The overall goal of this protocol is to facilitate the study of the immune system's role and its interaction with the microbiome in the development of Diversion Colitis. This method can help answer key questions regarding Diversion Colitis which occurs in both segments excluded from the fecal stream and affects up to 90%of patients with Diverting Enterostomy. The main advantage of this technique is that it does not require genetically modified animals, the use of chemically irritating substances or the transfer of specific cell populations to immune deficient mice to induce colitis.
Upon arrival at the animal facility, divide mice into groups of similar size. Cage each group together and keep the groups constant throughout the experiments. At least one week before surgery, switch to a high energy and high protein feed containing all the essential trace elements and vitamins.
To prepare for surgery after anesthetizing the animal according to the text protocol, use tape to stabilize the animal in a supine position on a heating pad to prevent loss of body heat. Then, check that the mouse tolerates mechanical stimulation e.g. toe pinch without a motor response.
Before starting surgery, disinfect the operation field three times using alcohol 70%and an iota 4. Drape the operation field to guarantee for aseptic conditions. Perform a 15 mm medium laparotomy by incising the abdominal muscles and the peritoneum along the linea alba thus minimizing blood loss.
Then, using two debakey atraumatic forceps, carefully pull the cecum, terminal ileum and descending and transverse colon from the peritoneal cavity. Be careful to limit the mechanical manipulation of the intestine in order to avoid injury to mesenteric structures and to maintain the optimal profusion of the intestine. Next, identify the cecal pole, the ascending colon and the small intestine.
If the anatomy of the ileocecal region is ambiguous, the presence of Peyer's patches identifies the small intestine and the presence of forms ature characterizes the colon. Use a ruler to determine the position of the future colostomy. It should be placed 20 mm distal to the ileocecal valve for a distal colostomy.
Make a second 3 mm incision in the abdominal wall in the upper right quadrant. Then, pull the previously identified colon segment through this incision to form a loop, taking care not to distort the loop. Carefully pass a 22 gauge, flexible IV canula through the mesocolon, taking care not to damage the mesenteric vascular structures.
Then, return the intestine to the peritoneal cavity. Using simple stitches and a resorbable suture, fix both ends of the flexible tube to the skin. Before closing the laparotomy, perform fluid resuscitation using an intra peritoneal injection of 0.5 mL of 0.9%saline.
Close the peritoneum and the muscle layer with a continuous resorbable suture. Then, close the skin in a similar manner. Using a fine scissor, open the exteriorized colon loop by performing a subtotal transection.
Avoid all injury to the mesentery and do not completely transect the colon. With a monofil absorbable suture, fix each colostomy opening using three single full thickness stitches to the peritoneum and skin. The afferent loop, which is a functional end colostomy and the efferent loop, which is a mucus fistula are clearly separated at this point.
To carry out a colotomy, after identifying the cecal pole, ascending colon and small intestine as demonstrated for the distal colostomy, use a ruler to determine the future colotomy position which should be positioned the same distance from the ileocecal valve as the colostomy. Using fine scissors, open the colon at least two-thirds its circumference. Using a monofil absorbable suture, close the colotomy with a full thickness interrupted suture before carrying out fluid resuscitation and closing of the peritoneum and skin as before.
Following the procedure, return animals to their cages and place them under an infrared lamp at 37 degrees celsius until they are fully awake before returning them to a temperature and humidity controlled environment. When animals show response to mechanical stimulation, start postoperative analgesia, by injecting 0.1 mg per kg body weight, buprenorphine SC.Carefully check for signs of respiratory depression. Supplement drinking water with 1 mg per mL of tramadol for continued analgesia during the first postoperative week.
To compensate for decrease fluid intake due to reduced mobility, supply a solid drink pad in the cage during the first postoperative week. Finally, weigh the animals and score animal behavior daily during the first week, every second day during the remainder of the first month, and every third day during the second month. Surgery is well tolerated both in the experimental and the sham groups.
Weight loss is more pronounced in the experimental group reaching 21.7%of initial body weight. In contrast, sham animals lose 10.8%of their initial body weight. Total mortality during the first 60 days postoperatively is around 40%in the colostomy group and about 10%in the sham group.
Most deaths occurred during the first postoperative week, with the causes of death shown in this figure. Distal colostomy in mice results in the development of predominantly lymphocytic colitis, reproducing the hallmark histological features of human DC.Crypt length is significantly shortened in excluded bowel segments and reaches statistical significance after 14 days of fecal diversion. The goblet cell-bearing crypt length is reduced after 30 days in excluded bowel segments.
Absolute goblet cell numbers are also significantly reduced in crypts in the excluded bowel segment after 14 postoperative days. The hallmark lesion of DC, the development of lymphoid follicles in the mucosa requires a longer duration of stool deviation. Although, an increased number of the lymphoid follicles can be observed as early as two weeks, differences become significant after two months.
After watching this video, you should have a good understanding of how to perform a Murine Distal Colostomy. Once mastered, this technique can be done in less than 20 minutes if performed properly. This model can be combined with other genetically modified models or transfer experiments in congenic mouse strains, in order to address the role of inflammatory mediators of specific cell populations, and the pathogenesis of Diversion Colitis.
