This method can help answer key questions about the healing mechanisms of colo-colonic anastomoses, and may provide a model for reducing anastomotic leak rates and their potentially devastating consequences. Our technique offers a relatively simple and reproducible method for performing colo-colonic or colo-rectal anastomosis in an animal model with similar clinical leak rates to human patients. Begin by securing the limbs of an anesthetized mouse to the surgical surface, and disinfecting the exposed abdomen with chlorhexidine.
Next place a sterile drape over the animal, and make a skin incision along the midline of the lower abdomen to the xiphoid. Use forceps to lift up the midline of the abdominal wall, and taking care not to injure the intra-abdominal structures incise the length of the midline abdominal fascia. 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.
Then cover the viscera with damp, sterile gauze. Coat a blunt probe tip with lubricating jelly, and carefully advance the probe into the rectum to aid the identification of the descending sigmoid colon. Once the colon has been identified, partially withdraw the probe, and use atraumatic forceps to gently grasp the sigmoid colon superior to the pelvic inlet.
Using sharp micro scissors, make a cut along the sigmoid colon, perpendicular to the bowel tissue, and extending 80 to 90%across the width of the colon. If a mesenteric blood vessel is injured during the course of the visceral rotation, or the colotomy, gently apply pressure to the sight of the injury for up to two minutes. To repair the sigmoid colotomy, use a castroviejo needle driver and five to six simple interrupted eight O non-absorbable polypropylene stitches to repair the colostomy.
Performing the anastomosis at the mesentery border and working your way bilaterally to the anti-mesentery border helps ensure a patent and non-stenosis anastomosis. As the repair progresses, advance the trans-anal, blunt tip dissection probe to span the repair before the initial sutures are placed to help prevent too narrow a repair, and to decrease rates of anastomotic stricture. When all of the sutures have been placed, use a 10 milliliter syringe filled with warmed sterile saline to irrigate the abdomen several times.
During the final irrigation fill the abdomen with sterile saline, and carefully advance a lubricated, 18 gage angiocatheter into the rectum to inflate the colon with 5 to one cubic centimeter of air, for the identification of an leaks within the anastomosis. Then place an additional suture for repair, and inflate the colon again to make sure there are no leaks. Deflate the colon with the angiocatheter before removal, and gently return the medialized viscera to its normal anatomic position.
Use four O absorbable braided sutures to close the abdominal incision, with 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. Then use a four O monofilament suture to close the skin, and monitor the mouse until it has fully recovered from the anesthesia. At the appropriate experimental end point, sterilize the abdomen with chlorhexidine and 70%ethanol, and use sharp scissors to re-incise the original midline incision.
Use the scissors to cut into the muscular layer, and carefully perform a left medial visceral rotation to expose the repaired sigmoid colon. Identify the repair sight by locating the tails of the polypropylene suture used for the repair. Gently and bluntly dissect any bowel to bowel adhesions away from the sight of repair, taking care not to disrupt the anastomosis.
Use a marking pen to demarcate the antimesenteric border of the colon, before removal of the colon segment. Next, use the scissors to transversely transect through the descending colon, one centimeter from the pair, up to one centimeter distal from the sight of anastomosis. The post operative tissues tend to be extremely fragile and surgical loops as well as gentle and meticulous handling of tissue, help to ensure against the disruption of the surgical anastomosis.
Use fine scissors to sharply dissect the colon from the attached mesentery along the posterior border of the colon to remove the sigmoidal segment. And cut along the length of the mesenteric border in a longitudinal fashion, to create a rectangular section of colon from the previously removed cylindrical specimen. And make a longitudinal cut along the length of the colon, opposite the marking.
Then fix, imbed, and cut the sample tissue as necessary for histology and or immunostaining. Histological analysis, seven days after injury, reveals a fibrotic healing response, mediated by smooth muscle alpha actin positive myofibroblasts. While attempting this procedure, it's important to remember to perform an insufflation test with the anastomosis submerged under normal saline, to confirm the patency of the anastomosis, and to ensure that no leak is present.
This procedure can be performed on genetically modified, or pathogenic mice with wound healing disorders, such as diabetic or immuno suppressed mice, to answer additional questions about the factors that contribute to an anastomotic leak. Following this procedure, immunohistochemistry and cell culture can be performed from anastomotic tissue harvested at specific time points following the surgery, to provide a better understanding of the cells involved, and their roles in the anastomotic healing process. Don't forget that working with Isoflurane can be extremely hazardous, and that precautions, such as using carbon filtration and ventilation hoods should always be taken while performing this procedure.
