Name: creation of colonic anastomosis in mice
Uploaded: 2019-01-17T14:00:00
Description: Watch this Scientific Journal Video about Creation of Colonic Anastomosis in Mice at JoVE.com

The following protocol has been approved by the University of Oklahoma Health Sciences Center Institutional Animal Care and Use Committee (IACUC) and complies with all institutional ethical guidelines regarding the use of research animals. Additionally, all experiments were performed in accordance with institutional, state and federal regulations regarding experimental animal research.
NOTE: For this protocol, we used female and male congenic in-bred C57BL6/J mice at 12 to 72 weeks of age. All...

<ol>
	<li>Murrell, Z. A., Stamos, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reoperation+for+anastomotic+failure.">Reoperation for anastomotic failure.</a> Clinics in colon and rectal surgery. 19, 213-216 (2006).</li><li>Shaban, F., Carney, K., McGarry, K., Holtham, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perforated+Diverticulitis:+To+Anastomose+or+Not+to+Anastomose?+A+Systematic+Review+and+Meta-Analysis.">Perforated Diverticulitis: To Anastomose or Not to Anastomose? A Systematic Review and Meta-Analysis.</a> International journal of surgery (London, England). , (2018).</li><li>Chambers, W. M., Mortensen, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postoperative+leakage+and+abscess+formation+after+colorectal+surgery.">Postoperative leakage and abscess formation after colorectal surgery.</a> Best practice, research. Clinical gastroenterology. 18, 865-880 (2004).</li><li>Ashkanani, F., Krukowski, Z. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgery+-+Oxford+International+Edition.">Surgery - Oxford International Edition.</a> Intestinal Anastomosis. 20, 104-107 (2002).</li><li>Raptis, D., Pramateftakis, M. G., Kanellos, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Our+20-year+experience+with+experimental+colonic+anastomotic+healing.">Our 20-year experience with experimental colonic anastomotic healing.</a> Journal of medicine and life. 11, 5-14 (2018).</li><li>Pommergaard, H. C., Rosenberg, J., Schumacher-Petersen, C., Achiam, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choosing+the+best+animal+species+to+mimic+clinical+colon+anastomotic+leakage+in+humans:+a+qualitative+systematic+review.">Choosing the best animal species to mimic clinical colon anastomotic leakage in humans: a qualitative systematic review.</a> European surgical research. Europaische chirurgische Forschung. Recherches chirurgicales europeennes. 47, 173-181 (2011).</li><li>Komen, N., 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=Colorectal+anastomotic+leakage:+a+new+experimental+model.">Colorectal anastomotic leakage: a new experimental model.</a> The Journal of surgical research. 155, 7-12 (2009).</li><li>Perry, T., Borowiec, A., Dicken, B., Fedorak, R., Madsen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+ileocolic+bowel+resection+with+primary+anastomosis.">Murine ileocolic bowel resection with primary anastomosis.</a> Journal of visualized experiments : JoVE. , e52106 (2014).</li><li>Pommergaard, H. C., Achiam, M. P., Rosenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colon+anastomotic+leakage:+improving+the+mouse+model.">Colon anastomotic leakage: improving the mouse model.</a> Surgery today. 44, 933-939 (2014).</li><li>Bosmans, J. W. A. M., Jongen, A. C. H. M., Bouvy, N. D., Derikx, J. P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorectal+anastomotic+healing:+why+the+biological+processes+that+lead+to+anastomotic+leakage+should+be+revealed+prior+to+conducting+intervention+studies.">Colorectal anastomotic healing: why the biological processes that lead to anastomotic leakage should be revealed prior to conducting intervention studies.</a> BMC Gastroenterology. 15, 180 (2015).</li></ol>

There are several key steps to ensure the success of and minimize the morbidity/mortality associated with this procedure. First, ensure careful handling of intestinal tissue and take care to prevent traction injury when rotating the viscera to expose the colon. Undue tension on the bowel or mesentery can injury the bowel or vasculature and cause bowel necrosis away from the site of anastomosis. Additionally, sharp forceps or forceps with teeth should not be used to handle the bowel.
Injury to ...

A highly feared complication of surgical anastomosis of the intestine is anastomotic breakdown or leak. Anastomotic leak, which causes spillage of the intraluminal contents into the abdominal cavity, is life threatening and can quickly lead to intra-abdominal sepsis; shock rates vary from 0.3% to 5.5% following small bowel anastomoses, and rise to between 0.5% and 26% for colonic anastomoses1,2. Mortality rates following leaks can be as high as 39% due to the swift onset of sepsis and rapid progression to death following intraabdominal contamination3. Preventative strategies and treatment modalities are currently based on pathophysiology that is not fully understood.
Currently, anastomotic healing is often compared to the more widely studied cutaneous wound healing, which is proving to be a relatively poor facsimile. Healing occurs in a series of overlapping phases, starting with the initial lag phase. During days 0-4 after creation of the intestinal anastomosis, the lag phase is defined by an acute inflammatory response that clears the wound of cellular debris. Next, over days 3-4, fibroblastic proliferation and production of collagen typifies the fibrophasia phase. Finally, after day 10, a prolonged period of collagen remodeling is known as the maturation phase. It is important to note that anastomotic strength is quite low and depends on the extrinsic support of surgically placed staples or suture until collagen is deposited4. Understanding the role and timing each layer of the bowel wall plays in anastomotic wound healing and the involvement in inflammatory-mediated cell types such as macrophages, as well as elucidating surrogate markers to predict anastomotic failure or success could greatly reduce morbidity, mortality, and healthcare expenditure following colon operations5.
The murine model has been shown in previous studies to be the useful in mimicking human anastomosis6. While there are popular, proven and well-studied examples of the murine colonic anastomotic model, particularly the method described by Komen et al.7, these models favor ileocecal or ascending colocolonic anastomotic techniques8. Previous studies of human patients have demonstrated significant difference in leak rates between ileocecal, colocolonic and colorectal anastomoses, highlighting the need for further experimental models at varying anatomic locations. Additional, popularly used methods are aimed at developing a model which intentionally creates an anastomotic leak rather than elucidate the cellular mechanisms required for normal anastomotic wound healing9. Rat models have been attempted in the past but have rates of anastomotic dehiscence and/or signs of leak (i.e., abscess formation) significantly lower than humans and mice6,7. Additionally, more genetically modified experimental lines are available in mice than rats. This makes the rat model less useful for an anastomotic model. In addition, porcine and canine models have had less clinical investigation than the murine model6,7. 
We propose a safe, technically simple, and easily and quickly reproducible procedure for the creation of colonic anastomoses in a murine model that should facilitate further investigation into the underreported mechanism of anastomotic wound healing10.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>C57BL6/J mice</td>
			<td>Jackson Labs</td>
			<td>#00664&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Absorbent Underpads, 20&#34; x 24&#34;</td>
			<td>Fisher Scientific</td>
			<td>14-206-62</td>
			<td></td>
		</tr>
		<tr>
			<td>Polylined Sterile Field, 18&#34; x 24&#34;</td>
			<td>Busse Hospital Disposables</td>
			<td>696</td>
			<td>Cut a rectangular hole&#160;</td>
		</tr>
		<tr>
			<td>Isothesia isoflurane</td>
			<td>Henry Schein&#160;</td>
			<td>50033</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Sterile cotton gauze pad, 4&#34; x 4&#34;</td>
			<td>Fisher Scientific</td>
			<td>22-415-469</td>
			<td></td>
		</tr>
		<tr>
			<td>Puralube petrolatum ophthalmic ointment, 1/8 oz. tube</td>
			<td>Dechra Veterinary Products</td>
			<td>NDC 17033-211-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Nair depilatory cream</td>
			<td>Church &#38; Dwight Co.</td>
			<td>22339-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenex buprenorphine&#160; 0.3 mg/ml</td>
			<td>Reckitt Benckiser Pharma Inc</td>
			<td>NDC 12496-0757-5</td>
			<td></td>
		</tr>
		<tr>
			<td>1 cc insulin syringe, 27&#160;G</td>
			<td>Becton Dickinson</td>
			<td>329412</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloraprep Shampoo</td>
			<td>Medline</td>
			<td>APL82287</td>
			<td></td>
		</tr>
		<tr>
			<td>Webcol alcohol prep swabs</td>
			<td>Covidien</td>
			<td>6818</td>
			<td></td>
		</tr>
		<tr>
			<td>BioGel PI surgical gloves</td>
			<td>M&#246;lnlycke Health Care</td>
			<td>ALA42675Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Forceps with teeth</td>
			<td>Roboz</td>
			<td>RS-5150</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine scissors- sharp</td>
			<td>Fine Science Tools</td>
			<td>14060-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight serrated forceps</td>
			<td>Fine Science Tools</td>
			<td>11050-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Castro-Viejo needle driver</td>
			<td>Fine Science Tools</td>
			<td>12565-14</td>
			<td></td>
		</tr>
		<tr>
			<td>0.9% Sodium Chloride Irrigation</td>
			<td>Baxter</td>
			<td>BHL2F7121</td>
			<td>Warm to 37 &#176;C prior to use</td>
		</tr>
		<tr>
			<td>10 ml syringe</td>
			<td>Becton Dickinson</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 Vicryl absorbable braided suture, 18&#34;, PS-2 needle</td>
			<td>Henry Schein&#160;</td>
			<td>6546037</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue monofilament suture 24&#8221; BV-1 needle</td>
			<td>Henry Schein&#160;</td>
			<td>8305H</td>
			<td>Usually comes double-armed. Cut the suture at the midway point to generate two usable sutures.</td>
		</tr>
		<tr>
			<td>Cole-Parmer Microscissors, Standard Grade, Straight, 4&#34;.</td>
			<td>Cole- Parmer</td>
			<td>EW-10818-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Medline Sterile lubricating jelly</td>
			<td>Medline</td>
			<td>MDS032273H</td>
			<td></td>
		</tr>
	</tbody>
</table>

creation of colonic anastomosis in mice

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

At 7 days following surgery, the anastomosis should be well healed. The anastomosis can be harvested at time points before and after seven days to better illustrate the stages of anastomotic healing. In Figure 9, the histological analysis shows a fibrotic (collagen visualized by trichrome staining) response mediated by smooth muscle alpha-actin positive myofibroblasts. However, we have found that results from histological analyses vary between mice, specifica...

Watch this Scientific Journal Video about Creation of Colonic Anastomosis in Mice at JoVE.com

Creation of Colonic Anastomosis in Mice

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

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

Research

JoVE Journal

Medicine

The C-seal: A Biofragmentable Drain Protecting the Stapled Colorectal Anastomosis from Leakage

Colorectal anastomotic leakage (AL) is a serious complication in colorectal surgery leading to high morbidity and mortality rates1. The incidence of AL ...

Creation of Reversible Cholestatic Rat Model

Cholestasis is a clinical condition commonly encountered by both surgeons and gastroenterologists. Cholestasis can cause various physiological changes and ...

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model ...

Ascending Aortic Constriction in Rats for Creation of Pressure Overload Cardiac Hypertrophy Model

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Murine Ileocolic Bowel Resection with Primary Anastomosis

Intestinal resections are frequently required for treatment of diseases involving the gastrointestinal tract, with Crohn&#8217;s disease and colon cancer ...

Creation of Abdominal Adhesions in Mice

Abdominal adhesions consist of fibrotic tissue that forms in the peritoneal space in response to an inflammatory insult, typically surgery or ...

Creation of a Rodent Model of Abdominal Aortic Aneurysm by Blocking Adventitial Vasa Vasorum Perfusion

The adventitial vasa vasorum (VV) provides oxygen and nourishment to the aortic wall. Hypoxia in the aortic wall can cause enlarged abdominal aortic ...

Application of End-to-end Anastomosis in Robotic Central Pancreatectomy

Central pancreatectomy is carried out for the treatment of benign or low-malignant potential tumors located in the pancreatic neck or proximal part of ...

Vessel-sparing Excision and Primary Anastomosis

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