Evaluating Therapeutic Interventions in the SHIP-deficient Mouse Model of Crohn Disease-like Ileitis and Fibrosis

We use SHIP-deficient mice to study the mechanisms driving intestinal inflammation and fibrosis in Crohn's disease and to evaluate new drugs or therapeutic strategies to treat Crohn's disease. So we have used this model in preclinical studies to show the efficacy of glycogen IBD drugs for targeted drug delivery. Our protocol uses SHIP-deficient mice, which naturally develop ileal inflammation and fibrosis, providing a more physiologically relevant model than traditional chemically-induced or injury-based models.

Our findings establish SHIP-deficient mice as a robust model of ileal inflammation and fibrosis, enabling mechanistic studies and preclinical testing of therapies for Crohn's disease. To begin, fast the mice for four hours before performing the assay. Dissolve four kilodalton FITC-dextran in sterile PBS to reach a final concentration of 80 milligrams per milliliter.

Next, gently restrain each mouse and administer 150 microliters of the FITC-dextran solution via oral gavage, then, keep the mice without food for another four hours after gavage. After euthanizing the mouse, perform cardiac puncture using a 25-gauge needle and a one milliliter syringe to collect blood. Immediately add 10 microliters of acid-citrate-dextrose solution to every 100 microliters of collected blood as an anticoagulant.

Mix thoroughly by inverting the tube several times. Centrifuge the collected blood samples at 1, 500 G for 10 minutes at four degrees Celsius. Carefully transfer the plasma to labeled 1.7 milliliter microcentrifuge tubes.

Keep all samples on ice and shielded from light until they are ready for analysis. Now, dilute each plasma sample at one to two and one to 10 ratios using PBS. Add 100 microliters of each plasma dilution into an opaque 96-well plate, placing them in duplicate wells.

Perform one to two serial dilutions of the original 80 milligrams per milliliter FITC-dextran stock using PBS to prepare different concentrations of the solution. Add 100 microliters of each dilution into duplicate wells of the 96-well plate. Then, measure fluorescence using a microplate reader set to 485 nanometers for excitation and 535 nanometers for emission.

Following blood collection For the FITC-dextran assay, carefully excise the entire small intestine from the mouse. Gently remove any attached fat and connective tissue from the intestine. Lay the cleaned intestine flat on a sheet of blank white paper to enhance visual contrast.

Examine the tissue visually for any macroscopic signs of disease, paying particular attention to the distal 10 centimeters in SHIP knockout mice, where inflammation commonly occurs. Align the intestine alongside a ruler to provide scale and photograph the entire specimen to document any gross pathological findings. Gently flush the intestinal lumen with PBS using a syringe to remove any intestinal contents while avoiding excessive force to prevent distortion of villus and crypt architecture.

Identify the region of the distal ileum most representative of gross pathology and excise approximately one centimeter of tissue. Lay the excised tissue flat in a histology cassette and sandwich it between two sponges to prevent folding during fixation. For hematoxylin and eosin staining and Masson's trichrome staining, fix the tissue in 10%neutral buffered formalin using 10 to 20 times the tissue volume overnight at four degrees Celsius.

After fixation, transfer the cassettes to 70%ethanol for storage and ensure they remain completely submerged until processing. For Alcian blue and periodic acid-Schiff staining, fix the tissue in Carnoy's solution overnight at four degrees Celsius. After fixation, transfer the cassettes to 100%ethanol for storage until processing.

Then, embed the fixed tissues in paraffin and section them into five micrometer slices for subsequent staining. Score the hematoxylin and eosin-stained ileal sections using a 16-point scale according to defined histopathological criteria. Evaluate the Masson's trichrome stained cross-sections using a six-point scale based on predefined criteria.

Calculate the final fibrosis score as the median of the scores assigned by two independent reviewers who are blinded to the experimental conditions. Following dissection and removal of the histology sample, collect the remaining portion of the distal 10 centimeters of the ileum. Blot the collected tissue dry with a paper towel or lint-free wipe, then record the tissue weight using a calibrated scale.

Flash freeze the tissue in liquid nitrogen before storing it at minus 80 degrees Celsius for future processing. When ready for processing, retrieve the frozen tissue and always maintain it on ice. Then, weigh the tissue samples again to confirm accurate mass.

Prepare the homogenization buffer by adding protease inhibitor to PBS. Use 10 microliters of buffer per milligram of tissue. Using a benchtop homogenizer, homogenize the full thickness ileal tissue in ice cold homogenization buffer.

Continue until the homogenate is uniform and free of visible tissue fragments. Rinse the homogenizer tip thoroughly with PBS between samples to prevent cross-contamination. Centrifuge the tissue homogenates at 10, 000 G for 10 minutes at four degrees Celsius.

Aliquot the clarified supernatants into smaller volumes to prevent freeze-thaw cycles, then store them at minus 80 degrees Celsius for future analysis. Histological analysis revealed significantly greater intestinal damage in SHIP-deficient mice compared to SHIP controls. Dexamethasone treatment reduced histological damage in SHIP-deficient mice, although the decrease was not statistically significant.

Masson's trichrome staining showed significantly increased collagen deposition in SHIP-deficient mice relative to SHIP controls, indicating higher fibrosis scores. Dexamethasone treatment markedly reduced fibrosis in SHIP-deficient mice, bringing scores closer to SHIP-positive controls. Alcian blue and PAS staining confirmed goblet cell hyperplasia in SHIP-deficient mice, which was visibly reduced following dexamethasone treatment.

IL-1 beta concentrations were significantly elevated in SHIP-deficient mice relative to SHIP controls. Dexamethasone significantly reduced IL-1beta concentrations in SHIP-deficient mice. MPO concentrations were significantly elevated in SHIP-deficient mice compared to SHIP controls, and this increase was reduced by dexamethasone without reaching statistical significance.

LCN-2 concentrations were also elevated in SHIP-deficient mice, but high values were not consistently reduced by dexamethasone. IL-1 beta concentrations positively correlated with histological damage scores and fibrosis scores, indicating a link between inflammation and tissue injury. Histological damage scores positively correlated with fibrosis scores, suggesting that tissue damage and fibrotic remodeling progressed in parallel.