressing complications. The selection of surgical procedures is contingent upon the specific conditions and complications that stem from these illnesses.
Here are some common surgical interventions for IBD:
<ol>
	<li>Strictureplasty</li>
	<li>Proctocolectomy or total colectomy with ileostomy</li>
	<li>Restorative proctocolectomy with Ileal pouch-anal anastomosis</li>
	<li>Continent ileostomy</li>
</ol>
Strictureplasty is a technique primarily used to treat strictures in the small bowel that often occur in Crohn&#39;s disease. This minimally invasive surgical procedure uses a laparoscope to widen obstructed or narrowed sections of the intestines. If the stricture is too severe or cannot be treated with strictureplasty, a small bowel resection may be performed to remove the affected segments of the intestine.
Proctocolectomy or Total Colectomy with Ileostomy:
<ol>
	<li>Proctocolectomy involves the surgical removal of both the colon and rectum and is commonly recommended for patients with ulcerative colitis when inflammation extends continuously from the rectum throughout the colon. Following this procedure, the surgeon must create an alternate pathway for waste elimination, such as an ileostomy or an ileoanal pouch.</li>
	<li>Total Colectomy involves the removal of the entire colon while preserving the rectum. In this procedure, the end of the small intestine (the ileum) is attached to the rectum, enabling waste elimination through the anus. This approach is typically used for ulcerative colitis when inflammation is confined to the colon, and the rectum remains healthy and functional.</li>
</ol>
Following either surgery, an Ileostomy is performed, creating a stoma in the ileum for the drainage of bowel contents.
Restorative Proctocolectomy with Ileal Pouch-Anal Anastomosis: The procedure is particularly beneficial for ulcerative colitis and familial adenomatous polyposis (FAP). It is less commonly performed for Crohn&#39;s disease due to the risk of disease recurrence in the pouch. It involves removing the diseased colon and rectum while preserving the anal sphincter to maintain voluntary defecation and anal continence. A key aspect of this procedure is the creation of an ileal pouch, or J pouch, from the small intestine to serve as a reservoir for intestinal contents, mimicking the function of the removed rectum. The ileal pouch is surgically connected to the anus to restore gastrointestinal continuity.
Continent Ileostomy (Kock Pouch) involves the construction of a J- or S-shaped continent ileal reservoir using 30 to 45 cm of the terminal ileum after a proctocolectomy. The design of the Kock pouch includes a nipple valve created by pulling a portion of the terminal ileal loop back into the ileum, functioning as a controlled outlet. It allows for the accumulation of gastrointestinal effluent in the pouch, which can be emptied through a catheter, offering a degree of continence and control over bowel movements without needing an external fecal collection bag. However, the Kock pouch is less commonly performed today due to advancements in other surgical techniques like the ileal pouch-anal anastomosis.
Surgical management in IBD aims to alleviate symptoms, address complications, and improve quality of life by balancing disease control with the preservation of normal bowel function. These surgeries are considered when medical treatments prove insufficient.

Surgical interventions for IBD include the following:
Strictureplasty treats small bowel strictures by widening narrowed sections while preserving ...

Education

JoVE Core

Medical-Surgical Nursing

Unit 1

Intestinal and Rectal Disorders

KEY TERMS AND CONCEPTS

A Technique to Generate a Murine Model of Dextran Sulfate Sodium-Induced Colitis

To induce dextran sulfate sodium, or DSS colitis, replace the regular drinking water with 3% DSS solution ad libitum for seven days, measuring each mouse&#39;s body weight, DSS solution consumption, and stool production daily. At the end of the seven-day treatment, place the mouse in the supine position, and use small scissors to make a three-centimeter-long midline incision in the abdomen.
Then, use forceps to lift the colon to allow it to be separated from the surrounding mesentery, and extract the whole colon until the cecum and rectum are visible. Transect the tissue at the colonocecal margin and deep within the pelvis, to free the proximal and distal colon respectively.
Measure and record the length of the isolated colon, and use a 10-milliliter syringe equipped with a gavage needle to flush the colon with 10 milliliters of ice-cold PBS to remove the feces and blood until the eluate runs clear. For histological identification, divide the tissue samples into equally-sized proximal, middle, and distal sections, and fix the tissues in 4% paraformaldehyde overnight at four degrees Celsius.

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

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