Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal model is a widely adaptable and physiologic way to measure how leaky the gut is and how leukocytes migrate in the intestine in mice, in vivo. This method helps to better understand mechanisms underlying increased intestinal permeability and pathologic intestinal inflammation. The technique can be employed to aid in our understanding mechanisms underlying gut barrier function, and pathological inflammation, as seen in ulcerative colitis and Crohn's disease.

This model employs a well vascularized, exteriorized bowel segment, either of the ileum or proximal colon, for the examination of the intestinal barrier function or immune cell recruitment in mouse. With this broad application, the intestinal loop model can provide insight in diseases resulting either from a leaky gut barrier as well as the inflammatory response. Struggles can be overcome by practicing the surgical steps.

It is critically important to exteriorize a fully vascularized intestinal segment and select appropriate locations for the ligations to avoid bleeding. The intestinal loop model is a microsurgical technique, thus, as with other surgical procedures, it benefits greatly from visual demonstration and a step-by-step presentation of the method. After achieving a surgical plane of anesthesia, begin by scrubbing the fur of the abdominal midline with alcohol swabs or a 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 horizontal incision in the middle of the abdomen, and expose the peritoneum, taking care to not injure intra-abdominal organs.

Place precut wet cotton gauze over the exposed intra-abdominal cavity. Use wet cotton swabs to mobilize and exteriorize the cecum and carefully place it on wet cotton gauze. Then, mobilize and gently exteriorize the ileum.

Deploy at least six centimeters of terminal ileum on the wet cotton gauze without disrupting the mesenteric vessels and the blood supply. Keep the exposed tissues moist at all times with warm HBSS. Identify the major artery supplying the ileum in the mesentery, close to the cecum.

Then, locate two ligation sites in the mesentery that are free of critical blood vessels. Firmly grab the terminal ileum with blunt tissue forceps and use fine tip forceps to fenestrate the mesentery, avoiding blood vessels. Place silk suture across the perforation, and tie a surgical knot to create the first ligation.

Use the ruler to measure four centimeters away from the first ligature, and create the second ligature. Carefully cut next to each ligation with fine scissors to isolate the four centimeter ileal loop, keeping the blood supply and mesenteric membrane intact. Gently flush the content of the ileal loop segment with warm HBSS 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. Ligate the two cut ends of the flushed ileal loop with silk suture. Use a one milliliter syringe with a 30 gauge needle to slowly inject 250 microliters of reagent, such as FITC-dextrans or chemokine, into the intestinal lumen.

The ileal loop will inflate, causing a moderate distension of the mucosa. Close the abdominal wall using a needle holder, anatomical forceps, and 3.0 non-absorbable silk sutures with a reverse cutting needle. After drying the animal to prevent hypothermia, place it in a temperature regulated anesthesia chamber for the incubation period.

Prepare the mouse for surgery and exteriorize the cecum as previously demonstrated. Using wet cotton swabs, exteriorize the entire ileum and place it on top of a wet cotton gauze. Identify the proximal colon and 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 cecum. Measure two 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 two 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 one milliliter syringe with a 30 gauge needle to slowly inject 200 microliters of reagent, such as FITC-dextran 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 cecum.

Close the abdominal wall using a needle holder, anatomical forceps, and 3.0 non-absorbable silk sutures with a reverse cutting needle. After the incubation period, euthanize the anesthetized mouse and collect tissues for analysis. In order to verify the accuracy of the I loop model for the assessment of intestinal permeability, an FITC-dextran pcLoop assay was performed to evaluate the role of tight junction associated protein Jam-a in the regulation of intestinal barrier function in vivo.

With the pcLoop model, a 2.5 fold increase in FITC-dextran serum levels was quantified in Jam-a null mice compared to controls. Similar results were obtained with mice harboring selective loss of Jam-a on intestinal epithelial cells. The pcLoop model was used to study polymorphonuclear neutrophil, or PMN, recruitment into the intestinal mucosa and subsequent transepithelial migration in vivo.

The number of PMN in the luminal content of the pcLoop was quantified by flow cytometry analysis. The number of PMN present in the segment of the proximal colon was low under physiological conditions. Pretreatment with pro-inflammatory cytokines, TNF alpha and IFN gamma, prior to surgery, resulted in augmented numbers of PMN recruited in the pcLoop lumen.

Administration of the PMN chemo attractant LTB4 led to a dramatic increase in PMN counts. Immunohistochemical staining of PMN in the colonic mucosa corroborate the elevated recruitment of PMN following stimulation with cytokines and LTB4. The contribution of Jam-a to PMN transepithelial migration was studied using the pcLoop model on mice harboring selective loss of Jam-a on intestinal epithelial cells.

Loss of epithelial Jam-a led to a reduced number of transmigrated PMN in the colonic lumen compared to littermate controls. A critical aspect for this technique is to remember to preserve blood supply to the exteriorized intestinal segment when performing the surgery. Following this protocol, other methods such as an intestinal permeability assay and a neutrophil transepithelial migration assay can be performed to investigate loss of barrier integrity and intestinal inflammation in vivo.