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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Generation of the ileal loop
- Skin preparation: Scrub fur of the abdominal midline with alcohol swabs or gauze sponge soaked with 70% Ethanol. Do not wet a wide area of fur with alcohol to prevent hypothermia.
- Using scissors, perform a midline laparotomy. Make a vertical incision in the middle of the abdomen (about 2 cm in length) and expose the peritoneum. Be careful to not injure intra-abdominal organs.
- Place pre-cut wet cotton gauze over the exposed intra-abdominal cavity.
- Use wet cotton swabs to mobilize and exteriorize the caecum. Carefully place the caecum on the wet cotton gauze.
NOTE: Caecum is localized in the left caudal quadrant of the abdominal cavity in most mice independent of the sex of the animal.
- Use wet cotton swabs to mobilize and gently exteriorize the ileum of which the terminal section (distal end) is attached to the caecum (Figure 1B).
- Deploy at least 6 cm of terminal ileum on the wet cotton gauze without disruption of the mesenteric vessels and blood supply. Blood supply is maintained if there is no bleeding, and the tissue maintains its pink color (Figure 1B).
NOTE: Avoid drying of exposed tissues by maintaining tissues moist all the time with warm HBSS (every 2 - 3 min) using 10 mL syringe pre-attached to a yellow feeding tube.
- Close to the caecum, identify the major artery in the mesentery, supplying blood to the ileum. Then locate two ligation sites in the mesentery that are free of critical blood vessels.
- Using blunt tissue forceps, firmly grab the terminal ileum (closest to the caecum) and using fine tip forceps, fenestrate the mesentery avoiding blood vessels. Place silk suture across the perforation and tie a surgical knot to create the first ligation (distal end of the loop).
- Use the ruler to measure 4 cm away from the first ligature and create the second ligature (proximal end of the loop) as mentioned in step 2.8 (Figure 1C).
- With fine scissors carefully cut next to each ligation to isolate the 4 cm ileal loop, keeping intact blood supply and mesenteric membrane.
NOTE: Cut off both ends of the exteriorized segment of the iLoop, then flush gently as a necessary step that prevents interference with luminal contents (fecal matter), thus facilitating even dispersion of FITC-dextran or chemotactic stimuli across the entire length of the isolated segment as well as allowing for more accurate quantification of leukocytes by flow cytometry. This procedure also allows uniform distension of the mucosa after injection of specified volumes of reagent and better reproducibility between animals.
- Gently flush the content of the ileal loop segment with warm HBSS using a flexible yellow feeding tube attached to a 10 mL syringe.
- Ligate the two cut ends of the flushed ileal loop using silk suture.
- Use a 1 mL syringe with 30 G needle to slowly inject 250 µL of reagent such as FITC-dextran or chemokine into the intestinal lumen. The ileal loop will inflate causing a moderate distension of the mucosa (Figure 1D).
NOTE: Inject reagent into the loop lumen on the opposite side of the mesenteric artery. Be careful not to pull out the ileal loop from the animal while injecting to avoid tearing blood vessels and induce bleeding.
- Using wet cotton swabs, gently put back the ileal loop, proximal ileum, and caecum.
- Use a needle holder, anatomical forceps and 3.0 non-absorbable silk sutures with reverse cutting needle to close the abdominal wall.
- Place the animal in a temperature-regulated anesthesia chamber for the incubation period.
