The authors would like to thank Benjamin J Weigler, D.V.M., Ph.D. and Walt Mandeville, D.V.M. (Animal Resources &#38; Campus Attending Veterinarian, University of Nevada, Reno) for their excellent animal services provided to the mice as well as their counsel on surgical procedures.

The following protocol has been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Nevada-Reno (UNR) Animal Resources and complies with all institutional ethical guidelines regarding the use of research animals.
1. Animals.
<ol>
	<li>Obtain mature (4-6 weeks old) C57BL/6 mice weighing between 20-30 g. House the colony of laboratory mice in a centralized animal facility at UNR Animal Resources.</li>
</ol>
2. Partial Obstruction Surgery
NOTE: Surgeries are performed in a room dedicated to surgical procedures. All surgical instruments are autoclaved prior to surgery. Sterile surgical gowns and gloves should be worn by all personnel in the surgical room at all times.
<ol>
	<li>Preparation for the surgical mouse
	<ol>
		<li>Check the anesthetic delivery system to ensure the supply of oxygen and isoflurane is adequate for procedure. Turn on the supply oxygen. Turn on the gas flowmeter and adjust it to 500-1000 mL/min. Place the animal in the induction chamber and seal the top.</li>
		<li>Turn on the isoflurane vaporizer to 5%, and monitor the animal until it becomes recumbent. Switch the anesthetic delivery system to the nose cone.</li>
		<li>Flush the induction chamber of any residual gas with oxygen, then turn off the induction chamber line while keeping the nose cone line open.</li>
		<li>Remove the animal from the chamber and carefully place ophthalmic ointment on the eyes of the animal.</li>
		<li>Place the nose cone on a preheated warm pad when gas continues to flow.</li>
		<li>Change the oxygen flow to 100-200 mL/min, with 2-3 % isoflurane. If the animal begins to move, gentlyrestrain the animal with the nose cone on until fully anesthetized again.</li>
		<li>Monitor the respiration and response to stimulation during the procedure and adjust the percentage of isoflurane (2-5 %) as needed. Animal anesthetization level is monitored by the lack of toe pinch reflex before surgery is performed.</li>
		<li>Inject pain medicine (buprenorphine, 1 &#181;g/g of body weight) intraperitoneally away from the incision site.</li>
		<li>Apply hair removal lotion on the abdomen using a clean cotton swab. Let the lotion sit for 3-5 min on mouse, then remove the hair using gauze and cotton swabs. Repeat this step until all hair has been removed from the abdomen of the mouse.</li>
		<li>Clean the skin with 70 % ethanol using gauze and cotton swabs. Apply swabsticks or povidone-iodine solution to clean the abdomen.</li>
	</ol>
	</li>
	<li>Partial obstruction surgery
	<ol>
		<li>Drape the surgical site using 25 x 50 cm sterile paper with a 2.5 x 2.5 cm opening in the middle for the surgery area. Secure the drape to the animal by placing sterile strips at the boundaries of the opening and skin.</li>
		<li>Make a ~3.0 cm abdominal incision longitudinally using a No. 15 blade scalpel, ensuring that only the skin is incised and avoiding cutting into the musculoperitoneal layer at this time.</li>
		<li>Using forceps and surgical scissors, carefully separate the skin from the musculoperitoneal layer without causing any incision to the musculoperitoneal layer. After the layers have been sufficiently separated (approximately 1 cm x 4 cm), identify the linea alba on the musculoperitoneal layer and cut ~2 cm along the linea alba to expose the intraperitoneal cavity using micro-forceps and scissors.</li>
		<li>Carefully locate and identify the cecum. Slowly and gently remove the cecum from the intraperitoneal cavity with micro-forceps, bringing the proximal colon and ileum with the cecum outside onto the sterile drape. Immediately moisten the intestinal tissue with 0.9 % sterile saline soaked gauze and keep exposed tissue moistened at all times while they are outside of the abdominal cavity.</li>
		<li>Locate and identify the mesentery between the ileum and proximal colon. Make an incision (~1 cm) parallel to, and just below, the ileum, in the mesentery and avoid cutting any vasculature.</li>
		<li>Take an autoclaved silicone ring (6 mm in length, 4 mm exterior diameter, 3.5 mm interior diameter). Cut longitudinally to open the tubing and open the ring with micro-forceps.</li>
		<li>Insert one end of the opened ring through the incision in the mesentery tissue. Return the ring to a completed ring shape by bringing one end into contact with another, with the ileum surrounded by the ring.</li>
		<li>Ensure that the silicone ring completely surrounds the ileum, close the ring with suture and carefully place the intestines back in the intraperitoneal cavity.</li>
	</ol>
	</li>
	<li>Surgery closure
	<ol>
		<li>Perform a simple continuous suture on the musculoperitoneal layer along the linea alba to close the musculoperitoneal wound with an absorbable suture. After the suture is complete, clean any bleeding with 0.9 % sterile saline soaked gauze.</li>
		<li>To completely close the wound, with a separate nylon suture, perform a simple continuous suture on the skin.</li>
		<li>After both sutures have been completed, clean the wound with new swabstick or povidone-iodine.</li>
		<li>Intraperitoneally inject antibiotics (gentamicin, 150 &#956;L per mouse based on 20-30 g body weight).</li>
		<li>After the completion of procedure, turn off the isoflurane vaporizer and allow the animal to breathe only flowing oxygen until it start gaining consciousness.</li>
		<li>Once the animal is awake, place the animal into a separate recovery area with thermal support until fully recovered.</li>
	</ol>
	</li>
</ol>
3. Post-operation observation.
<ol>
	<li>After the completion of surgery, move the animals to an incubator in a recovery room where temperature and humidity is regulated. Monitor the animals postoperatively every 15 min for the first hour then every 30 min for the second hour while the animals are in the incubator.</li>
	<li>As soon as the prescribed observation is complete, move the animals to their own individual cage and monitor them daily for clinical indications of pain18, and to ensure that the surgical wound is healing properly without any signs of complications (dehiscence) present.</li>
</ol>

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<li>Liu, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Voltage+dependent+potassium+channel+remodeling+in+murine+intestinal+smooth+muscle+hypertrophy+induced+by+partial+obstruction%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Voltage dependent potassium channel remodeling in murine intestinal smooth muscle hypertrophy induced by partial obstruction.</a> PLoS One. 9 (2), e86109(2014).</li>
<li>Guo, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Down-regulation+of+hydrogen+sulfide+biosynthesis+accompanies+murine+interstitial+cells+of+Cajal+dysfunction+in+partial+ileal+obstruction%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Down-regulation of hydrogen sulfide biosynthesis accompanies murine interstitial cells of Cajal dysfunction in partial ileal obstruction.</a> PLoS One. 7, e48249(2012).</li>
<li>Yang, J., Zhao, J., Chen, P., Nakaguchi, T., Grundy, D., Gregersen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interdependency+between+mechanical+parameters+and+afferent+nerve+discharge+in+hypertrophic+intestine+of+rats%5BTitle%5D)+AND+%22Am+J+Physiol-Gastr+L%22%5BJournal%5D)">Interdependency between mechanical parameters and afferent nerve discharge in hypertrophic intestine of rats.</a> Am J Physiol-Gastr L. 310, G376-G386 (2016).</li>
<li>Zhao, J., Liao, D., Yang, J., Gregersen, H. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2857539/">Biomechanical remodeling of obstructed guinea pig jejunum.</a> J Biomech. 43, 1322-1329 (2010).</li>
<li>Bowen, E. J., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/22129371">Duodenal Brunner's glade adenoma causing chronic small intestinal obstruction in a dog.</a> J Small Anim Pract. 53, 136-139 (2012).</li>
<li>Bettini, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypertrophy+of+intestinal+smooth+muscle+in+cats%5BTitle%5D)+AND+%22Res+Vet+Sci%22%5BJournal%5D)">Hypertrophy of intestinal smooth muscle in cats.</a> Res Vet Sci. 75, 43-53 (2003).</li>
<li>Macdonald, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Smooth+muscle+phenotypic+plasticity+in+mechanical+obstruction+of+the+small+intestine%5BTitle%5D)+AND+%22J+Neurogastroenterol+Motil%22%5BJournal%5D)">Smooth muscle phenotypic plasticity in mechanical obstruction of the small intestine.</a> J Neurogastroenterol Motil. 20, 737-740 (2008).</li>
<li>Ha, S. E., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/28806761">Transcriptome analysis of PDGFRα+ Cells identifies T-types Ca2+ channel CACNA1G as a new pathological marker for PDGFRα+ cell hyperplasia.</a> PLoS One. 12, e0182265(2017).</li>
<li>Park, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Serum+response+factor+is+essential+for+prenatal+gastrointestinal+smooth+muscle+development+and+maintenance+of+differentiated+phenotype%5BTitle%5D)+AND+%22J+Neurogastroenterol+Motil%22%5BJournal%5D)">Serum response factor is essential for prenatal gastrointestinal smooth muscle development and maintenance of differentiated phenotype.</a> J Neurogastroenterol Motil. 21, 589-602 (2015).</li>
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<li>Langford, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coding+of+facial+expressions+of+pain+in+the+laboratory+mouse%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Coding of facial expressions of pain in the laboratory mouse.</a> Nat Methods. 7, 447-449 (2010).</li>
<li>Terez, S. D., Notari, L., Sun, R., Zhao, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+of+smooth+muscle+responses+to+inflammation%5BTitle%5D)+AND+%22Neurogastroenterol+Motil%22%5BJournal%5D)">Mechanisms of smooth muscle responses to inflammation.</a> Neurogastroenterol Motil. 24, 802-811 (2012).</li>
<li>Chen, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Smooth+muscle+hyperplasia%2Fhypertrophy+is+the+most+prominent+histological+change+in+Crohn%27s+fibrostenosing+bowel+strictures%3A+A+semiquantitative+analysis+by+using+a+novel+histological+grading+scheme%5BTitle%5D)+AND+%22J+Crohns+Colitis%22%5BJournal%5D)">Smooth muscle hyperplasia/hypertrophy is the most prominent histological change in Crohn's fibrostenosing bowel strictures: A semiquantitative analysis by using a novel histological grading scheme.</a> J Crohns Colitis. 11, 92-104 (2017).</li>
<li>Huizinga, J. D., Chen, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interstitial+Cells+of+Cajal%3A+Update+on+Basic+and+Clinical+Science%5BTitle%5D)+AND+%22Curr+Gastroenterol+Rep%22%5BJournal%5D)">Interstitial Cells of Cajal: Update on Basic and Clinical Science.</a> Curr Gastroenterol Rep. 16, 363(2014).</li>
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</ol>

The authors have nothing to disclose.

We demonstrated that mice receiving the intestinal PO surgery consistently and reproducibly develop intestinal smooth muscle hypertrophy, which mimics human intestinal obstruction. Intestinal obstruction surgeries have been developed for different animals including mice7, rats10, guinea pigs11, dog12 and cats13. The mouse model of intestinal obstruction has time, cost, size, and phenotypic advantages over other larger animal models. The development of hypertrophy in mice or rats takes only 10-14 days10, compared to 2-4 weeks in guinea pigs, dogs and cats11,12,13. Cost for purchasing and maintaining mice is also a tremendous financial advantage when compared to other models. Additionally, mice are small and easy to handle for the PO surgery. Most importantly, mice receiving PO surgery progressively and extensively develop hypertrophy in the small intestine while other larger animals develop less extensive growth.
There are several key factors to consider when attempting to produce reliable hypertrophy through intestinal PO surgery. Several research groups have used rings of various sizes and placed a ring at various locations along the intestine to create partial obstructions7,9,10,11,12,13,14. However, a silicone ring of optimal size should be used for mice as a larger ring creates little or no blockage in the ileum where hypertrophy only partially, or never, developed (Figure 4A). Conversely, when a ring was too small, it created near complete blockage of the ileum which induced intestinal ischemia and/or damaged the mucosa causing sepsis, leading to early death within a week (Figure 4B). We used a silicone ring of a specific size (6 mm in length, 4 mm exterior diameter, 3.5 mm interior diameter) on one month old mice (Table of Materials). Different sizes of the rings should be tested and used for mice of different sizes in order to create the optimal blockage in the ileum. Another key factor is the location of the ring on the intestine. The ileum, close to ileocecal valve, is the best place to place the ring in order to robustly produce partial obstructions. Other regions, such as the jejunum or different regions of the ileum away from ileocecal valve, have had a similar ring placed on them in order to induce hypertrophy7. However, due to the serosal surface of the intestine being highly lubricated by peritoneal fluid, a ring placed on these parts of the intestine easily moves because of the contractile force of intestine pushing fecal material through. As the ring is pushed down the length of the intestine, it cuts off mesenteric arteries innervated into the myenteric region of intestine, causing hemorrhaging. When a ring is inserted at the end of ileum next to ileocecal valve, it is physically prevented from moving further by the bulky nature of the cecum. The cecum can be easily located and taken out of the abdomen in the surgery in order to locate the ileocecal valve region as the cecum connects to both the ileum and proximal colon. The ileum and colon connected at the cecum look quite similar and a ring can accidentally be placed on the proximal colon instead of the ileum. When a ring was misplaced on the proximal colon, as also seen on the jejunum, the ring was pushed downward and damaged mesenteric arteries, causing extensive hemorrhaging (Figure 4C). To avoid this misplacement, the ileum should always be properly located and identified before the ring is put into place. The ileum runs into the middle of cecum while the colon runs into one side of the cecum pouch.
This PO surgery protocol is considered as a major surgery for mice. All surgical instruments and materials should be sterilized before use, and surgery should be performed in the clean environment of a dedicated surgery room in order to minimize contamination, which can lead to infection and inflammation in mice. In addition, a pain medication should be provided to mice after surgery. We chose to use a slow release version of buprenorphine, which is efficacious up to 7-8 h22. Furthermore, after surgery, mice have difficulty chewing and swallowing solid food. For up to 5 days post-surgery, the mice should be provided with softened food (solid diet with a bit of water added to soften food) and switched to normal solid diet after 5 days.
Our intestinal PO surgery provides an in vivo hypertrophy model for intestinal obstruction in which SMC, ICC, and PDGFR&#945;+ cells are abnormally remodeled. This PO model is also ideally suited to understanding how enteric neurons are modified by PO and how the major motor activities are affected in the small and large bowel23. These cells can be dynamically remodeled in pathological conditions as well as under cultured conditions15,16. We observed that these cells in SO mice behaved a bit differently than normal mice without operation (Figure 2 and Figure 3). Most intestinal obstruction in humans occurs by abdominal adhesions developed after surgery, leading to smooth muscle hypertrophy3,4,5,21. After SO surgery, we also found that SMC, ICC, and PDGFR&#945;+ cells were slightly hypertrophic in the small intestine, compared to NO (Figure 3), suggesting that surgery itself can induce intestinal smooth muscle hypertrophy. If this is the case, SO surgery is not a complete negative control for PO surgery. NO as well as SO should be used as a comparison to PO intestine.
In summary, we have found a cost/time effective, reliable, and repeatable PO surgical protocol in mice that robustly generates intestinal obstructions. During the development of the obstruction, SMC, ICC, PDGFR&#945;+, as well as neuronal cells are dynamically remodeled within various tissue layers and locales. This in vivo obstruction model offers new insights into our knowledge of how phenotypic changes occur in obstructed intestine at the cellular level.

Intestinal obstructions are a partial or complete blockage in the small or large intestine which prevents digested food, fluids, and gas from moving through the intestines1. Due to the obstruction, the blockage induces the intestinal walls to become thickened, narrowing the lumen2. Intestinal obstruction can occur as a result of abdominal or pelvic surgeries that cause abdominal adhesion tissue formation or from GI disorders such as inflammatory bowel diseases (Crohn&#39;s disease), diverticulitis, hernias, volvulus, stricture, intussusception, constipation, fecal impaction, pseudo-obstruction, cancers and tumors3,4,5. Intestinal obstructions in these cases often lead to the hypertrophy of the tunica muscularis of the intestine6.
PO of the lumen induces intestinal distention, and increases smooth muscle layer thickness around the obstruction in response to the need to continue functional peristalsis7,8,9,10,11,12,13. Animal models of intestinal PO have been developed to study smooth muscle hypertrophy in mice7, rats10, guinea pigs11, dogs12, and cats13 that consistently develop similar hypertrophy within the intestinal muscle layers.
A mouse model of intestinal PO is the most cost effective way to generate and study intestinal obstructions in vivo. Small intestine obstructions are carried out in mice by using a silicone ring surgically placed surrounding the ileum. The PO mice showed an early increase in the number of cells (hyperplasia), and an increase in muscle layer thickness (hypertrophy) after PO surgery8,15. SMC are the primary plastic cells that are growing within smooth muscle layers in response to the hypertrophic conditions14, but other cells such as ICC and PDGFR&#945;+ cells that are closely associated with SMC, are also repopulated. We have previously reported that the PO mice develop hypertrophy in the small intestine, in which SMC are dedifferentiated into PDGFR&#945;+ cells that are highly proliferative7,15,16. Conversely, ICC are degenerated and lost within the hypertrophied smooth muscle layers during the development of intestinal obsruction7. Another major benefit of the PO model is its capacity to induce changes in the enteric nervous system and propagating neurogenic motor patterns. The major propagating neurogenic motor pattern in the mouse small bowel is the migrating motor complex (MMC), which is neurogenic and does not require ICC or electrical slow waves17. The PO model can provide clear insights into how MMCs and enteric nerves are remodeled by partial obstruction.
Here, we propose a murine protocol for intestinal PO surgery using a silicone ring. Mice receiving PO surgery reliably produce hypertrophy in the tunica muscularis of the small intestine. Within hypertrophic muscle, SMC, ICC, PDGFR&#945;+, and neuronal cells are dramatically remodeled.

<table><tbody><tr><td>Surgical drape</td><td>Medical and veterinary supplies</td><td>SMS40</td><td>40&amp;rdquo;X100 yards</td></tr><tr><td>Underpad, econ, pro plus</td><td>Medical and veterinary supplies</td><td>MSC281224</td><td>17x24&amp;rdquo;</td></tr><tr><td>Iris scissors</td><td>Braintree scientific, Inc</td><td>SC-i-130</td><td></td></tr><tr><td>Iris scissors</td><td>Vantage</td><td>V95-304</td><td></td></tr><tr><td>Dumont electronic &amp;amp; jeweler tweezers</td><td>Dumont</td><td>98-180-3</td><td></td></tr><tr><td>Braided absorbable suture</td><td>Covidien polysorb</td><td>SL-5687G</td><td>5-0, polyglactin</td></tr><tr><td>Nylon non-absorbable mono filament</td><td>AD surgical</td><td>S-N618R13</td><td>6-0, nylon</td></tr><tr><td>Surgical blade</td><td>Dynarex</td><td></td><td>No.15</td></tr><tr><td>Needle holder</td><td>Jacobson microvascular</td><td>36-1342TC</td><td>8.5 inch</td></tr><tr><td>Scalpel handle</td><td>Flinn scientific</td><td>AB1049</td><td></td></tr><tr><td>Microsurgical scissor</td><td>WPI</td><td>503305</td><td></td></tr><tr><td>Petrolatum ophthalmic ointment</td><td>Puralube VET</td><td></td><td>3.5 g</td></tr><tr><td>Fluriso (isoflurane)</td><td>Vetone</td><td>V1 502017</td><td>250 ml</td></tr><tr><td>Steri-strip reinforced skin closure</td><td>3M</td><td>R1547</td><td></td></tr><tr><td>Surgical gloves</td><td>Medline</td><td>MSG2270</td><td></td></tr><tr><td>Ear loop face mask</td><td>The safety zone</td><td>RS700</td><td></td></tr><tr><td>Avant gauze non-woven sponges</td><td>Caring</td><td>PRM25444</td><td></td></tr><tr><td>Surgical cup</td><td>Admiral&amp;nbsp; craft OYC-2</td><td>725-A42</td><td>2.5 oz</td></tr><tr><td>Swabstick</td><td>ChloraPrep</td><td>260103</td><td>2% w/v Chlorhexidine&amp;nbsp; Gluconate (CHG) and 70% v/v Isopropyl Alcohol (IPA)</td></tr><tr><td>Cotton tipped applicator</td><td>Puritan</td><td>806-WC</td><td></td></tr><tr><td>Buprenorphine</td><td>Zoo pharm</td><td>BZ8069317</td><td>1 mg/ml</td></tr><tr><td>Gentamycin sulfate</td><td>Vetone</td><td>G-6336-04</td><td>100 mg/ml</td></tr><tr><td>Fast acting gel cream remover</td><td>Veet</td><td>8111002</td><td></td></tr><tr><td>Syringe</td><td>AHS</td><td>AH01T2516</td><td>1 ml with needle</td></tr><tr><td>Silicon ring</td><td>VWR</td><td>60985-720</td><td>6 mm in length, 4 mm exterior diameter, 3.5 mm interior diameter</td></tr><tr><td>C57BL/6 mice</td><td>The Jackson Laboratory</td><td></td><td>4-6 weeks old</td></tr></tbody></table>

a mouse model of intestinal partial obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Partial obstruction (PO) was surgically induced in one month old mice by placing a silicone ring around the ileum close to the ileocecal sphincter. This ring created a partial blockage in the ileum. Sham operations (SO) were also performed without a ring on age/sex matched mice and these mice did not show any symptoms similar to those found in PO mice. Mice quickly recovered from PO surgery within a few hours. They showed no obvious behavioral changes or weakness within the first week, but after the first week, they progressively began to show signs of PO: a distended abdomen and the production of fewer and smaller fecal pellets. PO mice were sacrificed at 8 and 13 days post-PO surgery along with SO control mice. The small intestine was partially filled and distended at 8 days post-PO surgery and fully filled and distended at 13 days post-PO surgery, compared to SO control mice (Figure 1A). Fecal pellet formation in the colon of 8 and 13 days post-PO surgery mice was reduced in comparison to SO control mice (Figure 1B). Ileal tissue that was just upstream of the ring was dissected and the smooth muscle was analyzed by hematoxylin &amp; eosin (H&amp;E) staining. The smooth muscle layer was hypertrophied at 8 days post-PO surgery and much further hypertrophied at 13 days post-PO surgery (Figure 2). We also observed changes in SMC, ICC, PDGFRα+, and neuronal cells within the tunica muscularis in PO mice through the use of immunochemistry. Each cell was labeled with cell-type specific markers: MYH11 (SMC), KIT (ICC), PDGFRA (PDGFRα+ cells), and PGP9.5 (neuronal cells). SMC are found within three layers of tissue: longitudinal muscle (LM), circular muscle (CM), and the muscularis mucosae (MM) in the ileum. SMC were progressively growing at rapid rates in all three layers at 8 and 13 days post-PO surgery (Figure 3). As for ICCs, their subpopulations are located in the deep muscular plexus (DMP), myenteric region (MY), and subserosal region (SS). However, ICC-DMP, ICC-MY, and ICC-SS were degenerated within the intra/intermuscular layers (Figure 3). Similar to ICC subpopulations, subpopulations of PDGFRα+ cells (PαC) are located in the DMP, MY, and SS. In PO mice, PαC-DMP, PαC-MY, and PαC-SS were dynamically remodeled within the intermuscular layers of PO mice: they were growing at 8 days post-PO surgery and degenerated at 13 days post-PO surgery (Figure 3). Finally, myenteric plexus (MP), submucosal plexus (SP), subserosal neurons (SS), and enteric motor neurons (EMN) were significantly lost in the inter/intramuscular regions of PO mice at 8 and 13 days post-PO surgery (Figure 3).
<img alt="Intestinal tissue analysis; rodent dissection images, digestive tract study, labeled sections." class="xfigimg" src="/files/ftp_upload/57381/57381fig1v2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Intestinal partial obstruction surgically induced in a mouse model. PO was induced by PO surgery. One month old mice were anesthetized, their abdomen was opened by an incision, a silicone ring was placed surrounding the ileum, and the opening was closed by suturing. Mice that underwent surgery were allowed to recover for either 8 or 13 days. Age and sex matched SO mice were operated on in the same manner as PO mice except there was no placement of a ring. (A) Gross images of the GI tract in mice that underwent PO or SO surgery. Images are of mice at 8 and 13 days post-surgery (B) GI tract dissected from mice in A. Small intestine upstream of the ring was distended at both 8 and 13 days post-PO surgery and fecal pellets in the colon were also smaller at both 8 and 13 day post-PO surgery as compared to 13 days post-SO surgery mice due to the partially obstructive silicone ring. Scale bars are 0.5 cm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57381/57381fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Histology analysis comparing epithelial tissue over time; labeled NO, SO, PO 8D, PO 13D in micrographs." class="xfigimg" src="/files/ftp_upload/57381/57381fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. Smooth muscle layer changed in the surgical mouse model. Representative H&amp;E staining of ileal cross sections from intestinal PO, SO and no operation (NO) mice. Smooth muscle (SM) and mucosa (Mu) layers are thicker in PO mice at 8 and 13 days post-PO surgery than those in SO and NO mice. Scale bars are 50 µm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57381/57381fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Fluorescence microscopy images showing protein expression over time in mouse intestinal tissues." class="xfigimg" src="/files/ftp_upload/57381/57381fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Dynamic cellular remodeling in smooth muscle hypertrophy induced by intestinal partial obstruction. Representative confocal laser scanning images of hypertrophic ileal cross sections, from PO, SO and NO mice. Immunohistochemically staining with antibodies (red) of MYH11 (SMC), KIT (ICC), PDGFRA (PDGFRα+ cells) or PGP9.5 (NC; neuronal cells), co-stained with DAPI (blue). SMC in mucosa muscularis (MM), circular muscle (CM), and longitudinal muscle (LM) layers, as well as PDGFRα+ cells in deep muscular plexus (DMP), myenteric region (MY), and subserosal region (SS) were growing in hypertrophic ileum at 8 and 13 days post-PO surgery while ICC in DMP, MY, and SS, myenteric plexus (MP), submucosal plexus (SP), subserosal NC (SS), and enteric motor neurons (EMN) were degenerated. Scale bars are 50 µm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57381/57381fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Intestinal rings: colon and ileum positions in experiments; no hypertrophy, ischemia, hemorrhage." class="xfigimg" src="/files/ftp_upload/57381/57381fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4. Results of silicone rings that are too large, too small or misplaced on the colon. (A) No smooth muscle hypertrophy develops in the ileum if the ring is too large to cause obstruction. The ring made little to no blockage of the ileum. (B) Intestinal ischemia develops in the ileum if the ring is too small. The ring made an almost complete blockage of the ileum, which can damage the tissue and lead to early death before hypertrophy develops. (C) Smooth muscle hypertrophy developed in the colon due to a misplaced ring. The ring on the colon was moved down by lumenal content (feces), which damaged mesentery vasculature and caused massive hemorrhaging in the colon. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57381/57381fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Mouse Model of Intestinal Partial Obstruction at JoVE.com

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

a-mouse-model-of-intestinal-partial-obstruction

Nanyang Technological University

Research

JoVE Journal

Biology

21.1K Views.  University of Nevada, Reno School of Medicine. Intestinale obstakels zijn een gedeeltelijke of volledige verstopping van de darm die leiden ernstige buikpijn, misselijkheid tot kan, braken, en het voorkomen van de passage van ontlasting. Deze procedure voor het maken van intestinale gedeeltelijke obsructions in muizen is betrouwbaar in het bestuderen van de mechanismen die ten grondslag liggen aan de pathologische celgroei en dood in de darm.

Video: A Mouse Model of Intestinal Partial Obstruction

Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the complex cellular and molecular of intestinal pathology. Although the tunica mucosa is often the organ of interest in many inflammatory diseases, recent works demonstrated that the muscularis externa (ME) is also a highly immunocompetent organ that harbours a dense network of resident immunocytes.1,2 These works were performed within the standardized model of intestinal manipulation (IM) that leads to inflammation of the bowel wall, mainly limited to the ME. Clinically this inflammation leads to prolonged intestinal dysmotility, known as postoperative ileus (POI) which is a frequent and unavoidable complication after abdominal surgery.3 The inflammation is characterized by liberation of proinflammatory mediators such as IL-64 or IL-1&beta; or inhibitory neurotransmitters like nitric oxide (NO).5 Subsequently, tremendous numbers of immunocytes extravasate into the ME, dominated by polymorphonuclear neutrophils (PMN) and monocytes and finally maintain POI.2 Lasting for days, this intestinal paralysis leads to an increased risk of aspiration, bacterial translocation and infectious complications up to sepsis and multi organ failure and causes a high economic burden.6
In this manuscript we demonstrate the standardized model of IM and in vivo assessment of gastrointestinal transit (GIT) and colonic transit. Furthermore we demonstrate a method for separation of the ME from the tunica mucosa followed by immunological analysis, which is crucial to distinguish between the inflammatory responses in these both highly immunoactive bowel wall compartments. All analyses are easily transferable to any other research models, affecting gastrointestinal function.

Postoperative ileus (POI) is a complication of abdominal surgery leading to increased morbidity and a prolonged hospital stay. Because prophylactic or therapeutic strategies are lacking intensified research is necessary. Therefore we established a standardized and feasible mouse model to investigate the pathophysiology of POI and to study potential therapeutic options.

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the ...

Medicine

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 ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...

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 ...

Single-Anastomosis Duodeno-Ileal Bypass with Sleeve Gastrectomy Model in Mice

Obesity is a major health issue worldwide. As a response, bariatric surgeries have emerged to treat obesity and its related comorbidities (e.g., diabetes mellitus, dyslipidemia, non-alcoholic steatohepatitis, cardiovascular events, and cancers) through restrictive and malabsorptive mechanisms. Understanding the mechanisms by which these procedures allow such improvements often require their transposition into animals, especially in mice, because of the ease of generating genetically modified animals. Recently, the single-anastomosis duodeno-ileal bypass with sleeve gastrectomy (SADI-S) has emerged as a procedure that uses both restrictive and malabsorptive effects, which is being used as an alternative to gastric bypass in case of major obesity. Thus far, this procedure has been associated with strong metabolic improvements, which has led to a marked increase in its use in daily clinical practice. However, the mechanisms underlying these metabolic effects have been poorly studied as a result of a lack of animal models. In this article, we present a reliable and reproducible model of SADI-S in mice, with a special focus on perioperative management. The description and use of this new rodent model will be helpful for the scientific community to better understand the molecular, metabolic, and structural changes induced by the SADI-S and to better define the surgical indications for clinical practice.

Single-anastomosis duodeno-ileal bypass (SADI-S) is an emerging bariatric procedure with important metabolic effects. In this article, we present a reliable and reproducible model of SADI-S in mice.

Obesity is a major health issue worldwide. As a response, bariatric surgeries have emerged to treat obesity and its related comorbidities (e.g., diabetes ...

Rodent Model of Intestinal Ischemia-Reperfusion Injury via Occlusion of the Superior Mesenteric Artery

Intestinal ischemia-reperfusion injury (IRI) is associated with a myriad of conditions in both veterinary and human medicine. Intestinal IRI conditions, such as gastric dilatation volvulus (GDV), mesenteric torsion, and colic, are observed in animals such as dogs and horses. An initial interruption of blood flow causes tissues to become ischemic. Although necessary to salvage viable tissue, subsequent reperfusion can induce further injury. The main mechanism responsible for IRI is free radical formation upon reperfusion and reintroduction of oxygen into damaged tissue, but there are many other components involved. The resulting local and systemic effects often impart a poor prognosis. 
Intestinal IRI has been the subject of extensive research over the past 50 years. An in vivo rodent model in which the base of the superior mesenteric artery (SMA) is temporarily ligated is currently the most common method used to study intestinal IRI. Here, we describe a model of intestinal IRI utilizing isoflurane anesthesia in 21% O2 medical air that yields reproducible injury, as demonstrated by consistent histopathology of the small intestines. Tissue injury was also assessed in the colon, liver, and kidneys.

We describe how to generate a widely used surgical model of intestinal ischemia-reperfusion injury (IRI) in rodents. The procedure involves occlusion of the superior mesenteric artery followed by the restoration of blood flow. This model is useful for studies investigating occlusive causes of intestinal IRI in both veterinary and human medicine.

Intestinal ischemia-reperfusion injury (IRI) is associated with a myriad of conditions in both veterinary and human medicine. Intestinal IRI conditions, ...

Murine Ileal Resection Model: Studying Ileectomy-Induced Bile Metabolism in Murine Model

Source: Zhang, R., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/55728"> Ileectomy-induced Bile Overaccumulation in Mouse Intestine.</a> J. Vis. Exp. (2017)
This video describes the detailed protocol for intestinal resection in a murine model. This technique provides insights into ileectomy-induced bile malabsorption, overaccumulation, and toxicity in the mouse intestine.

Source: Zhang, R., et al.  Ileectomy-induced Bile Overaccumulation in Mouse Intestine. J. Vis. Exp. (2017)
This video describes the detailed protocol for ...

JoVE Encyclopedia of Experiments

Cancer Research

Colorectal Cancer

Animal model studies

Generation of Mouse Proximal Colon Loop Model: A Surgical Procedure to Generate Proximal Colon Loop and Measure Intestinal Permeability by Detection of Fluorescent Markers in Serum Following Intraluminal Injection

Source: Boerner, K. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/62093">Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model.</a> J. Vis. Exp. (2021).
In this video, we describe a surgical procedure to generate mouse proximal colon loop or pcLoop segment followed by intraluminal injection of fluorescent markers into the segment to study the intestinal epithelial permeability.

Generation of Mouse Proximal Colon Loop Model: A Surgical Procedure to Generate Proximal Colon Loop and Measure Intestinal Permeability by Detection of Fluorescent Markers in Serum Following Intralumi

Source: Boerner, K. et al. Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized ...

Rodent Models

Adult

Mouse Ileal Loop Model: A Surgical Model to Study Intestinal Permeability in the Mouse

After achieving a surgical plane of anesthesia, begin by scrubbing the fur of the abdominal midline with alcohol swabs, or a gauze sponge soaked with 70% ethanol. Do not wet a wide area of fur with alcohol to prevent hypothermia. Using scissors, perform a midline laparotomy. Make a horizontal incision in the middle of the abdomen, and expose the peritoneum, taking care to not injure intra-abdominal organs.
Place pre-cut wet cotton gauze over the exposed intra-abdominal cavity. Use wet cotton swabs to mobilize and exteriorize the caecum, and carefully place it on wet cotton gauze. Then, mobilize and gently exteriorize the ileum. Deploy at least 6 centimeters of terminal ileum on the wet cotton gauze without disrupting the mesenteric vessels and the blood supply.
Keep the exposed tissues moist at all times with warm HBSS. Identify the major artery supplying the ileum in the mesentery close to the cecum. Then, locate two ligation sites in the mesentery that are free of critical blood vessels. Firmly grab the terminal ileum with blunt tissue forceps and use fine tip forceps to fenestrate the mesentery, avoiding blood vessels.
Place silk suture across the perforation and tie a surgical knot to create the first ligation. Use the ruler to measure 4 centimeters away from the first ligature, and create the second ligature. Carefully cut next to each ligation with fine scissors to isolate the 4-centimeter ileal loop, keeping the blood supply and mesenteric membrane intact.
Gently flush the content of the ileal loop segment with warm HBSS using a flexible yellow feeding tube attached to a 10-milliliter syringe. Make sure to flush the luminal contents out of the abdominal cavity to keep the surgical site clean. Ligate the two cut ends of the flushed ileal loop with silk suture.
Use a 1-milliliter syringe with a 30-gauge needle to slowly inject 250 microliters of reagent, such as FITC-dextrans or chemokine into the intestinal lumen. The ileal loop will inflate, causing a moderate distension of the mucosa.
Close the abdominal wall using a needle holder, anatomical forceps, and 3.0 non-absorbable silk sutures with a reverse cutting needle. After drying the animal to prevent hypothermia, place it in a temperature-regulated anesthesia chamber for the incubation period.

A Mouse Model of Intestinal Partial Obstruction

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