Name: creation of abdominal adhesions in mice
Uploaded: 2016-08-27T15:00:00.000+00:00
Duration: 6 min 44 s
Description: Watch this Scientific Journal Video about Creation of Abdominal Adhesions in Mice at JoVE.com

C.D.M. was supported by the American College of Surgeons (ACS) Resident Research Scholarship. M.S.H. was supported by the California Institute for Regenerative Medicine (CIRM) Clinical Fellow training grant TG2-01159. M.S.H., H.P.L., and M.T.L. were supported by the American Society of Maxillofacial Surgeons (ASMS)/Maxillofacial Surgeons Foundation (MSF) Research Grant Award. H.P.L. was supported by NIH grant R01 GM087609 and a gift from Ingrid Lai and Bill Shu in honor of Anthony Shu. H.P.L. and M.T.L. were supported by the Hagey Laboratory for Pediatric Regenerative Medicine and The Oak Foundation. M.T.L. was supported by the Gunn/Olivier Fund.

The following protocol has been approved by the Stanford University Institutional Animal Care and Use Committee (IACUC) and complies with all institutional ethical guidelines regarding the use of research animals.
1. Creation of Abdominal Adhesions
<ol>
	<li>Start the mouse on antibiotic chow diet one week prior to the procedure.</li>
	<li>Autoclave the surgical instruments and pre-warm the saline irrigation solution.</li>
	<li>Anesthetize the mouse using 2% inhaled isoflurane.</li>
	<li>Using a hair clipper followed by a quick application (5 - 10 sec) of depilatory agent, remove the hair from the majority of the surface of the abdomen. Thoroughly remove the depilatory agent by gently wiping with wet gauze and, optionally, by carefully dunking the lower half of the mouse into warm water. Thoroughly dry the animal.</li>
	<li>Place the animal&#39;s snout into the anesthetic nose cone. During the entire surgery, carefully monitor the respiratory rate of the animal and titrate the anesthetic flow rate as needed.</li>
	<li>Use a warming device, such as a heating pad or a warming lamp, to prevent hypothermia.</li>
	<li>Prior to prepping the abdomen, secure the mouse with strips of tape placed above and below the abdomen to prevent the mouse from shifting during surgery. Disinfect the abdomen with betadine, then follow with 70% ethanol. Include as much area as possible in the prep, including the hair on the edges of the shaved area. Place a sterile drape over the mouse. (Figure 1)</li>
	<li>Subcutaneously inject 0.1 mg/kg buprenorphine, or 3 &#181;g for a 30 g mouse.</li>
	<li>Make the skin incision.
	<ol>
		<li>Starting in the lower abdominal midline, grasp the skin with forceps and make a shallow vertical cut in the skin using sharp scissors.</li>
		<li>Carry the cut superiorly to the xyphoid with the scissors. Take care to enter only the skin.</li>
	</ol>
	</li>
	<li>As the abdominal musculature is now visible covering the viscera, grasp the midline of the musculature with forceps and very carefully make a small cut into it using sharp scissors. Make sure not to accidentally cut into an abdominal organ.</li>
	<li>After entering the peritoneal space with a small cut and seeing the muscular layer pull away from the abdominal organs, extend the cut superiorly and inferiorly by inserting the scissors into the opening and carefully cutting in both directions. Extend the incision from the xyphoid superiorly to above the bladder inferiorly. (Figure 2)</li>
	<li>Now that the intestines are exposed, locate the cecum and gently exteriorize it. Avoid grasping the cecum with toothed or sharp forceps. Instead use an atraumatic forceps such as an Adson serrated forceps.</li>
	<li>Abrade the cecum with sandpaper.
	<ol>
		<li>Gently abrade the entire surface of both sides of the cecum with 100 grit sandpaper for 30 - 60 sec until the surface becomes less shiny and petechiae appear on the surface. If the surgeon is right-handed, it helps to orient the cecum with the tip facing to the right and drape the cecum over the surgeon&#39;s left index finger. (Figure 3)</li>
		<li>Perform the sanding gently, as it is easy to accidentally cause a perforation of the thin cecal wall. A small amount of grittiness should be felt as the sandpaper is moved along the cecal surface. If the sandpaper is felt repeatedly catching on the cecum, this will likely cause a tear in the cecal wall. Watch carefully to ensure that particles of sandpaper do not dislodge and remain in the abdomen, as this may cause excessive foreign body reaction.</li>
		<li>If a tear in the cecal wall occurs, terminate the procedure and euthanize the animal.</li>
		<li>If a cecal blood vessel is sheared by the sandpaper and causes bleeding, hold pressure for up to two minutes with gauze. However, if bleeding persists after this time, place a figure-of-eight 7-0 monofilament suture around the point of bleeding. This will reliably stop the bleeding in almost all cases. Take care not to incorporate a large amount of cecal wall into the suture, as this risks causing necrosis of the involved part of the wall.</li>
	</ol>
	</li>
	<li>Injure the right abdominal sidewall.
	<ol>
		<li>Use a strip of sandpaper to abrade the peritoneal surface of the muscle of the right abdominal sidewall. Be more aggressive with the sanding here than on the cecum and continue until the entire surface of the right abdominal sidewall appears roughed up. Avoid sanding so hard that skin is visible through openings in the muscular layer. (Figure 4)</li>
		<li>Using a Castro-Viejo needle driver, place between two and four figure-of-eight 4-0 silk stitches into the muscular layer of the right abdominal sidewall. Leave the tails about 5 mm long. Take care not to accidentally catch bowel wall into the suture. (Figure 5)</li>
	</ol>
	</li>
	<li>Using a 10 ml syringe filled with warmed saline, irrigate the intestines several times. Direct the stream of saline into the abdominal cavity to irrigate the interior as well. If the surface underneath the mouse becomes soaked, move the mouse over or replace the surface, in order to avoid hypothermia.</li>
	<li>Use a sterile gauze placed over the incision to soak up excess irrigation.</li>
	<li>Take a pinch of starch and sprinkle it onto the surface of the right abdominal sidewall and onto both sides of the cecum. (Figure 6)</li>
	<li>Make sure that there is no active bleeding. If there is, use a gauze sponge to apply pressure directly onto the point of bleeding until it stops. If the bleeding does not stop readily, place a hemostatic suture as described above.</li>
	<li>Using the blunt end of the forceps and a finger, gently push the intestines back into the abdominal cavity. Position the cecum next to the sutures in the right abdominal sidewall in order to maximize adhesion formation.</li>
	<li>Close the abdominal incision.
	<ol>
		<li>Using 6-0 absorbable braided suture, place a running stitch into the muscular layer at the top of the incision.</li>
		<li>Run the suture down toward the bottom of the incision, bringing together the muscular layer. Travel by about 3 mm and take 3 mm bites with each new stitch. Take care not to accidentally take a bite of intestine while placing sutures.</li>
		<li>At the bottom of the incision, leave a loop of suture from the previous bite and use this to instrument tie the suture. Cut the suture leaving 5 mm tails.</li>
		<li>Repeat the above three steps with 6-0 nylon monofilament suture to close the skin.</li>
	</ol>
	</li>
	<li>Administer a 20 ml/kg subcutaneous saline bolus (roughly 0.5 ml for a 30 g mouse).</li>
	<li>Using a dry gauze, thoroughly dry the entire animal, as the dorsal fur tends to get wet during irrigation.</li>
	<li>Loosely wrap an adhesive dressing around the abdomen to cover the incision. Take care not to constrain the animal&#39;s legs or breathing mechanics with the dressing.</li>
	<li>Carefully monitor the animal as it recovers from anesthesia.&#160;Administer buprenorphine every 12 hr for 2-3 days after the procedure for pain control.</li>
</ol>
2. Harvesting Adhesion Tissue
<ol>
	<li>Wait a minimum of seven days after the initial surgery. Again, autoclave surgical instruments and anesthetize the mouse with isoflurane.</li>
	<li>Sterilize the abdomen with betadine followed by 70% ethanol. Shave and depilate additional hair from the right side of the abdomen so that the adhesion specimen containing skin will not be covered in hair, which makes histology more difficult.</li>
	<li>Use a left paramedian incision, since bowel is often stuck to the original incision. Starting at the lower end of the abdomen, about 5 mm to the left of the bottom of the original incision, grasp the skin with small toothed forceps and cut into the skin with sharp scissors. Extend this cut superiorly to the ribcage. (Figure 7)</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. There will be adhesive tissue causing the cecum and small bowel to adhere to the right sidewall of the abdomen and possibly also to the original incision. (Figure 8)</li>
	<li>Using the scissors and starting on the edge of the incision, above the level of the cecum, start cutting into the abdominal wall in a circle around the area where the bowel is adherent.</li>
	<li>Alternating above and below the level of the cecum, gradually cut all the way around the adhesion and complete the circle, yielding an &#34;island&#34; of skin and abdominal wall adherent to the bowel. (Figure 9)</li>
	<li>Again using the scissors, cut into the bowel and separate the adherent part from the rest of the bowel, yielding a sandwich of tissue that consists of skin and abdominal wall on one side, bowel on the other side, and adhesive tissue connecting the two in between. (Figure 10)</li>
	<li>Optionally, use a pointed scissors to sharply separate the skin from the abdominal wall muscle. For histology consider leaving the skin attached, but for digestion and cell isolation the skin can be removed. If the tissue will be used for histology, make sure to remove as much suture material as possible without excessively disturbing the adhesive interface. For digestion and cell isolation it is acceptable to leave the sutures in place as long as the tissue digest is filtered.</li>
	<li>After excising adhesion tissue, euthanize the mouse using a humane method approved by your institutional animal use committee.&#160;</li>
</ol>

<ol>
	<li>Ellis, H., 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=Adhesion-related+hospital+readmissions+after+abdominal+and+pelvic+surgery:+a+retrospective+cohort+study.">Adhesion-related hospital readmissions after abdominal and pelvic surgery: a retrospective cohort study.</a> Lancet. 353 (9163), 1476-1480 (1999).</li><li>Brochhausen, 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=Current+strategies+and+future+perspectives+for+intraperitoneal+adhesion+prevention.">Current strategies and future perspectives for intraperitoneal adhesion prevention.</a> J Gastrointest Surg. 16 (6), 1256-1274 (2012).</li><li>diZerega, G. S., Campeau, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peritoneal+repair+and+post-surgical+adhesion+formation.">Peritoneal repair and post-surgical adhesion formation.</a> Hum Reprod Update. 7 (6), 547-555 (2001).</li><li>Hellebrekers, B. W., Kooistra, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+postoperative+adhesion+formation.">Pathogenesis of postoperative adhesion formation.</a> Br J Surg. 98 (11), 1503-1516 (2011).</li><li>Herrick, S. 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=Human+peritoneal+adhesions+are+highly+cellular,+innervated,+and+vascularized.">Human peritoneal adhesions are highly cellular, innervated, and vascularized.</a> J Pathol. 192 (1), 67-72 (2000).</li><li>Beyene, R. T., Kavalukas, S. L., Barbul, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-abdominal+adhesions:+Anatomy,+physiology,+pathophysiology,+and+treatment.">Intra-abdominal adhesions: Anatomy, physiology, pathophysiology, and treatment.</a> Curr Probl Surg. 52 (7), 271-319 (2015).</li><li>ten Broek, R. P., 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=Benefits+and+harms+of+adhesion+barriers+for+abdominal+surgery:+a+systematic+review+and+meta-analysis.">Benefits and harms of adhesion barriers for abdominal surgery: a systematic review and meta-analysis.</a> Lancet. 383 (9911), 48-59 (2014).</li><li>Becker, J. M., 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=Prevention+of+postoperative+abdominal+adhesions+by+a+sodium+hyaluronate-based+bioresorbable+membrane:+a+prospective,+randomized,+double-blind+multicenter+study.">Prevention of postoperative abdominal adhesions by a sodium hyaluronate-based bioresorbable membrane: a prospective, randomized, double-blind multicenter study.</a> J Am Coll Surg. 183 (4), 297-306 (1996).</li><li>Cassidy, M. R., Sherburne, A. C., Heydrick, S. J., Stucchi, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+intraoperative+administration+of+a+histone+deacetylase+inhibitor+and+a+neurokinin-1+receptor+antagonist+synergistically+reduces+intra-abdominal+adhesion+formation+in+a+rat+model.">Combined intraoperative administration of a histone deacetylase inhibitor and a neurokinin-1 receptor antagonist synergistically reduces intra-abdominal adhesion formation in a rat model.</a> Surgery. 157 (3), 581-589 (2015).</li><li>Thaler, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coincidence+of+connective+tissue+growth+factor+expression+with+fibrosis+and+angiogenesis+in+postoperative+peritoneal+adhesion+formation.">Coincidence of connective tissue growth factor expression with fibrosis and angiogenesis in postoperative peritoneal adhesion formation.</a> Eur Surg Res. 37 (4), 235-241 (2005).</li><li>Hong, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+macrophage-dependent+peroxisome+proliferator-activated+receptor+gamma+signalling+on+adhesion+formation+after+abdominal+surgery+in+an+experimental+model.">Effects of macrophage-dependent peroxisome proliferator-activated receptor gamma signalling on adhesion formation after abdominal surgery in an experimental model.</a> Br J Surg. 102 (12), 1506-1516 (2015).</li><li>Whang, S. H., 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=In+search+of+the+best+peritoneal+adhesion+model:+comparison+of+different+techniques+in+a+rat+model.">In search of the best peritoneal adhesion model: comparison of different techniques in a rat model.</a> J Surg Res. 167 (2), 245-250 (2011).</li><li>Buckenmaier, C. C., Pusateri, A. E., Harris, R. A., Hetz, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+antiadhesive+treatments+using+an+objective+rat+model.">Comparison of antiadhesive treatments using an objective rat model.</a> Am Surg. 65 (3), 274-282 (1999).</li><li>Rajab, T. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+model+for+the+induction+of+experimental+adhesions.">An improved model for the induction of experimental adhesions.</a> J Invest Surg. 23 (1), 35-39 (2010).</li></ol>

The authors declare that they have no competing financial interests.

The critical steps in this procedure are: thoroughly abrading the cecum without causing perforation, placing sutures in the abdominal sidewall, and applying the right amount of starch. Only apply sandpaper to the cecum, or to a small specific portion of the bowel. Wide use of sandpaper on large amounts of small bowel tends to cause significant ileus. Take care to abrade the cecum with enough force that the surface becomes rough, but not so much that the wall tears. Finding this balance can take some time. Always handle t...

Abdominal adhesions are a form of scar tissue that form in the abdomen in response to inflammation, typically following surgery or intraabdominal infection. Adhesions are a major cause of chronic abdominal pain and infertility, and are the most common cause of small bowel obstruction1. The presence of adhesions makes performing a second abdominal operation more difficult and increases the likelihood of complications2.
Despite years of research, the mechanisms underlying the formation of adhesions remain poorly understood. It is known that an initial injury to the peritoneal surface causes an exudation of fibrin-rich fluid, which then forms a clot that binds the surfaces of bowel and the abdominal wall together3. Later, fibroblasts and other cells migrate into the adhesive space and secrete connective tissue4. Over months to years the adhesion matures by developing blood vessels and nerves5.
Several commercial products exist that are designed to reduce the formation of adhesions after abdominal surgery (e.g., Seprafilm). All of these products act as mechanical barriers and stop adhesion formation by preventing physical contact between loops of bowel and the abdominal wall6,7. Despite evidence from a controlled trial that a surgical adhesion barrier reduces the formation of adhesions8, many surgeons anecdotally have been disappointed with the effectiveness of mechanical barrier products.
Currently there are no drug-based anti-adhesion therapies, which reflects the fact that the precise processes involved in adhesion formation are poorly understood. Developing a therapy that specifically targets cellular or molecular agents involved in the formation of adhesions will require an improved understanding of the events that are involved in the formation of the adhesion. Several groups have identified molecular pathways that may be important for adhesion formation9-11. Animal models provide a superb environment for studying the formation of adhesions. Many studies have been published describing the surgical creation of adhesion in several animals, particular the rat and mouse6,12-14. Given our experience with studying fibrosis in the mouse and the wide availability of transgenic mice and mouse-based antibodies, we chose the mouse as our model for the study of adhesions. Herein, we report the technique that we have developed to reproducibly and reliably create abdominal adhesions in the mouse.

<table><tbody><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 of the appropriate size</td></tr><tr><td>Isothesia isoflurane</td><td>Henry Schein&amp;nbsp;</td><td>050033</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 Pharmaceuticals 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>Povidone Iodine Prep Solution</td><td>Medline</td><td>MDS093944H</td><td></td></tr><tr><td>Webcol alcohol prep swabs</td><td>Covidien</td><td>6818</td><td></td></tr><tr><td>General-Purpose Labarotory Labeling tape</td><td>VWR</td><td>89097-912</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>100 grit 1/4 sheet sandpaper</td><td>ACE Hardware</td><td>1010446</td><td>Cut into strips</td></tr><tr><td>4-0 silk suture, 30&quot;, SH needle</td><td>Ethicon</td><td>K831</td><td></td></tr><tr><td>7-0 PDS II absorbable monofilament suture, 30&quot;, BV-1 needle</td><td>Ethicon</td><td>Z135</td><td>Usually comes double-armed. Cut the suture at the midway point to generate two usable sutures.</td></tr><tr><td>Rice starch</td><td>MP Biomedicals</td><td>102955</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>6-0 Vicryl absorbable braided suture, 18&quot;, RB-1 taper needle</td><td>Ethicon</td><td>J212H</td><td></td></tr><tr><td>6-0 Ethilon nylon monofilament&amp;nbsp; suture, 18&quot;, P-3 needle,&amp;nbsp;</td><td>Ethicon</td><td>1698G</td><td></td></tr><tr><td>Tegaderm Transparent Film Dressing Frame Style, 6 cm x 7 cm</td><td>3M</td><td>1624W</td><td>Cut in half lengthwise</td></tr></tbody></table>

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 intraabdominal infection. The precise mechanisms underlying adhesion formation are poorly understood. Many compounds and physical barriers have been tested for their ability to prevent adhesions after surgery with varying levels of success. The mouse and rat are important models for the study of abdominal adhesions. Several different techniques for the creation of adhesions in the mouse and rat exist in the literature. Here we describe a protocol utilizing abrasion of the cecum with sandpaper and sutures placed in the right abdominal sidewall. The mouse is anesthetized and the abdomen is prepped. A midline laparotomy is created and the cecum is identified. Sandpaper is used to gently abrade the surface of the cecum. Next, several figure-of-eight sutures are placed into the peritoneum of the right abdominal sidewall. The abdominal cavity is irrigated, a small amount of starch is applied, and the incision is closed. We have found that this technique produces the most consistent adhesions with the lowest mortality rate.

At seven days after surgery, the cecum and possibly ascending colon, liver, and loops of small bowel should be adherent to the right-sided abdominal wall. (Figure 8) Excised tissue can be embedded and sectioned and will yield excellent histological slides. (Figure 11, 12)
When the procedure is performed properly, 100% of mice should have substantial adhesions at seven days. Mortality should be l...

Watch this Scientific Journal Video about Creation of Abdominal Adhesions in Mice at JoVE.com

Creation of Abdominal Adhesions in Mice

Abdominal adhesions that form after surgery are a major cause of pain, infertility, and hospitalization and reoperation for small bowel obstruction. Our surgical procedure for creating abdominal adhesions in mice is a reliable tool to study the mechanisms underlying the formation of adhesions.

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

Research

JoVE Journal

Medicine

12.6K Views.  Stanford University School of Medicine. Abdominal adhesions that form after surgery are a major cause of pain, infertility, and hospitalization and reoperation for small bowel obstruction. Our surgical procedure for creating abdominal adhesions in mice is a reliable tool to study the mechanisms underlying the formation of adhesions.

Video: Creation of Abdominal Adhesions in Mice

Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase 

Transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta is the most widely used animal model of abdominal aortic aneurysm (AAA) ever since it was first described in rats by Anidjar and colleagues.1 The rationale for its development was based on the disrupted nature of elastin observed in AAAs. This rat model has been modified to produce AAAs in the infrarenal aortic region of mice.2 The model has the ability to add broad insight into the pathobiology of AAA due to the emergence of numerous transgenic and gene knockout mice. Moreover, it is a viable platform to test potential therapeutic agents for AAA. In this video, we demonstrate the elastase infusion AAA procedure used in our laboratory. 
Mice are anesthetized using 2.5% isoflurane, and a laparotomy is performed under sterile conditions. The abdominal aortais isolated with the assistance of an operating stereomicroscope (Leica). After placing temporary ligatures around the proximal and distal aorta, an aortotomy is created at the bifurcation with the tip of a 30-gauge needle. A heat-tapered segment of PE-10 polyethylene tubing is introduced through the aortotomy and secured. The aortic lumen is subsequently perfused for 5-15 minutes at 100 mm Hg with saline containing type I porcine pancreatic elastase (4.5 U/mL; Sigma Chemical Co.). After removing the perfusion catheter, the aortotomy is repaired without constriction of the lumen.

Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase

This video shows how to induce abdominal aortic aneurysms (AAA) in mice via transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta. The model has the ability to add broad insight into the pathobiology of AAA due to the emergence of numerous transgenic and gene knockout mice.

Transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta is the most widely used animal model of ...

Biology

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine balloon denudation model, which is comparable with established vessel injury models in large animals. The aorta denudation model with balloon catheters mimics the clinical setting and leads to comparable pathobiological and physiological changes. Briefly, after performing a horizontal incision in the aorta abdominalis, a balloon catheter will be inserted into the vessel, inflated, and introduced retrogradely. Inflation of the balloon will lead to intima injury and overdistension of the vessel. After removing the catheter, the aortic incision will be closed with single stiches. The model shown in this article is reproducible, easy to perform, and can be established quickly and reliably. It is especially suitable for evaluating expensive experimental therapeutic agents, which can be applied in an economical fashion. By using different knockout-mouse strains, the impact of different genes on MH development can be assessed.

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine ...

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.

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 ...

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 ...

Establishing In Situ Closed Circuit Perfusion of Lower Abdominal Organs and Hind Limbs in Mice

Ex vivo perfusion is an important physiological tool to study the function of isolated organs (e.g. liver, kidneys). At the same time, due to the small size of mouse organs, ex vivo perfusion of bone, bladder, skin, prostate, and reproductive organs is challenging or not feasible. Here, we report for the first time an in situ lower body perfusion circuit in mice that includes the above tissues, but bypasses the main clearance organs (kidney, liver, and spleen). The circuit is established by cannulating the abdominal aorta and inferior vena cava above the iliac artery and vein and cauterizing peripheral blood vessels. Perfusion is performed via a peristaltic pump with perfusate flow maintained for up to 2 h. In situ staining with fluorescent lectin and Hoechst solution confirmed that the microvasculature was successfully perfused. This mouse model can be a very useful tool for studying pathological processes as well as mechanisms of drug delivery, migration/metastasis of circulating tumor cells into/from the tumor, and interactions of immune system with perfused organs and tissues.

A protocol is described for in situ perfusion of the mouse lower body, including the bladder, the prostate, sex organs, bone, muscle and foot skin.

Ex vivo perfusion is an important physiological tool to study the function of isolated organs (e.g. liver, kidneys). At the same time, due to the small ...

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 ...

Immunology and Infection

Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile duct proliferation. However, long-term experiments can lead to increased morbidity and mortality. In select mouse strains with underlying liver disease, meaningful comparisons can be made even with ligation of a single lobe of the liver, which can reduce animal losses and expenses. Here, we describe partial bile duct ligation (pBDL) in the mouse, in which only the left hepatic bile duct is ligated, causing biliary obstruction in the left lobe but not the remaining lobes. With careful microsurgical technique, pBDL experiments can be cost-effective, since the unligated lobe serves as an internal control to the ligated lobes, when subjected to the same conditions in the same animal. Unlike cBDL, a separate sham-operated control group is not necessary. pBDL is highly useful to directly compare localized versus systemic effects of cholestasis and other retained bile components. pBDL can also be repurposed as a novel method to investigate mechanisms related to medications and cell migration.

Here, we present partial bile duct ligation as a surgical model of liver injury and regeneration in rodents.

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile ...

Investigating the Molecular Mechanisms Underlying Murine Abdominal Aortic Aneurysm

A Mouse Abdominal Aortic Aneurysm Model by Periadventitial Calcium Chloride and Elastase Infiltration

Abdominal aortic aneurysm (AAA) is a life-threatening disease associated with high mortality rates. It is characterized by the permanent dilation of the abdominal aorta with at least a 50% increase in arterial diameter. Various animal models of AAA have been introduced to mimic the pathophysiological changes and study the underlying mechanisms of AAA. Among these models, the calcium chloride (CaCl2)- and elastase-induced AAA models are commonly used in mice. However, these methods have certain limitations. Traditional intraluminal porcine pancreatic elastase (PPE) perfusion is associated with high technical difficulty and a high rupture rate, while periadventitial administration of PPE yields inconsistent results. In addition, the CaCl2-induced AAA model lacks human AAA features, such as atherothrombosis and aneurysm rupture. Therefore, the combined application of CaCl2 and PPE has been proposed as an approach to enhance success rates and induce greater diameter increases in AAA animal models. This manuscript presents a comprehensive protocol for establishing a mouse AAA model through periaortic infiltration of PPE and CaCl2 in the infrarenal segment of the abdominal aorta. By following this protocol, we can achieve an AAA formation rate of approximately 90% with technical simplicity and reproducibility. Further ultrasound and histological experiments confirm that this model effectively replicates the morphological and pathological changes observed in human AAA.

Author Spotlight: Development and Characterization of a Mouse Model for Abdominal Aortic Aneurysm

This manuscript presents a protocol for establishing a mouse abdominal aortic aneurysm model using calcium chloride and elastase, combining the advantages of previous modeling methods. This model can be utilized to investigate the pathophysiological mechanisms underlying abdominal aortic aneurysms.

Abdominal aortic aneurysm (AAA) is a life-threatening disease associated with high mortality rates. It is characterized by the permanent dilation of the ...

Fixation and Operational Method for Abdominal Massage in T2DM Mice

Type 2 diabetes mellitus (T2DM) is a rapidly growing global public health issue, affecting over 500 million people worldwide. Although abdominal massage has shown potential benefits in managing T2DM, its effectiveness remains unclear, particularly in animal studies, where challenges such as animal compliance and the need for precise pressure control complicate implementation. To address these challenges, this study introduces a novel abdominal massage simulation device specifically designed for mice. This device provides a practical solution for conducting abdominal massage interventions in a controlled manner while minimizing stress and tissue damage to the animals. It securely restrains the mice's limbs, allowing them to remain conscious during the massage, and offers precise control over both the pressure and frequency of the massage applied to the abdomen. The device's ability to simulate manual abdominal massage with accuracy opens new possibilities for experimental studies assessing its effects on T2DM. The primary goal of this protocol is to investigate the impact of abdominal massage on key T2DM markers such as blood glucose levels, lipid metabolism, and insulin sensitivity in mice. By providing a reliable and reproducible method for abdominal massage, this device can offer valuable insights into its potential as a non-invasive therapeutic intervention for T2DM. The findings from this research may contribute to advancing clinical strategies for diabetes prevention and treatment, particularly in enhancing the understanding of traditional therapies like abdominal massage in modern medical practice.

This study introduces an abdominal massage device for mice that replicates manual massage while minimizing stress and tissue damage. The device significantly lowers blood glucose levels, improves lipid metabolism, and enhances insulin sensitivity in type 2 diabetes mice, offering a promising, non-invasive therapeutic approach with clinical potential.

Type 2 diabetes mellitus (T2DM) is a rapidly growing global public health issue, affecting over 500 million people worldwide. Although abdominal massage ...

Creation of Abdominal Adhesions in Mice

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Chapters in this video

Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

Creation of Colonic Anastomosis in Mice

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

Establishing In Situ Closed Circuit Perfusion of Lower Abdominal Organs and Hind Limbs in Mice

Postoperative Ileus Murine Model

Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis

A Mouse Abdominal Aortic Aneurysm Model by Periadventitial Calcium Chloride and Elastase Infiltration

Fixation and Operational Method for Abdominal Massage in T2DM Mice