Name: a mouse model of intestinal partial obstruction
Uploaded: 2018-03-05T14:30:00
Description: Watch this Scientific Journal Video about A Mouse Model of Intestinal Partial Obstruction at JoVE.com

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 Surge...

<ol>
	<li>Millat, B., Guillon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiopathology+and+principles+of+intensive+care+in+intestinal+obstructions.">Physiopathology and principles of intensive care in intestinal obstructions.</a> Rev Prat. 43, 667-672 (1993).</li><li>Tonelli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+developments+in+Crohn's+disease:+solution+of+doctrinal+mysteries+and+reinstatement+as+a+surgically+treatable+disease.+1.+The+process+is+not+a+form+of+enteritis+but+lymphedema+contaminated+by+intestinal+contents.">New developments in Crohn's disease: solution of doctrinal mysteries and reinstatement as a surgically treatable disease. 1. 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E., 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=Transcriptome+analysis+of+PDGFR&#945;++Cells+identifies+T-types+Ca2++channel+CACNA1G+as+a+new+pathological+marker+for+PDGFR&#945;++cell+hyperplasia.">Transcriptome analysis of PDGFR&#945;+ Cells identifies T-types Ca2+ channel CACNA1G as a new pathological marker for PDGFR&#945;+ cell hyperplasia.</a> PLoS One. 12, e0182265 (2017).</li><li>Park, 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=Serum+response+factor+is+essential+for+prenatal+gastrointestinal+smooth+muscle+development+and+maintenance+of+differentiated+phenotype.">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><li>Spencer, N. J., Sanders, K. M., Smith, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migrating+motor+complexes+do+not+require+electrical+slow+waves+in+the+mouse+small+intestine.">Migrating motor complexes do not require electrical slow waves in the mouse small intestine.</a> J Physiol. 553, 881-893 (2003).</li><li>Langford, D. 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=Coding+of+facial+expressions+of+pain+in+the+laboratory+mouse.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+smooth+muscle+responses+to+inflammation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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. 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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 ov...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Surgical drape</td>
			<td>Medical and veterinary supplies</td>
			<td>SMS40</td>
			<td>40&#8221;X100 yards</td>
		</tr>
		<tr>
			<td>Underpad, econ, pro plus</td>
			<td>Medical and veterinary supplies</td>
			<td>MSC281224</td>
			<td>17x24&#8221;</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 &#38; 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&#160; 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&#160; 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, bu...

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 ...

The overall goal of this surgical procedure is to reliably and consistently produce intestinal obstructions in mice that facilitate the study of the molecular and phenotypic changes in gastrointestinal tissue that occur in response to the obstructions. This method can help answer key questions in the gastrointestinal field surrounding the cellular mechanisms underlying the phenotypic changes that result from intestinal obstruction. The main advantages of this technique is that it's reliable, replicable, and cost effective.The implication of this technique extend toward the therapy of intestinal obstruction as they mimic phenotypic conditions observed in human pathologies caused by bowel obstruction. Demonstrating this procedure will be Se Eun Ha, a post doc, and Lai Wei, a graduate student, both from our laboratory. After confirming a lack of response to toe pinch, apply ointment to the animal's eyes and place the mouse onto a warming pad.Remove the hair from the abdomen with depilatory cream and clean the exposed skin with 70%ethanol. Disinfect the surgical site with povidone-iodine solution and drape the animal with a 25 by 50 centimeter sterile paper with a 2.5 by 2.5 centimeter opening for the surgical area. Secure the drape with sterile strips at the boundaries of the opening and the skin and use a number 15 scalpel blade to make a three centimeter longitudinal skin incision.Using forceps and surgical scissors, carefully separate the skin from the musculoperitoneal layer without incising the tissue and locate the linea alba. Then use micro forceps and scissors to cut two centimeters along the linea alba to expose the intraperitoneal cavity and carefully locate the cecum. Use the micro forceps to slowly and gently move the cecum, proximal colon, and ileum from within the intraperitoneal cavity onto the sterile drape and immediately moisten the intestinal tissue with 0.9%sterile saline soaked gauze.Locate the mesentery between the ileum and proximal colon and make a one centimeter incision in the mesentery parallel to and just below the ileum taking care to avoid cutting any vasculature. Cut longitudinally to open the mesenteric vessel and use micro forceps to open an appropriately sized autoclaved sterilized silicone ring. Insert one end of the ring through the incision and bring the end of the ring into contact with the first end to return the instrument to its original shape.Confirm that the ring is completely surrounding the ileum and close the ring with a suture. The incisions in the mesentery should be large enough for the ring to fit, but small enough that no blood vessels are incised. Also make sure the ring is placed on the ileum, not on the colon.Carefully return the intestines to their original anatomical location and use an absorbable suture in a simple continuous stitch to close the musculoperitoneal layer along the linea alba. Clean any bleeding with 0.9%sterile saline soaked gauze and use a new nylon suture and a simple continuous stitch to close the skin. Then disinfect the wound site with povidone-iodine and inject antibiotics before placing the animal in a warming gate with monitoring until full recumbency.In this experiment, the small intestine was partially filled and distended at eight days post partial obstruction surgery and fully filled and distended at 13 days post partial obstruction surgery compared to control sham operated animals. Further, fecal pellet formation in the colon of day eight and 13 post partial obstruction surgery mice was reduced compared to that in control animals. The smooth muscle layer of the ileum just upstream of the ring was hypertrophied at eight days post partial obstruction surgery with further hypertrophy observed at day 13.The smooth muscle cells grew at progressively rapid rates in all three layers of tissue at eight and 13 days post partial obstruction surgery as assessed by immunochemical analysis while the interstitial cells of Cajal degenerated within the intra and intermuscular layers. Platelet-Derived Growth Factor Receptor alpha positive cell subsets also known as PDGFR alpha cells were dynamically remodeled within the intermuscular layers of partial obstruction mice with growth observed at eight days post surgery that had degenerated by day 13. In addition, neuronal cells were also significantly lost in the inter and intramuscular regions of partial obstruction mice at both days eight and 13 post surgery.Once mastered, this technique should be completed within 30 minutes to ensure the survival of the mouse. While attempting this procedure, it is important to treat the tissue quickly and delicately. Following this procedure, other methods like gastrointestinal trend testing can be performed to answer additional questions about how obstructions alter intestinal peristalsis.After its development, this technique paved the way for researchers in the field of gastrointestinal research to explore the consequences of intestinal obstruction in mice. After watching this video, you should have a good understanding of how to quickly and properly place a ring around the mouse ileum for the creation of intestinal partial obstruction. Don't forget that working with surgical tools and needles can be extremely hazardous so take the proper precautions.Also, be sure to wear sterile gloves and practice aseptic techniques while performing this procedure.

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Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

Transgenic mouse models are powerful for understanding the critical genes controlling osteoclast differentiation and activity, and for studying mechanisms ...

A Mouse Model of Lumbar Spine Instability

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding ...

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...

Partial Hepatectomy in Adult Zebrafish

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy ...