Medicine
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Summary August 27th, 2016
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.
Transcript
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.
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