Challenge Whole Body Plethysmography TCP_CHL_003

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Purpose

The purpose of this procedure is to record the respiratory function of mice after sensitization with an inert allergen (ovalbumin; OVA) in response to challenge with a non-allergen airway agonist (Methacholine; MCh) using unrestrained whole-body plethysmography and assess the ability of the immune system to mount an acquired Type I allergic hypersensitivity immune response (allergic airway disease).

Experimental Design

Minimum number per strain:

One cohort of 6M mutants + 2M WT controls
One cohort of 6F mutants + 2F WT controls
Age at start of test: 6-8 weeks
Sex: Both (sexually dimorphic)

Equipment

  1. Scale/balance
  2. Flow chambers
  3. Plethysmographs
  4. Nebulizers
  5. Computer connected to flow chamber
  6. Challenge reagents
  7. Personal safety equipment (masks, gloves, etc.)

Procedure

Sensitization:

  • Day 0: administrate OVA (20 µg) + Alum (2mg ) ip
  • Day 7: administrate OVA (20 µg) + Alum (2mg ) ip
  • Day 14: administrate aerosol 2.5% OVA (40min/day)
  • Day 15/16: administrate aerosol 2.5% OVA (40min/day)
  • Day 17/18: administrate aerosol 2.5% OVA (40min/day)
  • Day 20: administrate aerosol 2.5% OVA (40min/day)
On the day of the Whole Body Plethysmography procedure:
  1. Transfer the cohort to be tested to the test room.
  2. Weigh each mouse in the cohort and record weight.
  3. Turn on amplifier, nebulizer and computer, and calibrate the equipment.
  4. Place each mouse in the cohort in an individual plethysmograph chamber and acclimatize for 30 minutes.
  5. Methacholine (MCh) challenge:
    • Measure and record baseline data for 5 minutes
    • Nebulize with PBS for 2 minutes
    • Measure and record response to PBS for 5 minutes
    • Nebulize with 12.5mg/ml MCh for 2 minutes
    • Measure and record response to 12.5mg/ml for 5 minutes
    • Nebulize with 25mg/ml MCh for 2 minutes
    • Measure and record response to 25mg/ml for 5 minutes
    • Nebulize with 50mg/ml MCh for 2 minutes
    • Measure and record response to 50mg/ml MCh for 5 minutes
  6. Remove each mouse from its chamber and place back in the home cage.
  7. Sacrifice the mouse and collect samples:
    • Whole blood by cardiac punture to prepare for serum analysis of IgE and IgG
    • Bronchoalveolar lavage (BAL) fluid into 1.5ml eppendorf tube for analysis of inflammatory cells and cytokines
    • Whole lung tissue immersion fixation in 10% NBF for histopathology

 

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