Methods of Inducing Inflammatory Bowel Disease in Mice

Byoungwook Bang1, Lenard M. Lichtenberger1

1 Department of Integrative Biology and Pharmacology, The University of Texas Medical School at Houston, Houston, Texas
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 5.58
DOI:  10.1002/0471141755.ph0558s72
Online Posting Date:  March, 2016
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Animal models of experimentally induced inflammatory bowel disease (IBD) are useful for understanding more about the mechanistic basis of the disease, identifying new targets for therapeutic intervention, and testing novel therapeutics. This unit provides detailed protocols for five widely used mouse models of experimentally induced intestinal inflammation: chemical induction of colitis by dextran sodium sulfate (DSS), hapten‐induced colitis via 2,4,6‐trinitrobenzene sulfonic acid (TNBS), Helicobacter‐induced colitis in mdr1a−/− mice, the CD4+ CD45RBhi SCID transfer colitis model, and the IL‐10−/− colitis model. © 2016 by John Wiley & Sons, Inc.

Keywords: mouse; IBD; model; colitis

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Table of Contents

  • Introduction
  • Basic Protocol 1: Induction of Dextran Sodium Sulfate (DSS) Colitis in Mice
  • Support Protocol 1: Measurement of Fecal Hemoglobin Concentration
  • Support Protocol 2: Necropsy and Collection of Intestinal Tissue
  • Basic Protocol 2: Induction of TNBS Colitis in Mice
  • Basic Protocol 3: Induction of Colitis in MDR1A–/– Mice by Inoculation with Helicobacter Bilis
  • Basic Protocol 4: Induction of Colitis in SCID Mice by Adoptive Transfer of CD4+ CD45RBhi T Cells
  • Induction of Colitis IL‐10−/− Mice with Inoculation of Piroxicam or Bacteria
  • Basic Protocol 5: Piroxicam‐Associated Colitis (PC) in IL‐10−/− Mice
  • Basic Protocol 6: Bacterial‐Induced Colitis in IL‐10−/− Mice
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Induction of Dextran Sodium Sulfate (DSS) Colitis in Mice

  • Age‐ and sex‐matched C57BL/6 mice, 8‐ to 16‐weeks old (Taconic or other preferred vendor)
  • Autoclaved or sterile drinking water
  • Dextran sodium sulfate (DSS, mol. wt. 35,000 to 50,000 Da; MP Biomedicals)
  • Test compound: e.g., cyclosporin A (Sigma)
  • Olive oil or polyethylene glycol 400 vehicle for cyclosporin A (Sigma)
  • Animal balance, accurate to 0.1 g
  • Animal cages fitted with a water bottle
  • Additional reagents and equipment for measurement of fetal hemoglobin concentration ( protocol 2) and necropsy and tissue collection ( protocol 3)

Support Protocol 1: Measurement of Fecal Hemoglobin Concentration

  • Fecal sample, frozen
  • 2 mg/ml hemoglobin (Hb; Sigma, cat. no. H3760)
  • Benzidine (dihydrochloride) reagent (Sigma, cat. no. B3383)
  • Acetic acid (Fisher Scientific)
  • 1% (v/v) hydrogen peroxide (Fisher Scientific), diluted immediately before use
  • Balance
  • Centrifuge
  • Spectrophotometer: Genesys 10 μv (Thermo Scientific)

Support Protocol 2: Necropsy and Collection of Intestinal Tissue

  • Mice (C57BL/6 mice and other preferred strain)
  • Carbon dioxide source
  • Phosphate‐buffered saline (PBS; appendix 2A; optional)
  • 10% neutral buffered formalin (Thermo Scientific)
  • 70% ethanol
  • Hematoxylin and eosin (H&E) stains
  • Anesthetizing box (e.g., AB‐1; Braintree Scientific)
  • Dissection tools: Scissors and forceps
  • Ruler, accurate to nearest mm
  • Balance, accurate to 0.01 g (optional)
  • Blunt needle attached to syringe (optional)
  • Tissue cassettes (Thermo Scientific)
  • Additional reagents and equipment for paraffin embedding and sectioning (appendix 3d; Zeller, )

Basic Protocol 2: Induction of TNBS Colitis in Mice

  • Female 8 to 10 week old SJL/J mice (Taconic or other preferred vendor)
  • TNBS (2,4,6‐trinitrobenzene sulfonic acid, aka picrylsulfonic acid; Sigma)
  • 50% (v/v) ethanol
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Isoflurane (Sigma) or other inhalable anesthetic
  • Surgilube or other surgical lubricant
  • Test compounds
  • Dexamethasone (Sigma), dissolved in water at a stock concentration of 10 mg/ml, stored at 4°C
  • Animal cages
  • Animal balance, accurate to 0.1 g
  • Posi‐seal mouse induction chamber (Molecular Imaging Products Company,
  • Polyethylene catheter (Becton Dickinson 1‐ml syringe fitted with an Intramedic PE‐20 tubing polyethylene cannula)
  • Additional reagents and equipment for necropsy and tissue collection ( protocol 3)
CAUTION: TNBS is a hazardous chemical with carcinogenic potential. Safety glasses and gloves should always be worn when handling this substance. The ethanol diluent for TNBS is flammable. Precautions must be taken to avoid an open flame, and appropriate handling and storage conditions must be used. TNBS is light sensitive and labile. It should be kept in the dark at 4°C and reagents greater 6 months old should be discarded.

Basic Protocol 3: Induction of Colitis in MDR1A–/– Mice by Inoculation with Helicobacter Bilis

  • Female mdr1a−/− mice (FVB.129P2‐Abcb1atm1Bor), 8‐ to 10‐weeks‐old (Taconic)
  • Brucella broth, lyophilized (Remel)
  • Fetal bovine serum (FBS), heat‐inactivated at 56°C
  • Brain Heart Infusion (BHI) broth (Remel)
  • Brucella agar plates with 5% sheep blood (Remel)
  • Compressed gas containing 90% N 2, 5% H 2, and 5% CO 2
  • Frozen vial of Helicobacter bilis (ATCC)
  • 30% hydrogen peroxide (Sigma‐Aldrich)
  • BactiDrop Oxidase (Remel)
  • Urea agar slant, 15 × 103–mm tube (Remel)
  • BBL Gram Stain Kit (BD Difco)
  • Female FVB mice, 8 to 10 weeks old (Taconic)
  • Test compounds
  • Positive control: CTLA4‐Fc (R&D Systems)
  • Inactive control such as distilled water or vehicle
  • Mouse cages
  • Animal balance, accurate to 0.1 g
  • 50‐ml conical polypropylene centrifuge tubes
  • Bacterial culture spreaders
  • Anaerobic jars (Oxoid)
  • Vacuum pump to create microaerophilic conditions
  • 250 ml PETG Erlenmeyer flasks with baffled bottom and vented cap (Nalgene)
  • Sterile bacterial loops
  • 37°C bacterial incubator with orbital shaker
  • Centrifuge with swinging‐bucket rotor
  • Glass microscope slides (Colormark Plus; Thermo Scientific)
  • Spectrophotometer capable of reading 600 nm
  • 20‐G disposable gavage needles (Cadence Science;
  • Additional reagents and equipment for necropsy and tissue collection ( protocol 3)

Basic Protocol 4: Induction of Colitis in SCID Mice by Adoptive Transfer of CD4+ CD45RBhi T Cells

  • Recipient female CB17 SCID mice, 8‐ to 10‐weeks‐old (e.g., Taconic)
  • Donor female BALB/c mice, 8‐10 weeks old (e.g., Taconic)
  • Phosphate‐buffered saline (PBS; appendix 2A), pH 7.2, containing 2% (v/v) fetal bovine serum (FBS; heat‐inactivated)
  • RoboSep Buffer (StemCell Technologies; PBS/2% FBS can be used as an alternative) with and without 5% (v/v) normal rat serum
  • EasySep Mouse CD4+ T Cell Enrichment Kit (StemCell Technologies) including:
  • Negative Selection Mouse CD4+ T Cell Enrichment Cocktail
  • EasySep Biotin Selection Cocktail (StemCell Technologies)
  • EasySep Magnetic Nanoparticles (StemCell Technologies)
  • Normal rat serum (NRS)
  • Fc blocking reagent: Anti‐mouse Fc Receptor II/III, clone 2.4G2, rat IgG2b (BD Biosciences)
  • Anti‐CD4‐FITC: fluorescein isothiocyanate (FITC)‐conjugated anti‐mouse CD4; clone H129.9, rat IgG2a (BD Biosciences)
  • Anti‐CD45RB‐PE: phycoerythrin (PE)‐conjugated anti‐mouse CD45RB; clone 16 A, rat IgG2a (Caltag/Invitrogen)
  • Anti‐mouse CD3‐FITC; clone 145‐2C11, Armenian hamster IgG1 (BD Biosciences)
  • Anti‐mouse CD3‐PE; clone 145‐2C11, Armenian hamster IgG1 (BD Biosciences)
  • FITC‐rat IgG2a isotype control; clone R35‐95 (BD Biosciences)
  • PE‐rat IgG2a isotype control, clone R35‐95 (BD Biosciences)
  • Phosphate‐buffered saline (PBS)
  • Test compounds
  • Positive control: CTLA4‐Fc (R&D Systems)
  • Mouse cages
  • Animal balance, accurate to 0.1 g
  • Mouse dissection tools: forceps and scissors
  • 100 × 20–mm petri dishes
  • Frosted, beveled slides
  • 70‐μm nylon mesh cell strainers (BD Biosciences)
  • 15‐ and 50‐ml conical polypropylene tubes (e.g., Falcon)
  • Centrifuge with swinging bucket rotor
  • Hemacytometer and microscope, or automated cell counter
  • Polystyrene round‐bottom tubes: 5‐ml tubes for FACS staining; 5‐ or 10‐ml tubes for magnetic separation)
  • EasySep Magnet or “Big Easy” EasySep Magnet (StemCell Technologies)
  • Fluorescence activated cell sorter (e.g., BD FACScan)
  • Additional reagents and equipment for euthanasia, necropsy, and tissue collection ( protocol 3)

Basic Protocol 5: Piroxicam‐Associated Colitis (PC) in IL‐10−/− Mice

  • IL‐10‐deficient mice back‐crossed with C57BL/6J background (The Jackson Laboratory)
  • Male 8‐ to 10 week old C57BL/6J mice (The Jackson Laboratory or other preferred vendor)
  • Standard mouse diet
  • Autoclaved mouse bedding
  • Piroxicam (Sigma‐Aldrich)
  • Test compounds
  • Animal micro‐isolator cages
  • Animal balance, accurate to 0.1 g
  • Additional reagents and equipment for fecal hemoglobin analysis ( protocol 2) and necropsy and tissue collection ( protocol 3)

Basic Protocol 6: Bacterial‐Induced Colitis in IL‐10−/− Mice

  • IL‐10−/− 129/SvI or C57BL/6 mice, 8 weeks of age (Taconic or other preferred vendor)
  • Antibiotic cocktail: e.g., 2 g/liter streptomycin, 1 g/liter bacteriocin, 0.5 g/liter gentamycin and 0.125 g/liter ciprofloxin in distilled water
  • C. jejuni 81‐176 (ATCC, cat. no. BAA‐2151)
  • Campylobacter selective blood plates (Remel, Thermo Scientific)
  • Columbia broth (U.S. Biological, cat. no. C7600‐02)
  • Animal micro‐isolator cages
  • Animal balance, accurate to 0.1 g
  • GasPak Jar (GasPak EZ Campy container system).
  • PS20 gavage needles
  • Spectrophotometer
  • Additional reagents and equipment for necropsy and tissue collection ( protocol 3)
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