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 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.
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.
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
2. Harvesting Adhesion Tissue
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 less than 5%.
Figure 1: Preparation for Surgery. (a) The animal has been secured with tape and the abdomen prepped with betadine. (b) The betadine has been cleared with an alcohol swab and a sterile drape has been placed. Please click here to view a larger version of this figure.
Figure 2: Incision. The incision should extend from the level of the bladder to the xyphoid. Please click here to view a larger version of this figure.
Figure 3: Abrasion of the Cecum. (a) Draping the cecum over the left index finger with the tip pointing to the right allows the base of the cecum to be stabilized by the left thumb. (b) An appropriate level of sanding leaves the cecum with a rough surface that is less shiny than before, and with several petechial points of bleeding. Extra care should be taken while sanding near the mesenteric side, as the vessels here will bleed the most briskly. Please click here to view a larger version of this figure.
Figure 4: Abrasion of the Abdominal Wall. The viscera are swept aside and the peritoneal surface of the muscle wall is sanded until it appears rough. The blood vessel seen just above the sandpaper in the abdominal wall is frequently encountered but will not bleed substantially. Please click here to view a larger version of this figure.
Figure 5: Placement of the Figure-of-eight Suture. A silk figure-of-eight suture placed into the muscle wall, prior to being tied down. Please click here to view a larger version of this figure.
Figure 6: Application of Starch. A light layer of starch is sprinkled over the abdominal sidewall and bowel. Two figure-of-eight sutures are seen in the muscle wall. Please click here to view a larger version of this figure.
Figure 7: Incision for the Second Surgery. (a) The incision is started just to the left of the original incision. (b) The new incision should extend from the level of the bladder to the ribcage. Please click here to view a larger version of this figure.
Figure 8: The Adhesion. Loops of small bowel (SB) are adherent to the right sidewall and to each other. The original midline incision, now healed, is seen (Inc). Please click here to view a larger version of this figure.
Figure 9: Excising Adhesion Tissue. (a) and (b) An "island" of skin and abdominal wall adherent to the underlying bowel is freed by cutting in a complete circle around the adherent area. Please click here to view a larger version of this figure.
Figure 10: The Specimen. Cutting across the bowel will yield a sandwich of tissue with bowel on one side (a) and skin on the other side (b). The pieces of suture visible should be removed if the specimen is to be used for histology. Please click here to view a larger version of this figure.
Figure 11: Histology. A hematoxylin and eosin (H&E) stained section of an adhesion at seven days after surgery showing skin and abdominal wall (bottom) attached via the adhesive interface to the cecum (top). At this early time point the cecum is likely adhered to the abdominal wall mainly through fibrin and other molecules, and substantial scar tissue has not yet formed. Please click here to view a larger version of this figure.
Figure 12: Immunofluorescent Imaging. A section of adhesion at seven days after surgery immunofluorescently stained for α-smooth muscle actin, showing true staining of the skin hair follicles (left) and cecal muscle wall (middle), and non-specific staining of the cecal luminal surface (right). Please click here to view a larger version of this figure.
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 the intestine carefully and avoid using sharp or toothed forceps to manipulate bowel. It is easy to accidentally cause bleeding by grabbing the small bowel mesentery with forceps or by pulling on the bowel too forcefully.
If a tear occurs in the cecal wall, the procedure should be terminated and the animal euthanized. In our experience, cecal tear results in death of the animal after a few days, even if the tear is repaired well. In addition, tear of the cecal wall will cause spillage of stool that will affect the inflammatory response in an unpredictable way.
Hemostasis must be complete at the end of the procedure. Some bleeding vessels will re-open and start to bleed after appearing to have stopped. When in doubt, place an absorbable monofilament figure-of-eight suture around the point of bleeding. This type of suture is preferable to silk or braided absorbable suture because it can be pulled though the tissue with minimal resistance. However we have found that silk or braided absorbable suture is most effective in the abdominal sidewall for inducing adhesions, as monofilament is less inflammatory.
Application of starch at the end of the procedure helps to increase the formation of adhesions, but we have discovered that an excessive amount of starch will cause an inflammatory reaction that causes death. The starch should be sprinkled on lightly. There should not be so much that it forms a solid layer.
When first learning this procedure, it is common to lose several mice. In our experience the most common causes of death are dehydration due to ileus caused by overly vigorous abrasion of intestine, and sepsis due to intestinal perforation. Death caused by bleeding can occur if complete hemostasis is not achieved at the end of the surgery.
If adhesion formation is inadequate, consider using more aggressive abrasion of the cecum and right sidewall, leaving more starch, or placing more sutures on the abdominal sidewall. In our experience, waiting for less than a week before reopening the abdomen results in less than adequate adhesion formation. On the other hand, if the mortality rate of mice after surgery is high, consider using less aggressive abrasion of the cecum, inspecting more closely for hemostasis, and applying less starch. It is also important to carefully monitor the respiratory rate of the mouse during surgery. Once the surgeon gains experience with this procedure, mortality should be less than 10%.
This technique is designed to primarily form adhesions between the cecum and the right abdominal sidewall. Adhesions will also often form between small bowel, liver, and the midline incision. Using this technique will usually not cause adhesions on the left side of the abdomen. A limitation of this technique is that adhesions between bowel and abdominal wall are more likely to form than adhesions between loops of bowel. In addition, it is not likely to result in a totally frozen abdomen full of adhesive tissue, which is often seen in humans who have had multiple surgeries.
As discussed in the introduction, many techniques for producing abdominal adhesions in mice and other species, particularly rat, have been described in the literature6,12-14. There are far fewer published protocols for the creation of adhesions in mice. We speculate that because the creation of adhesions in mice is more difficult than in rats, most investigators chose to develop their technique in the rat. However, because of the greater availability of transgenic mice and anti-mouse antibodies, we believe that it is valuable to have a robust model for adhesion creation in mice, despite the greater technical difficulty. We developed this protocol after trying many of the published rat protocols. We found that methods for creating adhesions in the rat, such as the use of electrocautery on the cecum, often cause mice to die. We were not satisfied with the density of adhesions produced by techniques that use a single intervention, such as placing sidewall ischemic buttons alone, or only abrading the cecum. The technique we present here represents a combination of interventions that we found, through trial and error, to produce adhesions consistently while minimizing mortality.
Because this technique consistently causes adhesive tissue to form between the cecum and the sidewall, we believe that it is ideal to test interventions designed to reduce adhesion formation. Also this technique can be used to explore the molecular pathways and cell types that are involved in adhesion formation. The adhesion tissue can easily be excised and prepared for histology, and yields excellent histological images (Figures 11, 12).
Some researchers may desire a model in which adhesions form less than 100% of the time. Omitting the application of starch will reduce the adhesion rate to roughly 80%. Decreasing the number of silk sutures placed into the right abdominal sidewall will reduce the adhesion rate further.
In our experience, day seven after surgery is the first time point where the bowel is consistently adherent to the abdominal wall. However, other time points may be more relevant depending on the focus of the project. For example, researchers interested in the migration of neutrophils and macrophages into the adhesion space may want to harvest tissue in the first five days after surgery. On the other hand, the deposition of collagen by fibroblasts takes place for weeks after the initial injury, and for this it will be more appropriate to examine tissue at time points spaced out for several weeks after surgery.
Surgical loupes are very helpful for magnification of the surgical field while performing this procedure. An operative microscope can also be used but limits the surgeon's ability to inspect the operative field from different angles. The procedure can also be done with no magnification at all.
The authors have nothing to disclose.
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.
Fisherbrand Absorbent Underpads, 20" x 24" | Fisher Scientific | 14-206-62 | |
Polylined Sterile Field, 18" x 24" | Busse Hospital Disposables | 696 | Cut a rectangular hole of the appropriate size |
Isothesia isoflurane | Henry Schein | 050033 | |
Fisherbrand Sterile cotton gauze pad, 4" x 4" | Fisher Scientific | 22-415-469 | |
Puralube petrolatum ophthalmic ointment, 1/8 oz. tube | Dechra Veterinary Products | NDC 17033-211-38 | |
Nair depilatory cream | Church & Dwight Co. | 22339-05 | |
Buprenex buprenorphine 0.3 mg/mL | Reckitt Benckiser Pharmaceuticals Inc. | NDC 12496-0757-5 | |
1 cc insulin syringe, 27G | Becton Dickinson | 329412 | |
Povidone Iodine Prep Solution | Medline | MDS093944H | |
Webcol alcohol prep swabs | Covidien | 6818 | |
General-Purpose Labarotory Labeling tape | VWR | 89097-912 | |
BioGel PI surgical gloves | Mölnlycke Health Care | ALA42675Z | |
Micro Forceps with teeth | Roboz | RS-5150 | |
Fine scissors- sharp | Fine Science Tools | 14060-09 | |
Straight serrated forceps | Fine Science Tools | 11050-10 | |
Castro-Viejo needle driver | Fine Science Tools | 12565-14 | |
100 grit 1/4 sheet sandpaper | ACE Hardware | 1010446 | Cut into strips |
4-0 silk suture, 30", SH needle | Ethicon | K831 | |
7-0 PDS II absorbable monofilament suture, 30", BV-1 needle | Ethicon | Z135 | Usually comes double-armed. Cut the suture at the midway point to generate two usable sutures. |
Rice starch | MP Biomedicals | 102955 | |
0.9% Sodium Chloride Irrigation | Baxter | BHL2F7121 | Warm to 37° C prior to use |
10 mL syringe | Becton Dickinson | 309604 | |
6-0 Vicryl absorbable braided suture, 18", RB-1 taper needle | Ethicon | J212H | |
6-0 Ethilon nylon monofilament suture, 18", P-3 needle, | Ethicon | 1698G | |
Tegaderm Transparent Film Dressing Frame Style, 6 cm x 7 cm | 3M | 1624W | Cut in half lengthwise |