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

All animal experiments were conducted in accordance with the guidelines and policies of the National Institutes of Health and approved by the Institutional Animal Care & Use Committee at the University of Michigan.

1. Preoperative preparation

NOTE: This method was generated employing adult mice from C57BL/6 genetic background, aged 8 – 12 weeks. All mice were kept under strict specific pathogen free conditions with ad libitum access to normal chow and water. Results were obtained using C57BL/6, Jam-a – null mice (Jam-a^-/-) or mice harboring selective loss of JAM-A on IECs (Villin-cre;Jam-a^fl/fl) and littermate Jam-a^fl/fl controls as previously described³⁰.

1. Area preparation
   1. Perform surgery in clean area. The intestinal Loop model, however, is a non-survival surgery that does not require aseptic/sterile technique. Observe veterinary sanitation practices and use cleaned surgical instruments (i.e., scrubbed with soap, rinsed with water followed by 70% ethanol).
   2. Turn on a temperature-controlled surgical board (or heating pads) and adapted light source to keep animal from hypothermia during the anesthesia and surgery.
   3. Prepare ligatures by cutting 6 cm segments of non-absorbable 4-0 silk surgical sutures.
   4. Prepare cotton gauzes (5 cm x 5 cm) that are cut in the center following an ellipsoid shape. These will be used to cover the midline laparotomy and prevent direct contact between the exteriorized iLoop and animal fur. Soak the cut gauzes in warm Hanks' Balanced Salt Solution (HBSS) in a petri dish container.
   5. Prepare wet cotton swabs soaked in warm HBSS that will be used to handle organs and exteriorized iLoop.
   6. Prepare 10 mL syringe filled with warm HBSS and attach to a yellow feeding tube. This syringe will be used for moisturizing exposed tissues during surgery and to gently flush the iLoop of fecal content.
2. Animal preparation
   1. Anesthetize the animal in accordance with the approved animal protocol. In this protocol a mix of isoflurane and oxygen is administered through an anesthesia vaporizer. According to manufacturer's instructions, adjust oxygen flow rate to 1 L/min. Set the vaporizer to 5% and pre-charge the induction chamber. After 5 min, reduce isoflurane vaporizer to 2% – 2.5%.
   2. Place the animal in the induction chamber for 3 min – 5 min, then transfer animal to a heated surgery board and connect an anesthesia nosecone plug. Restrain the animal in supine position by the four limbs using adhesive tape.
      NOTE: As an anesthesia alternative, a mixture of ketamine (80 mg/kg – 100 mg/kg) and xylazine (5 mg/kg – 10 mg/kg) diluted in saline solution (0.9% NaCl) can be administrated by intraperitoneal injection. Anesthesia should be maintained throughout the surgery by intramuscular administration of ketamine/xylazine (at 0.1 – 0.25 times of initial doses) to ensure anesthetic depth. If available, an isoflurane anesthesia vaporizer is highly recommended to assure better reproducibility, survivability and prevent animal pain.
   3. Apply ophthalmic ointment to both eyes to prevent corneal desiccation.
   4. Perform a physical exam that includes heart rate (around 500 beats/min) and rhythm, mucous membrane color (pink), capillary refill time (< 2 s), respiratory rate (not lower than 40 – 60 breaths/min), and temperature (36.5 °C)³².
   5. Before proceeding to the next steps, assess anesthetic depth by pedal withdrawal reflex. Employ a painful stimulus (pinch) of the skin between the toes and/or toe pads. Mouse will respond by contracting and removing its leg. This pedal reflex disappears when animal is anesthetized deeply.
      ​NOTE: Monitoring of vitals and pedal reflex are recommended throughout anesthesia at minimum every 15 min. Institutional Animal Care and Use Committee (IACUC) guidelines for evaluation of anesthetic depth recommend monitoring of the following: (a) color of tail, foot and mucous membrane (such as tongue). Color pink as normal and pale or blue as indicative of decreased blood perfusion or respiratory distress; (b) evaluation of the breathing pattern as regular versus irregular breaths. A rectal temperature probe, rodent oximeter and heart rate monitors can be used for the assessment of body temperature, heart and respiratory rates, respectively.

2. Generation of the ileal loop

1. 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.
2. 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.
3. Place pre-cut wet cotton gauze over the exposed intra-abdominal cavity.
4. 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 a majority of mice independent of the sex of the animal.
5. 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).
6. 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 at all times with warm HBSS (every 2 – 3 min) using 10 mL syringe attached to a yellow feeding tube (step 1.1.6).
7. Close to the caecum, identify the major artery supplying the ileum in the mesentery. Then locate two ligation sites in the mesentery that are free of critical blood vessels.
8. 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).
9. 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).
10. 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-dextrans 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.
11. Gently flush the content of the ileal loop segment with warm HBSS using a flexible yellow feeding tube attached to a 10 mL syringe (see step 1.1.6).
12. Ligate the two cut ends of the flushed ileal loop using silk suture.
13. Use a 1 mL syringe with 30 G needle to slowly inject 250 µL of reagent such as FITC-dextrans (step 4.2) or chemokine (step 5.3) 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.
14. Using wet cotton swabs, gently put back the ileal loop, proximal ileum and caecum.
15. Use a needle holder, anatomical forceps and 3.0 non-absorbable silk sutures with reverse cutting needle to close the abdominal wall.
16. Place the animal in a temperature-regulated anesthesia chamber for the incubation period.

3. Generation of the proximal colon loop (pcLoop)

NOTE: For details about mice that were used for the generation of the pcLoop, see the information provided at the beginning of the protocol section.

1. Perform steps 2.1. – 2.4. as described above for the ileal loop.
2. Using wet cotton swabs, exteriorize the entire ileum and place it on the top of a wet cotton gauze. Identify the proximal colon and the blood supply located in the mesocolon. Mobilize the proximal colon and by using fine tip forceps create the first ligature in an area free of vessels in the mesocolon at about 0.5 cm distal from the caecum (Figure 2B).
3. Measure 2 cm from the first ligature and create a second ligature at an area free of blood supply in the mesocolon (Figure 2C).
4. Using fine scissors carefully cut next to each ligation to isolate a 2 cm long pcLoop.
   NOTE: As mentioned in the note under step 2.10, it is important to cut off both ends to isolate a pcLoop that is gently cleaned of luminal contents. Carefully cut through the colonic tissue and mesocolon to prevent small vessels from bleeding into the intestinal lumen. If necessary, use thermal cautery to limit bleeding at the incision site.
5. Gently flush the pcLoop with warm HBSS to remove feces using a flexible yellow feeding tube attached to 10 mL syringe (see step 1.1.6).
6. Ligate the two cut ends of the flushed pcLoop using silk suture.
7. Use a 1 mL syringe with 30G needle to slowly inject 200 μL of reagent such as FITC-dextrans (step 4.2) or chemokine (step 5.3) into the intestinal lumen. The pcLoop will inflate causing a moderate distension of the mucosa (Figure 2D).
   NOTE: Inject the reagent into the pcLoop lumen on the opposite side of the mesenteric artery. Ensure consistency between animals and create a 2 cm long pcLoop to ensure equal distension of the mucosa.
8. Utilize wet cotton swabs to gently place back the ligated pcLoop, ileum and caecum into the abdominal cavity.
9. Use a needle holder, anatomical forceps and 3.0 non-absorbable silk sutures with reverse cutting needle to close the abdominal wall.
10. Place the animal in a temperature-regulated anesthesia chamber for the incubation period.

4. Quantitative assessment of intestinal permeability: 4 kDa FITC-dextran assay

1. Perform an ileal loop or pcLoop (as described above).
2. Using a 1 mL syringe with 30 G needle, inject into the intestinal lumen either 250 µL (ileum – step 2.13) or 200 µL (colon – step 3.7) of 4 kDa FITC-dextran solution (1 mg / mL in HBSS). Keep the unused FITC-dextran solution protected from light to prepare the standard curve after serum collection.
3. For the ileal loop follow steps 2.14 to 2.16, for the pcLoop steps 3.8 to 3.10. Briefly, put organs and iLoop back in place into the abdominal cavity, close the abdominal wall.
4. Place the animal for 120 min in a heated anesthesia chamber.
5. After the incubation period open the abdominal wall, gain access to the heart and perform cardiac puncture using a 1 mL syringe with 25G needle to collect the blood. Transfer blood into a 1.3 mL serum clot activator tube, mix gently, and keep on ice protected from light. Collect at least 500 µL of blood per mouse.
   ​NOTE: Animals are euthanized when being under anesthesia by using a physical method such as decapitation or cervical dislocation, and in accordance with the approved animal protocol.
6. Centrifuge serum clot activator tube for 5 min at 10,000 x g at room temperature according to manufacturer's recommendations. Collect the serum (supernatant) and transfer into a 1.7 mL centrifuge tube. Keep the tube on ice and protected from light.
7. Quantification of fluorescence in blood serum
   1. Prepare a standard curve of FITC-dextran 4 kDa in serum of control mice (saline or HBSS is a valid alternative). Create a two-fold serial dilution with a starting concentration of 1 mg/mL FITC-dextran. FITC-dextran 4 kDa concentrations that are measured range between 0.25 mg/mL and 2 µg/mL.
   2. Transfer equal volume of the sample and standards to a black 96-well plate (flat bottom) and measure FITC in a fluorescence plate reader (Excitation 490 nm, Emission 520 nm), according to manufacturer's instructions and published protocols³³. Calculate the FITC concentration based on the standard curve or present permeability values as fold-change normalized to the experimental control group.

5. Quantitative assessment of migrated PMN into the intestinal lumen after intraluminal stimulation with chemokines

NOTE: Very few PMN reside in the intestinal mucosa at the baseline level. Pretreatment of animals with pro-inflammatory cytokines results in an inflammatory environment that facilitates PMN recruitment from bloodstream into the intestinal mucosa.

1. Using a 1 mL syringe with 30 G needle, perform intraperitoneal (i.p.) injection of a sterile solution of 100 ng of Tumor Necrosis Factor-α (TNFα) and 100 ng of Interferon-γ (INFγ) in 200 µL of Phosphate Buffered Saline (PBS).
2. After 4 – 24 h of pretreatment with pro-inflammatory cytokines, perform an ileal loop or pcLoop (as described above).
3. Using a 1 mL syringe with 30 G needle, inject into the intestinal lumen either 250 µL (ileum – step 2.13) or 200 µL (colon – step 3.7) of chemoattractant solution Leukotriene B4 (LTB₄) 1 nM in HBSS.
   ​NOTE: Leukotriene B4 (LTB₄) is used in this protocol as a potent chemoattractant for PMN. Other chemoattractants such as N-Formylmethionyl-leucyl-phenylalanine (fMLF) or chemokine (C-X-C motif) ligand 1 (CXCL1/KC) can also be used to induce significant recruitment of PMN into the colonic lumen³⁰.
4. For the ileal loop follow steps 2.14 to 2.16. For the pcLoop follow steps 3.8 to 3.10. Briefly, put organs and iLoop back in place into the abdominal cavity, close the abdominal wall.
5. Place the animal for 60 min in a heated anesthesia chamber.
6. Collection of the intestinal loop content
   1. Prepare solutions and store on ice: For the ileal loop and pcLoop, prepare a wash buffer containing 2 mM Ethylenediaminetetraacetic acid (EDTA), 5 mM Dithiothreitol (DTT) and 2% FBS in sterile PBS without calcium and magnesium.
   2. After the incubation period and under anesthesia maintenance, open the abdominal wall and pull out the iLoop (ileal loop or pcLoop). Euthanize animals when being under anesthesia by using a physical method such as decapitation or cervical dislocation, and in accordance with the approved animal protocol.
   3. Rinse loop with cold PBS to remove any residual blood contaminant and absorb excess of PBS with tissue wipes. Carefully collect the loop content into a 1.7 mL centrifuge tube (about 250 µL for ileal loop and 200 µL for pcLoop). Flush the loop with 500 µL cold wash buffer and, immediately after collection, place the tube on ice.
      ​NOTE: DTT helps to dissolve mucus. If iLoop luminal content is very viscous (depending on the mouse genetic background), dilute it 1:2 or 1:3 with wash buffer containing DTT.
   4. Pass the luminal content solution through a 35 µm nylon mesh filter using a 5 mL round-bottom tube with cell strainer cap. This step helps to remove tissue fragments and cell aggregates. Rinse the cell strainer with 1 mL of wash buffer.
   5. Centrifuge tube at 400 x g for 5 min at 4 °C. Discard supernatant, rinse pellet with 500 µL – 1 mL wash buffer, then centrifuge at 400 x g for 5 min, 4 °C.
   6. Resuspend iLoop luminal cell pellet in 200 µL of flow cytometry buffer (FCB) containing 2% FBS in sterile PBS without calcium and magnesium. Cells can be kept in a tube or transferred to a 96-well round bottom plate for flow cytometry staining and analysis.
7. Flow Cytometry staining and analysis
   1. Compensation controls: white blood cells
      1. Prepare a 1 mL syringe with 25 G needle pre-filled with sterile 0.5 M EDTA (pH 8.0). 10% EDTA per expected blood volume (100 µL of EDTA for 1 mL blood).
      2. Collect blood under anesthesia by cardiac puncture. Transfer blood into a 1.7 mL tube, then centrifuge at 400 x g for 10 min, 4 °C.
         NOTE: While the mouse is under anesthesia use a physical method to confirm death in accordance with the approved animal protocol (such as cervical dislocation).
      3. Aspirate the supernatant. Resuspend the pellet in 1 mL of Ammonium-Chloride-Potassium (ACK) lysis buffer for the lysis of red blood cells. Incubate for 3 min – 5 min on ice. Centrifuge 400 x g for 5 min, 4 °C. If the pellet is still red, repeat this ACK lysis buffer step until pellet turns white.
      4. Resuspend the pellet in 1 mL FCB and plate 0.5 x 10⁶– 1 x 10⁶ of cells per well. Prepare five wells of a 96-well round-bottom plate. Place the plate on ice.
         ​NOTE: Use the same 96-well plate that contains the loop luminal contents (see step 5.6.6).
   2. Flow cytometry staining
      1. Centrifuge the 96-well plate for 5 min at 400 x g, 4 °C. Discard the supernatant and resuspend the pellets with 50 µL of Purified Rat Anti-Mouse CD16/CD32 as an Fc-Block (1 μg per 100 µL of FCB). Incubate for 5 min – 10 min on ice.
      2. Immunostaining of the iLoop luminal content: Prepare a mixture containing all fluorochrome-conjugated antibodies (1:50 dilution in FCB): anti-CD45-PerCP, anti-CD11b-PE and anti-Ly-6G-Alexa Fluor 647. Add 50 µL of the combination per well, for a final volume of 100 µL.
      3. Immunostaining of the white blood cells for compensations (1:50 dilution in FCB): Use 50 µL of FCB alone (unstained sample, well 1), 50 µL of each individual fluorochrome-conjugated antibody (wells 2 – 4), 50 µL of the combination of all fluorochrome-conjugated antibodies (well 5). Final volume of 100 µL.
      4. Incubate the plate for 30 min on ice protected from light.
      5. Centrifuge the plate for 5 min at 400 x g, 4 °C. Discard the supernatant and wash with 200 µL of FCB. Repeat this washing step twice.
      6. Add FCB 150 µL/well to the blood samples.
      7. Add 100 µL/well FCB to the iLoop luminal content sample. Then 50 µL/well of fluorescent counting beads.
   3. Flow cytometry analysis
      1. Gate for CD45 positive events and for the expression of Ly-6G-/Gr-1 and CD11b³⁰.
      2. Use 100 µL of the sample volume as a stop condition.
      3. Calculate the absolute number of PMN that has migrated into the iLoop lumen following the information provided by the manufacturer of the fluorescent counting beads.
         NOTE: Data may be presented as (1) total number of PMN in the lumen^30,34,35, (2) number of PMN per gram of tissue as well as (3) number of PMN per mm³ by using the formula for volume of a cylinder: V= π(pi) r 2 h (V for volume, r for radius and h for height).