Murine Asthma Models

Jean F. Regal1

1 School of Medicine, University of Minnesota, Duluth, Minnesota
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 18.3
DOI:  10.1002/0471140856.tx1803s21
Online Posting Date:  September, 2004
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Abstract

Immunotoxicity can take the form of an enhanced immune response or hypersensitivity. Asthma is one possible consequence of hypersensitivity in the lung, with characteristics that include reversible airway obstruction, eosinophil infiltration into the lung, and airway hyperresponsiveness to agonists such as methacholine. In toxicology, two primary areas of investigation prompt the measurement of the asthmatic response in an animal: (1) identification of chemicals or proteins that cause asthma, i.e., respiratory allergens, and (2) identification of exposures that will exacerbate existing asthma. An ovalbuminā€induced asthma model can be used to identify exposures that exacerbate existing asthma. A protocol for the sensitization and challenge of mice with ovalbumin is described; it leads to the asthma symptoms of airway hyperresponsiveness and eosinophil infiltration. Assessment of airway hyperresponsiveness to methacholine uses whole body plethysmography in conscious unrestrained mice. Bronchoalveolar lavage of the mouse determines the extent of cellular infiltration into the airspace. Removal of lung lobes and assay of eosinophil peroxidase and myeloperoxidase provides a measure of the numbers of eosinophils and neutrophils, respectively, in the lung. Depending on the experimental goals, bronchoalveolar lavage fluid and lung tissue can also be used for isolation of RNA, and measurement of cytokines, chemokines, antibodies, and inflammatory mediators.

Keywords: asthma; occupational asthma; mouse; lung; eosinophil; neutrophil

     
 
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Table of Contents

  • Basic Protocol 1: Mouse Sensitization and Intranasal Challenge
  • Support Protocol 1: Measuring Airway Hyperresponsiveness in a Conscious Unrestrained Mouse
  • Support Protocol 2: Measurement of Cellular Infiltration into the Airspace
  • Support Protocol 3: Measuring Cellular Infiltration into the Lung
  • Support Protocol 4: Eosinophil Peroxidase (EPO) Assay
  • Support Protocol 5: Myeloperoxidase (MPO) Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Mouse Sensitization and Intranasal Challenge

  Materials
  • Ovalbumin for sensitization (see recipe)
  • Imject Alum aluminum hydroxide solution (Pierce Chemical)
  • Normal saline solution (endotoxin‐free)
  • Mice of selected strain, age, and sex (e.g., Balb/c, 6 to 8 weeks old)
  • Ovalbumin for challenge (see recipe)
  • Ketamine/xylazine (see recipe)
  • Water (endotoxin‐free)
  • 2‐ml polypropylene screw‐cap vials
  • Micro stir bar
  • Animal balance (accurate to 0.1 g)
  • 1‐ml syringes and 27‐G × 1/2‐in. needles
  • 100‐µl pipet and tips

Support Protocol 1: Measuring Airway Hyperresponsiveness in a Conscious Unrestrained Mouse

  Materials
  • Methacholine (see recipe)
  • Mice sensitized and challenged with ovalbumin and appropriate control animals (see protocol 1)
  • Endotoxin‐free saline
  • Buxco whole‐body plethysmograph with Softmax XA software for whole‐body flow‐derived parameters (Buxco Electronics); four chambers monitored simultaneously
  • Balance (accurate to 0.1 g)

Support Protocol 2: Measurement of Cellular Infiltration into the Airspace

  Materials
  • Mice sensitized and challenged with ovalbumin and appropriate control animals (see protocol 1)
  • Phosphate‐buffered saline (PBS; appendix 2A; or from GIBCO)
  • 50 mg/ml pentobarbital sodium (Abbott)
  • Unopette Microcollection System solution (Becton Dickinson)
  • Normal guinea pig serum (Sigma)
  • Methanol
  • Modified Wrights' stain (Diff Quik, VWR)
  • Balance for weighing mice (accurate to 0.1 g)
  • 10‐ml beaker
  • 3‐ml syringes
  • Thread (4‐0 silk)
  • Forceps (blunt, curved tip, serrated micro‐dissecting forceps, Roboz Surgical Instruments)
  • Tracheal cannula (20‐G needle, bevel filed off)
  • Dissecting scissors (5 1/2‐in. blunt, Roboz Surgical Instruments)
  • Iris scissors (3‐in., Roboz Surgical Instruments)
  • 1.5‐ml siliconized eppendorf tubes (low adhesion, Dot Scientific)
  • Refrigerated, low‐speed centrifuge
  • Analytical balance (accurate to 0.001 g)
  • 1.5‐ml microcentrifuge tube
  • Neubauer hemacytometer (or automated cell counter such as a Coulter counter)
  • 96‐well U‐bottomed microtiter plates (Sarstedt)
  • Shandon Cytospin 3 centrifuge (Shandon/Lipshaw)

Support Protocol 3: Measuring Cellular Infiltration into the Lung

  Materials
  • Mice sensitized and challenged with ovalbumin and appropriate control animals (see protocol 1)
  • 50 mg/ml pentobarbital sodium (Abbott)
  • Cetrimide (hexadecyltrimethylammonium bromide; see recipe)
  • Balance (accurate to 0.1 g)
  • 2‐ml glass vials (12 × 32–mm; Fisher)
  • Analytical balance (accurate to 0.0001 g)
  • Small hemostat (4 1/2‐in.)
  • Dissecting scissors (5 1/2‐in. blunt, Roboz Surgical Instruments)
  • Kimwipes in petri dish
  • Luxo magnifier (Daigger)
  • 12 × 75–mm polystyrene round‐bottomed tubes (Falcon)
  • Drying oven (Carbolite)
  • Homogenizer (Janke Kunkel Ultra‐Turrax T‐25, 24,000 rpm) with 8‐mm generator probe for aqueous and organic solutions
  • 250‐ and 50‐ml beakers
  • Ear plugs
  • Forceps
  • 1.0‐ml pipet
  • Refrigerated, low‐speed centrifuge
  • Sonicator (e.g., Daigger VC‐130 ultrasonic processor) with microtip
  • Refrigerated, high‐speed centrifuge and 12 × 75–mm high‐speed centrifuge tubes
  • 1.5‐ml microcentrifuge tubes

Support Protocol 4: Eosinophil Peroxidase (EPO) Assay

  Materials
  • Homogenized left lung (see protocol 4)
  • 0.1% and 1% (v/v) Triton/Tris buffers (see reciperecipes)
  • o‐Phenylenediamine dihydrochloride (OPD; see recipe)
  • 30% H 2O 2 (Sigma)
  • Spectrophotometer with visible light and kinetics software
  • Sonicator (Daigger) with microtip
  • Vortex
  • 96‐well U‐bottomed microtiter plates (Sarstedt)
  • Glass cuvettes
  • Small squares of Parafilm (∼2 × 2–cm)
CAUTION: Wear gloves throughout the procedure because OPD is a potential carcinogen. Hazardous waste containing OPD should be collected for proper disposal. Cover the work area with plastic‐backed absorbent paper and wipe down all surfaces at the end with water.

Support Protocol 5: Myeloperoxidase (MPO) Assay

  Materials
  • Frozen homogenized inferior lung lobe sample (see protocol 4)
  • 0.05 M potassium phosphate buffer, pH 6.0 (see recipe)
  • o‐Dianisidine dihydrochloride (ODA; see recipe)
  • Aminotriazole (AMT; see recipe)
  • 0.5 mM H 2O 2 (see recipe)
  • Spectrophotometer with visible light and kinetics software
  • Glass cuvettes
  • Small squares of parafilm (∼2 × 2–cm)
CAUTION: Wear gloves throughout the procedure because ODA and AMT are potential carcinogens. Hazardous waste should be collected for proper disposal. Cover the work area with plastic‐backed absorbent paper and wipe down all surfaces at the end with water.
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Figures

Videos

Literature Cited

Literature Cited
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