Murine Appendectomy Model of Chronic Colitis Associated Colorectal Cancer by Precise Localization of Caecal Patch

The overall protocol describes easily performed surgical procedures of removing the appendix known as caecal patch in mice. The murine appendectomy model can be used to study the biological roles of appendix in pathogenesis of the human gastrointestinal disease. The human appendix is a disputable organ.

Recent studies have suggested that the appendix plays an important role in maintaining the gut health. To study the biological function of appendix, generating a disease-associated appendectomy animal model is critical. In this protocol, we will describe a simple and a safe surgical steps of removing appendix, known as caecal patch in mice.

To ensure the success of this model, there are three key steps as you will see in the following video. First, locate the caecum of the mice before the surgery. Second, make a small incision in the abdomen.

Third, disinfect and close the caecum stump. The first part, mice appendectomy. Prepare sterile surgical instruments and cleaning solutions before the surgery.

Anesthetize and secure the mouse on a platform in a supine position by placing four strips of medical adhesive tape across the limbs to prevent postural movement during the surgery. Gently touch the whole abdomen of the mouse to find the feeling of bump. Cover the drape and disinfect the shaved area of mouse abdomen as wide as possible using two times of entoiodine.

Make a longitudinal incision ranging from 0.5 centimeter to one centimeter at the midline of abdomen. According to the predetermined location of the caecum, seek the caecum and gently exteriorize it for approximately one centimeter long to identify the caecal patch. Use a pre-warmed and sterile physiological saline to hydrate the intestine.

To prevent potential complications of post-operative bleeding and infection, ligate the mesenteric blood vessels of caecum using the 8-0 suture. Hydrate the caecum with the saline. Mark the resection region using the 4-0 suture with open loop near the apex of the caecum at the proximal colon.

Cut off the caecal patch below the marked resection using micro-scissors and wipe out the residual caecal content with medical cotton swabs. Disinfect the stump of caecum with entoiodine. Close the stump with running suture using the 8-0 suture.

Carefully remove the 4-0 thread loop, previously used for marking the resection at the stump of caecum. Sterilize the sutured position using entoiodine. Rehydrate the cutting area with the saline.

Close the musculature layer with running suture using 8-0 suture thread. Close the skin layers using interrupted sutures with 4-0 suture thread. Disinfect the surgical cut twice with entoiodine.

Then, remove iodine using 75%medical alcohol. Gently flip the mouse back. Subcutaneously inject 0.1 milligram per kilogram body weight of buprenorphine, and 0.4 milliliters of physiological saline.

And rest the mouse on the heat pad, until it returns to consciousness. Put the mouse back to the sterile cage, and closely monitor the mouse for the signs of pain for three days post the surgery to ensure its recovery. The second part:Induction of Colitis-Associated Colorectal Cancer with Azoxymethane, AOM, and Dextran Sodium Sulfate, DSS, seven days post-appendectomy.

Thaw one aliquot of AOM and dilute it to the 1 milligram per milliliter using 0.9%normal saline. The entire preparation process is carried out in the fume hood. Dissolve four grams DSS powder in 200 milliliters autoclaved water solution to prepare 2%DSS.

Each cage contains five mice. Make an intraperitoneal injection of AOM with 0.01 milliliter per gram per mouse basing on its weight. Replace the autoclaved water with 2%DSS for five days.

The mice will be fed with autoclaved water for additional two cycles. The third part:Results. Flow and ELISA results indicate the successful removal of the caecal patch.

This procedure can contribute to significant reduction of IgA-specific plasma cells, an obvious change of secretory IgA in large bowel, compared to 3%DSS, which has high mortality rate and significant weight change because of severe bloody stool, anus prolapse, and abdominal abcess. The 2%DSS is suitable for the whole process for its high survival rate to nearly 80%and reasonable weight change in both sham and appendectomy groups. As shown in these images, we have successfully induced the colitis-associated colorectal tumors.

In H&E staining images, black arrow indicates mucosal ulcer, arrowhead indicates adenocarcinoma, and a white arrow indicates neutrophil infiltration between glands. Different stages of adenocarcinoma can be seen in both sham and appendectomy groups. This cost-effective Murine Appendectomy Model can be used for answering some important questions such as:what's the role of appendix in the regulation of gut bacteria and how it participates in the immune response in the gut-associated lymphoid tissue.

Remember, to avoid the damage in adhesion of intestine, the pre-location of caecum and small skin incision are important. In addition, we used the 2%DSS to induce colitis. But a higher concentration of DSS may be used for other mice strains.