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>Usi...

<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.

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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 ...

The overall goal of this procedure is to consistently produce abdominal adhesions in the mouse while minimizing mortality. This method can help answer key questions in the field of abdominal adhesions, such as what cells, macromolecules, signaling pathways and cytokines are involved in adhesion formation. The main advantages of this technique are that the mice produce adhesions 100%of the time and the mortality rate is very low when the procedure is performed correctly.Generally individuals new to this procedure will struggle with applying the appropriate amount of force when using the sandpaper and also when applying the correct amount of starch. Visual demonstration of this technique is critical because too much force will perforate the cecum, but too little force may not result in the formation of adhesions. On the day of the surgery, use hair clippers to trim the abdominal fur of the anesthetized animal.Then transfer the animal to a warming device and secure the limbs with tape. Next, disinfect the abdomen with betadine, followed by 70%ethanol, taking care to include the hair on the edges of the shaved area. Place a sterile drape over the mouse.Then grasp the lower abdominal skin with a forceps and make a shallow vertical cut. Continue the incision up to the xiphoid process. Then grasp the abdominal musculature at the midline and use sharp scissors to carefully make a small incision, taking care to avoid the internal organs.When the abdominal organs are visible, insert the scissors into the incision and carefully cut in both directions to extend the cut from the xiphoid process to above the bladder. Once the intestines have been exposed, locate the cecum and use an atraumatic forceps to gently exteriorize the tissue. Orient the cecum with the tip facing toward the dominant hand and drape the cecum over the non-dominant index finger.Using 100 grit sandpaper, gently abrade the entire surface of both sides of the externalized intestine for 30 to 60 seconds. A small amount of grittiness should be felt as the sandpaper's moved along the cecal surface. If a cecal blood vessel is sheared during the abrasion, gently press gauze onto the site of bleeding for up to two minutes.If the bleeding continues after two minutes, place a figure of 870 monofilament suture around the vessel. When the cecal surface becomes less shiny and petechia appear, use a new strip of sandpaper to abrade the peritoneal surface of the right abdominal side wall muscle until the entire surface of the side wall appears rough. Then use a castroviejo needle driver to place two to four figure of 840 silk stitches into the muscular layer of the right abdominal side wall, leaving fine millimeter long tails.Use a 10 milliliter syringe of warmed saline to irrigate the intestines and the abdominal cavity several times. If the area underneath the mouse becomes soaked, move the mouse to a dry surface to avoid hypothermia. When the intestines have been well-irrigated, place sterile gauze over the incision to soak up the excess saline.Then position the cecum next to the sutures in the right abdominal side wall. Use a gauze sponge to apply pressure onto any points of active bleeding. Then sprinkle a pinch of rice starch onto the surface of the right abdominal side wall and the cecum.To close the abdomen, use a 60 absorbable braided suture to place a running stitch into the muscular layer at the top of the incision. Run the suture down toward the bottom of the incision, bringing together three by three millimeter sections of the musculature with each new stitch. At the bottom of the incision, leave a loop of suture from the previous stitch to instrument tie the suture and cut the suture, leaving five millimeter tails.Use a 60 nylon monofilament suture to close the skin in the same manner. Then administer a 20 milliliter per kilogram subcutaneous saline bolus and thoroughly dry the entire animal with fresh gauze. Finally, loosely wrap an adhesive dressing around the incision, taking care not to constrain the animal's legs or breathing mechanics and monitor the mouse until it is fully recovered.At seven days after the surgery, the cecum and possibly the ascending colon, liver and loops of small bowel should be adherent to the right-sided abdominal wall. Embedding and sectioning of the excised abdominal tissue yields excellent slides for histological and immunohistochemical analyses. Once this procedure is mastered, it can be performed in under 30 minutes.Following this procedure, other methods such as quantitative PCR and fiberglass culture can be used to answer questions about gene expression within the adhesion tissue.

Research

JoVE Journal

Medicine

12.4K visualizações.  Stanford University School of Medicine. The overall goal of this procedure is to consistently produce abdominal adhesions in the mouse while minimizing mortality. This method can help answer key questions in the field of abdominal adhesions, such as what cells, macromolecules, signaling pathways and cytokines are involved in adhesion formation. The main advantages of this technique are that the mice produce adhesions 100%of the time and the mortality rate is very low when the procedure is performed correctly.Generally indivi...