This study presents a method to assess intestinal permeability using a proximal colon loop (pcLoop) model in mice. The procedure involves surgical isolation of a segment of the proximal colon to evaluate the transport of fluorescent markers into the bloodstream.
During pathological conditions, impaired intestinal epithelial tight junction barrier causes increased permeation of harmful luminal contents into systemic circulation, resulting in increased systemic inflammation.
To assess intestinal permeability by generating an intestinal proximal colon loop or pcLoop model, prep an anesthetized mouse in the supine position. Perform a midline abdominal incision. Gently exteriorize the cecum, terminal ileum, and proximal colon, placed over wet gauze to maintain organ hydration.
Locate the proximal colon’s mesocolon — membranous folds of peritoneum containing blood vessels. Identify two sites appropriately apart in the mesocolon free of blood vessels, distal from the cecum, to be ligated. Perforate the sites.
Creating the ligature in the selected sites, make incisions close to the ligated sites to isolate a pcLoop segment without disturbing the blood supply. Flush the segment with buffer to remove any fecal matter.
Surgically tie up the cut ends of the flushed segment. Inject desired fluorescent-marker solution into the colon segment lumen. Return the organs into the abdominal cavity. Close the surgical incision and allow the mouse to recover.
Following injection, reduced intestinal barrier integrity facilitates increased paracellular transport of the fluorescent marker molecules from the intestinal lumen into the blood circulation, which can be detected in the serum fraction of blood to measure the intestinal permeability.
Prepare the mouse for surgery and exteriorize the caecum. Using wet cotton swabs, exteriorize the entire ileum and place it on top of a wet cotton gauze. Identify the proximal colon into the blood supply located in the mesocolon. Mobilize the proximal colon, and create the first ligature in an area free of vessels in the mesocolon, at about 0.5 centimeters distal from the caecum.
Measure 2 centimeters from the first ligature, and create a second ligature at an area free of blood supply in the mesocolon. Using fine scissors, carefully cut next to each ligation to isolate a 2-centimeter-long pcLoop.
Gently flush the pcLoop with warm HBSS to remove feces using a flexible yellow feeding tube attached to a 10-milliliter syringe. Make sure to flush the luminal contents out of the abdominal cavity to keep the surgical site clean. Then, ligate the two cut ends of the flushed pcLoop using silk suture.
Use a 1-milliliter syringe with a 30-gauge needle to slowly inject 200 microliters of reagent such as FITC-dextrans or chemokine into the intestinal lumen. The pcLoop will inflate, causing a moderate distension of the mucosa. Use wet cotton swabs to gently put back the ligated pcLoop, ileum, and caecum. Close the abdominal wall using a needle holder, anatomical forceps, and 3.0 non-absorbable silk sutures with a reverse cutting needle.
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