Name: creation of colonic anastomosis in mice
Uploaded: 2019-01-17T14:00:00.000+00:00
Duration: 7 min 22 s
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 mice used for this procedure were kept at our non-barrier facility for at least one week prior to the operation to adapt to the local biome. Animals were allowed to feed up to time of operation and we used neither oral mechanical bowel preparation nor rectal flushing prior to procedure.
1) Creation of Anastomoses
<ol>
	<li>Autoclave the surgical instruments, warm the saline irrigation solution and thoroughly clean all operative surfaces with 70% ethanol.</li>
	<li>To induce anesthesia, place the mouse in an induction box that is connected to a vaporizer. Deliver 2-3% isoflurane at 1-2 L/min until mice are sedated.</li>
	<li>Place the animal on a warming device, such as a heating pad, to prevent hypothermia.</li>
	<li>Subcutaneously inject a one time dose of 0.1 mg/kg buprenorphine (3 &#181;g for a 30 g mouse) for postoperative analgesia and a one time dose of 10 mg/kg of enrofloxacin for preoperative antibiotic prophylaxis. 
	NOTE: Though pre- and postoperative antibiotics are not required for this operation, institutional guidelines and the animal care and use committee at our institution recommended their use at our facility.</li>
	<li>Place the anesthetic nose cone around the mouse&#39;s snout. Throughout the operation, carefully monitor the respiratory rate of the animal and adjust the anesthetic flow rate as needed. Maintain a respiratory rate at 30-40 breaths per minute.</li>
	<li>Use a hair clipper followed by a depilatory agent to remove the hair from the entire skin surface of the abdomen. Thoroughly remove the depilatory agent with gauze wetted with water or 70% ethanol. Thoroughly dry the animal.</li>
	<li>Prior to prepping the abdomen, tape the mouse&#39;s limbs to the heating pad or operative surface at the end of each leg to prevent the mouse from shifting during surgery. Disinfect the abdomen with chlorhexidine. Include the entire shaved area as well as the hair on the edges of the shaved area. Place a sterile drape over the mouse (Figure 1A).
	<ol>
		<li>Prior to draping, cut a small ovoid or rectangular window into the drape to expose the prepped abdomen. 
		NOTE: The window should expose the entire abdomen of the mouse but cover the limbs, tail, head and thorax. We cut a 4 x 3 cm2 window for our operations (Figure 1B).</li>
		<li>Make a small slit along the drape midline, from the bottom edge towards the ovoid or rectangular window, to allow access to the rectum during the procedure (Figure 1C).</li>
	</ol>
	</li>
	<li>Make a skin incision. Start along the midline of the lower abdomen and incise skin with scissors vertically to the xiphoid. Incise only the skin and avoid cutting the abdominal wall musculature (Figure 2A).</li>
	<li>Grasp the midline of the abdominal wall with forceps and lift away from the abdominal contents. Taking care not to injure intraabdominal structures, incise the length of the midline abdominal fascia vertically with sharp scissors. Extend the length of the skin incision (Figure 2B).</li>
	<li>When the intestines are exposed, locate the cecum and small bowel, and rotate the left-sided viscera medially to expose the descending and sigmoid colon. Coat the blunt probe tip with lubricating jelly and carefully advance the probe into the rectum to aid in the identification of the descending/sigmoid colon (Figure 3). 
	NOTE: Unlike humans, the mouse cecum is generally found in the left upper quadrant of the abdomen. Take care not to perforate the bowel wall with the probe in this step. If bowel wall is perforated, terminate the procedure and euthanize the mouse.
	<ol>
		<li>Avoid grasping the cecum with toothed or sharp forceps. Instead use atraumatic forceps, cotton tipped applicator, or gentle finger manipulation to rotate viscera.</li>
		<li>Use warmed sterile saline to ensure the exposed bowel is kept moist throughout the procedure.</li>
		<li>The colon will be on the left of midline and attached to the retroperitoneum via the mesentery. After identification, withdraw the blunt probe partially to facilitate colotomy.</li>
	</ol>
	</li>
	<li>Perform transverse colotomy of the sigmoid colon.
	<ol>
		<li>Gently grasp the sigmoid colon superior to the pelvic inlet with atraumatic forceps (Figure 4A).</li>
		<li>Using sharp micro-scissors, make a cut across the sigmoid colon perpendicular to the direction of the colon and extending across 80-90% the width of the colon. This will help prevent retraction of one end of the colon out of the surgical field, and facilitate repair (Figure 4B).</li>
		<li>If a mesenteric blood vessel is injured during the course of the visceral rotation or colotomy, gently hold pressure for up to 2 min with gauze or a cotton-tipped applicator. If bleeding persists after this time, place a figure-of-eight 8-0 monofilament suture around the point of bleeding. 
		NOTE: If the bowel shows signs of necrosis or injury after this, terminate the procedure and euthanize the mouse using a humane method approved by your institutional animal use committee.</li>
	</ol>
	</li>
	<li>Repair the sigmoid colotomy.
	<ol>
		<li>Using a Castroviejo needle driver, place 5-6 simple interrupted 8-0 nonabsorbable polypropylene stitches to repair the colostomy. Place sutures 1 mm apart from each other with 0.5 mm bites.
		<ol>
			<li>Take care to avoid rolling the edges of bowel into the repair by taking slightly more serosa than mucosa with each stitch. Leave the tails 5 mm long. Take care not to accidentally catch surrounding bowel into the suture (Figure 4C). 
			NOTE: It is helpful to begin suturing at the near the mesenteric border of the colotomy, saving the anti-mesenteric-most portion of the colotomy for last.</li>
			<li>As the repair progresses, advance the trans-anal blunt-tipped dissection probe to span the repair before the final sutures are placed. This will help prevent too narrow a repair and decrease rates of anastomotic stricture.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Using a 10 mL syringe filled with warmed saline, irrigate the abdomen several times. If the surface underneath the mouse becomes soaked, dry the mouse or move the mouse to a dry surface in order to avoid hypothermia.
	<ol>
		<li>Use a sterile gauze placed over the incision to soak up excess irrigation.</li>
		<li>At the final irrigation, fill the abdomen with sterile saline. Carefully advance a lubricated 18 G angiocatheter into the rectum and inflate with 0.5-1 cm3 of air. This will demonstrate any leaks in the anastomosis than can be repaired with another interrupted polypropylene suture using the sterile probe to span the anastomosis.</li>
		<li>Deflate the colon using the angiocatheter before removal.</li>
	</ol>
	</li>
	<li>Make sure that there is no active bleeding. If there is, apply gentle pressure directly onto the point of bleeding for 1-2 min. Place hemostatic suture as described above if bleeding continues.</li>
	<li>Gently return the medialized viscera to its normal anatomic position (Figure 5A).</li>
	<li>Close the abdominal incision (Figure 5B).
	<ol>
		<li>Using 4-0 absorbable braided sutures, place a running suture along the muscular layer of the abdominal wall starting at the top of the incision and finishing at the inferior aspect of the incision. Install a tie and cut the suture leaving 5 mm tails. 
		NOTE: Do not accidentally incorporate intestine in the abdominal wall closure.</li>
		<li>Repeat with 4-0 monofilament suture to close the skin (Figure 5C).</li>
	</ol>
	</li>
	<li>Ensure that the entire animal is warm and dry, to prevent postoperative hypothermia.</li>
	<li>Carefully monitor the animal as it recovers from anesthesia. Administer 0.1 mg/kg buprenorphine every 72 h for 2-3 days after the procedure for pain control.&#160; During the recovery period, mice should be carefully examined daily for signs of abscess or peritoneal sepsis.&#160; Post-operative observation for pain and distress should include: restriction of movement including hunching type of posture, lack of eating or drinking, not proper grooming, excessive inflammation around wound, lack of normal healing at the site of incision, or not normal nesting.&#160; Any animal demonstrating any of these behaviors should be immediately euthanized by CO2 asphyxiation for a minimum of 10 min&#160;followed by bilateral thoracotomy.</li>
	<li>Mix 0.1 mg/mL of enrofloxacin with the mouse&#39;s drinking water for 7 days following surgery to prevent intra-abdominal infection or sepsis due to fecal contamination associated with the operation.</li>
</ol>
2) Harvesting Anastomotic Tissue
<ol>
	<li>Just before excising anastomotic tissue, euthanize the mouse using a humane method approved by the institutional animal use committee. 
	NOTE: Based on institutional policy, we euthanize our animals by placing them in a carbon dioxide chamber with 100% carbon dioxide gas infusion at 2 L/min for 5 min before performing bilateral thoracotomy.</li>
	<li>Sterilize the abdomen with chlorhexidine followed by 70% ethanol.</li>
	<li>Re-incise the original midline incision with sharp scissors. Start at the lower pole of the previous incision and extend this cut superiorly to the xiphoid (Figure 6A).</li>
	<li>Using scissors, cut into the muscular layer, and after entering the peritoneal space, extend the cut to the top and bottom of the incision. Take care to avoid injuring the bowel when entering the abdomen or disrupting the anastomosis, as there may be bowel adhered to the incision or anastomotic site.</li>
	<li>Carefully perform a left medial visceral rotation exposing the repaired sigmoid colon (Figure 6B). There will likely be bowel to bowel adhesions surrounding the repair. Gently and bluntly dissect these adhesions away from the site of repair. Take great care not to disrupt the anastomosis.
	<ol>
		<li>Identify the repair site by locating the tails of the polypropylene suture used for the repair. Using scissors and starting 1 cm proximal to the repair, transversely transect through the descending colon. Repeat this step 1 cm distal to site of anastomosis.</li>
	</ol>
	</li>
	<li>Remove the sigmoidal segment by sharply dissecting the colon from the attached mesentery along the posterior border of the colon with fine scissors (Figure 7).</li>
	<li>Identify the mesenteric border of the colon specimen and use fine scissors to cut along the length of the mesenteric border in a longitudinal fashion. This will create a square or rectangular section of colon from the cylindrical specimen previously removed (Figure 8).</li>
	<li>Use a marking pen to demarcate the anti-mesenteric border of the colon prior to removal of the colon segment. After removal, make a longitudinal cut along the length of the colon opposite the marking. Fix, embed and cut sample tissue as needed for histology and/or immunostaining.</li>
</ol>

<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>

The authors declare that they have no competing financial interests.

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 the bowel separate from the site of planned anastomosis, either during visceral rotation or by perforation with the blunt probe, should result in euthanasia of the animal. In our experience, bowel necrosis or injury can lead to intra-abdominal sepsis or death, and a greatly increased inflammatory response that makes recovery of a healed anastomotic specimen prohibitively difficult.
Transection of the colon should span as much of the descending colon as possible, but care should be taken to not transect the bowel serosa in contact with the mesentery. In our experience, transection that includes the serosa-and especially the mesentery-can lead to retraction of the proximal section of the colon that makes anastomosis more difficult and can lead to further injury trying to recover the retracted segment.
Anastomosis can be repaired with 5-6 interrupted sutures. More may be needed for large mice. Sutures should be 1 mm apart. Place sutures on either side of the mesenteric border and then on alternating sides of the transection to meet in the middle at the anti-mesenteric border facilitates an easier and quicker anastomosis. Before the final suture, spanning the anastomosis with the blunt probe ensures a patent anastomosis and prevents &#34;back-walling&#34; the anastomosis, which would effectively ligate the colon.
Ensure hemostasis before abdominal closure. Despite postoperative antibiotic coverage, hemoperitoneum in the recovery period after intraabdominal contamination can lead to an unacceptably high rate of intra-abdominal infection and adhesive disease.
When first performing this procedure, several mice may be lost in the postoperative period. Our initial mortality rate approximated 30%. We have found this to be mainly due to anastomotic stricture leading to obstruction and bowel necrosis, breakdown of anastomotic repair, or bowel wall necrosis away from site of anastomosis likely due to ischemia after mesenteric injury. All of these can lead to intra-abdominal infection, sepsis and death. Once experienced in this procedure, operating surgeon should experience a mortality rate of under 10%. Evidence of subclinical anastomotic site leak, illustrated by purulence and dense adhesions at the site of anastomosis, were present in a further 10%. While this procedure can be done without the aid of magnification, its largest drawback as a reproducible experimental model is the relative technical challenge when compared to ileocecal or ascending colon anastomoses. We found surgical loupes exceedingly helpful in performing the anastomosis while minimizing trauma to the bowel wall. A surgical microscope may also be used, but we did not find that necessary.
Many techniques have been used to describe animal models for colonic anastomosis; particularly utilizing rat and rabbit models. We have found there to be a paucity of reproducible procedures of descending colon anastomosis in mouse models; we suspect this is due to the smaller size of mice increasing the technical difficulty of anastomotic procedures. However, mice have greater selection and wider availability of transgenic lines and antibodies, which to this point were unavailable to be used in a colon anastomosis model. We developed the current model to unify the mouse model with a model of colon anastomosis.
Our experience with this procedure includes both male and female mice of varying sizes and ages. At this point, we have not compared anastomotic wound healing results between ages, sizes or sexes of mice, though differences may certainly exist, and this model would allow that comparison.
We believe that this model can be easily and consistently reproduced and used to study anastomotic wound healing. Although we recommend first specimen harvests at seven days, earlier and later time points may be helpful in illustrating a timeline of anastomotic healing; demonstrating, for example, early migration of inflammatory cells and mediators into the anastomotic site and the deposition of collagen by fibroblasts in the following weeks.

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><tbody><tr><td>C57BL6/J mice</td><td>Jackson Labs</td><td>#00664&amp;nbsp;</td><td></td></tr><tr><td>Fisherbrand Absorbent Underpads, 20&quot; x 24&quot;</td><td>Fisher Scientific</td><td>14-206-62</td><td></td></tr><tr><td>Polylined Sterile Field, 18&quot; x 24&quot;</td><td>Busse Hospital Disposables</td><td>696</td><td>Cut a rectangular hole&amp;nbsp;</td></tr><tr><td>Isothesia isoflurane</td><td>Henry Schein&amp;nbsp;</td><td>50033</td><td></td></tr><tr><td>Fisherbrand Sterile cotton gauze pad, 4&quot; x 4&quot;</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 &amp;amp; Dwight Co.</td><td>22339-05</td><td></td></tr><tr><td>Buprenex buprenorphine&amp;nbsp; 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&amp;nbsp;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&amp;ouml;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 &amp;deg;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&quot;, PS-2 needle</td><td>Henry Schein&amp;nbsp;</td><td>6546037</td><td></td></tr><tr><td>Blue monofilament suture 24&amp;rdquo; BV-1 needle</td><td>Henry Schein&amp;nbsp;</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&quot;.</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, specifically the degree of re-epithelialization and concentration of myofibroblasts at the anastomotic site. We report mortality following this procedure to be less than 10%, with more than 90% of surviving mice having well-healed anastomoses at 7 days.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58742/58742fig1.jpg" /> 
Figure 1: Draping and surgical preparation. (A) A Sterile drape is placed over the mouse after the abdomen has been prepped. (B) A small oval or rectangle is cut into the drape to expose the abdomen. (C) A small slit should be made along the midline of the drape near the ovoid or rectangular window, to allow access to the rectum during the procedure. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58742/58742fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58742/58742fig2.jpg" /> 
Figure 2: Incisions. (A) A skin incision is made while taking care not to injure the peritoneum.(B) The peritoneum is elevated away from the abdominal contents and incised exposing the intra-abdominal contents.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58742/58742fig3.jpg" /> 
Figure 3: Probe placement. A blunt probe is gently placed in the rectum. After locating the cecum, the contents of the abdomen are rotated medially visualizing the rectal probe previously placed.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58742/58742fig4.jpg" /> 
Figure 4: Colotomy and repair. (A) The sigmoid is grasped with atraumatic forceps. (B) Using sharp micro-scissors, make a cut across the sigmoid colon perpendicular to the direction of the colon and extending across 80-90% the width of the colon. (C) Using a Castro-Viejo needle driver, place 5-6 simple interrupted 8-0 polypropylene stitches to repair the colotomy. Sutures should be about 1 mm apart from each other with 0.5 mm bites. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58742/58742fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58742/58742fig5.jpg" /> 
Figure 5: Wound closure. (A) The visceral organs are returned to their normal anatomic position. (B) The abdominal incision is closed with 4-0 braided suture. (C) The skin is closed with 4-0 monofilament suture. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58742/58742fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58742/58742fig6.jpg" /> 
Figure 6: Incision post-surgery. (A) Re-open the original midline incision revealing the intra-abdominal contents. Be sure to extend the incision superiorly and laterally for better exposure to the sigmoid. (B) Rotate the viscera medially revealing the sigmoid anastomosis.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58742/58742fig7.jpg" /> 
Figure 7: Harvesting the anastomosis. Remove the segment of bowel proximal and distal to the anastomosis by 1 cm on each side.
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/58742/58742fig8.jpg" /> 
Figure 8: Orientation for tissue sections. Cut along the length of the mesenteric border in a longitudinal fashion. This leaves a rectangular specimen. This depicts specimen of sigmoid anastomosis.
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/58742/58742fig9.jpg" /> 
Figure 9: Histology and immunohistochemical analysis of a fully healed colonic anastomosis. Histological analysis shows fibrotic (collagen visualized by trichrome staining) response mediated by smooth muscle alpha-actin positive myofibroblasts.

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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 ...

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Medicine

11.5K Views.  University of Oklahoma Health Sciences Center. 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.

Video: Creation of Colonic Anastomosis in Mice

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Genetically engineered mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in large organs such as the colon in which only a single tumor is desired.
As a result, an immunocompetent, genetically engineered mouse model of colorectal cancer was developed which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor development is initiated by surgical, segmental infection of the distal colon with adeno-cre virus in compound conditionally mutant mice. The tumors can be easily detected and monitored via colonoscopy. We here describe the surgical technique of segmental adeno-cre infection of the colon, the surveillance of the tumor via high-resolution colonoscopy and present the resulting colorectal tumors.

A protocol for the establishment of a genetically engineered mouse model of colorectal cancer by segmental adeno-cre infection and its surveillance via high-resolution colonoscopy is presented.

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations ...

Cancer Research

Orthotopic Mouse Model of Colorectal Cancer

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature cancer cells growing in their natural location, replicate human disease with high fidelity. Two techniques can be used to establish this model. Both techniques are similar and require mouse anesthesia and laparotomy for exposure of the cecum. One technique involves injection of a colorectal cancer cell suspension into the cecal wall. Cancer cells are first grown in culture, harvested when subconfluent and prepared as a single cell suspension. A small volume of cells is injected slowly to avoid leakage. The other technique involves transplantation of a piece of subcutaneous tumor onto the cecum. A mouse with a previously established subcutaneous colorectal tumor is euthanized and the tumor is removed using sterile technique. The tumor piece is divided into small pieces for transplantation to another mouse. Prior to transplantation, the cecal wall is lightly damaged to facilitate tumor cell infiltration. The time to developing primary tumors and liver metastases will vary depending on the technique, cell line, and mouse species used. This orthotopic mouse model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

Two techniques can be used to establish this model: injection of a cancer cell suspension into the cecal wall or transplantation of a piece of subcutaneous tumor onto the cecum. This model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature ...

Biology

Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology, with the system option to differentiate distal from proximal endoscopic colitis (D-PECS).

Over the past few years, new generation endoscopes have emerged as important diagnostic research aids for evaluating murine colitis and colorectal tumors. We present herein a detailed protocol for endoscopic assessment of inflammation and colorectal tumors in mice, as well as a novel scoring system that uses decimal identifiers to document the endoscopic severity of colitis and colorectal tumors.

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental ...

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 being two common examples. Despite the frequency of these procedures, a significant knowledge gap remains in describing the inherent effects of intestinal resection on host physiology and disease pathophysiology. This article provides detailed instructions for an ileocolic resection with primary end-to-end anastomosis in mice, as well as essential aspects of peri-operative care to maximize post-operative success. When followed closely, this procedure yields a 95% long-term survival rate, no failure to thrive, and minimizes post-operative complications of bowel obstruction and anastomotic leak. The technical challenges of performing the procedure in mice are a barrier to its wide spread use in research. The skills described in this article can be acquired without previous surgical experience. Once mastered, the murine ileocolic resection procedure will provide a reproducible tool for studying the effects of intestinal resection in models of human disease.

Ileocolic resection is commonly performed in several human diseases; however, little is known regarding the impact of intestinal resection on surgical illnesses. This article provides instruction on executing the procedure in mice with high success, providing a means to study the effects of ileocolic resection in models of disease.

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

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 ...

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 Appendectomy Model of Chronic Colitis Associated Colorectal Cancer by Precise Localization of Caecal Patch

The human appendix has been recently implicated to play important biological roles in the pathogenesis of various complex diseases, such as colorectal cancer, inflammatory bowel disease, and Parkinson&#8217;s disease. To study the function of the appendix, a gut disease-associated murine appendectomy model has been established and its step-by-step protocol is described here. This report introduces a facile protocol for caecal patch removal in mice followed by the chemical induction of chronic colitis-associated colorectal cancer using a combination of dextran sulfate sodium (DSS) and azoxymethane (AOM). IgA specific cells and IgA concentration were significantly reduced upon removal of the caecal patch in male C57BL/6 mice&#160;compared to those in the sham group. Simultaneously administering 2% DSS and AOM resulted in nearly 80% mice survival in both sham and appendectomy groups without significant body weight loss. Histological results confirmed colonic inflammation and different degrees of adenocarcinoma. This model can be used for the study of the functional role of the appendix in maintaining gut microbiota homeostasis and pathogenesis of gut colitis and malignancies, as well as for the potential development of drug targeting therapies.

The presented protocol describes a facile surgical removal of the appendix (caecal patch) in a mouse followed by the induction of inflammatory bowel disease-associated colorectal cancer. This murine appendectomy model enables investigation of the biological role of the appendix in the pathogenesis of human gastrointestinal disease.

The human appendix has been recently implicated to play important biological roles in the pathogenesis of various complex diseases, such as colorectal ...

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 ...

Evaluation and Quantification of Micro Epithelial Gaps in the Colonic Mucosa using Immunofluorescence Staining

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier impairment has been associated with the development of various pathologies such as inflammatory bowel diseases (IBD). In the inflamed mucosa, superficial erosions or micro-erosions that corrupt epithelial monolayers correspond to sites of high permeability. Several mechanisms have been implicated in the formation of micro-erosions including cell shedding and apoptosis. These micro-erosions often represent microscopic epithelial gaps randomly distributed in the colon. Visualization and quantification of those epithelial gaps has emerged as an important tool to investigate intestinal epithelial barrier function. Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa. Additional use of cell death markers revealed that HPR encompass apoptotic foci where epithelial extrusion/shedding occurs. The protocol described here provides a simple but effective approach to visualize and quantify micro-erosions in the intestine, which is a very useful tool in disease models, in which the intestinal epithelial barrier is compromised.

Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa.

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier ...

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

Source: Boerner, K., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/62093">Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model.</a> J. Vis. Exp. (2021)
This video demonstrates the generation of an ileal loop model to study intestinal permeability in a mouse model. This method provides a live model to understand the epithelial barrier function and consequent immune response.