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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
Equipment
- Scale/balance
- Flow chambers
- Plethysmographs
- Nebulizers
- Computer connected to flow chamber
- Challenge reagents
- 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:
- Transfer the cohort to be tested to the test room.
- Weigh each mouse in the cohort and record weight.
- Turn on amplifier, nebulizer and computer, and calibrate the equipment.
- Place each mouse in the cohort in an individual plethysmograph chamber and acclimatize for 30 minutes.
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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
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Remove each mouse from its chamber and place back in the home cage.
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Sacrifice the mouse and collect samples:
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Whole blood by cardiac punture to prepare for serum analysis of IgE and IgG
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Bronchoalveolar lavage (BAL) fluid into 1.5ml eppendorf tube for analysis of inflammatory cells and cytokines
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Whole lung tissue immersion fixation in 10% NBF for histopathology
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