Animal Models of Airway Sensitization

Muriel Pichavant1, Sho Goya1, Eckard Hamelmann2, Erwin W. Gelfand3, Dale T. Umetsu1

1 Children's Hospital, Harvard Medical School, Boston, Massachusetts, 2 University Hospital Charité, Berlin, Germany, 3 National Jewish Medical and Research Center, Denver, Colorado
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 15.18
DOI:  10.1002/0471142735.im1518s79
Online Posting Date:  November, 2007
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Abstract

Asthma is a complex phenotype that involves multiple mechanisms, including adaptive and innate immunity as well as physiological and mechanical changes in the airways. A cardinal feature of asthma is airway hyperreactivity (AHR), a multifaceted reaction that can only be assessed in vivo. Mouse models of asthma replicate many of the features of human asthma, including AHR, which can be assessed using standard protocols. Examination of AHR in mice has provided important information about human asthma, primarily because the immunology of allergy is easily studied in mice, especially with the availability of reagents including genetically modified mice. In this unit we discuss the induction and measurement of AHR and the two most common methodologies: noninvasive measurement using a whole‐body plethysmograph (WBP) and invasive measurement of lung resistance and dynamic compliance. Curr. Protoc. Immunol. 79:15.18.1‐15.18.19. © 2007 by John Wiley & Sons, Inc.

Keywords: AHR; noninvasive measurement; invasive measurement; OVA; methacholine

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Induction of Airway Hyperresponsiveness by Systemic Sensitization and Airway Challenge with Ovalbumin
  • Support Protocol 1: Noninvasive Measurement of Airway Responsiveness by Barometric Whole‐Body Plethysmography (WBP)
  • Support Protocol 2: Invasive Measurement of Airway Responsiveness by Pulmonary Resistance and Dynamic Compliance Assessment
  • Alternate Protocol 1: Induction of Airway Hyperresponsiveness by Airway Sensitization with Ovalbumin
  • Support Protocol 3: Assessment of T Cell Function and Airway Inflammation by Bronchoalveolar Lavage
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Induction of Airway Hyperresponsiveness by Systemic Sensitization and Airway Challenge with Ovalbumin

  Materials
  • OVA/Alum sensitizing solution (see recipe)
  • BALB/c mice, female, 6 to 12 weeks old, specific pathogen free (see unit 1.1)
  • 10 mg/ml ovalbumin stock solution (see recipe)
  • Saline (0.9% w/v sodium chloride), sterile
  • Gas anesthesia delivery setup
  • Isoflurane
  • Additional reagents and equipment for intraperitoneal injection of mice (unit 1.6), noninvasive ( protocol 2) and invasive ( protocol 3) measurement of airway responsiveness, collection of bronchoalveolar lavage samples ( protocol 5), and removal of lymphoid organs (unit 1.9)

Support Protocol 1: Noninvasive Measurement of Airway Responsiveness by Barometric Whole‐Body Plethysmography (WBP)

  Materials
  • Acetyl‐β‐methyl choline chloride (methacholine, Sigma, cat. no. A‐2251)
  • Saline (0.9% w/v NaCl), sterile
  • Ovalbumin‐sensitized/challenged and control mice ( protocol 1)
  • Buxco whole‐body plethysmography (WBP) system (http://www.buxco.com) including (also see Fig. ):
    • Whole‐body plethysmography (WBP) chamber for unrestrained animals
    • Bias Flow Supply
    • Flow transducers
    • Preamplifier
    • Data‐acquisition card
    • BioSystem XA software
  • Personal computer (not Macintosh)
  • Ultrasonic nebulizer (Lumiscope, model 6610; http://www.lumiscope.net)
  • Plastic chamber for nebulization (e.g., 20 cm diameter, 20 cm height)

Support Protocol 2: Invasive Measurement of Airway Responsiveness by Pulmonary Resistance and Dynamic Compliance Assessment

  Materials
  • Acetyl‐β‐methyl choline chloride (methacholine, Sigma, cat. no. A‐2251)
  • Ovalbumin‐sensitized/challenged mice ( protocol 1)
  • 70% ethanol
  • Saline (0.9% w/v NaCl), sterile
  • Buxco whole‐body plethysmography (WPB) system (http://www.buxco.com) including (also see Fig. ):
    • Whole‐body plethysmography (WBP) chamber for anesthetized animals
    • Ventilator (Lumiscope; http://www.lumiscope.net)
    • Transducers (both for flow and pressure)
    • In‐line aerosol delivery system
    • Preamplifier
    • Data‐acquisition card
    • BioSystem XA software with R/C analyzer
  • Personal computer (not Macintosh)
  • Dissection instruments including fine scissors and forceps
  • 18‐G or 19‐G tracheal tube with bevel
  • Sutures
  • Manometer
  • 1‐ml and 5‐ml syringe
  • Additional reagents and equipment for pentobarbital anesthesia of the mouse (unit 1.4)

Alternate Protocol 1: Induction of Airway Hyperresponsiveness by Airway Sensitization with Ovalbumin

  • 1% (w/v) ovalbumin in saline
  • Saline (0.9% v/v NaCl), sterile

Support Protocol 3: Assessment of T Cell Function and Airway Inflammation by Bronchoalveolar Lavage

  Materials
  • Ovalbumin‐sensitized/challenged mice ( protocol 1) after measurement of airway responsiveness ( protocol 32 or protocol 53)
  • 70% ethanol
  • Phosphate‐buffered saline (PBS; appendix 2A), cold
  • 1% FBS/PBS: PBS ( appendix 2A) containing 1% fetal bovine serum (FBS)
  • Dissection board
  • Dissection tools including forceps and scissors
  • Sutures
  • 20‐G to 24‐G angiocatheter
  • 1‐ml plastic syringe
  • 15‐ml conical tube
  • Cytospin equipment (Thermo Scientific; http://www.thermo.com):
    • Funnels
    • Clips
    • Filter cards
    • Glass slides
    • Cytocentrifuge
  • Diff‐Quik staining kit (e.g., Fisher)
  • Additional reagents and equipment for euthanasia of the mouse via pentobarbital overdose (unit 1.8) and counting cells ( appendix 3A)
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Figures

  •   FigureFigure 15.18.1 Schematic diagram of the whole‐body plethysmograph. (A) Main chamber containing the mouse; (B) reference chamber; (C) pressure transducer connected to analyzer; (D) pneumotachograph; (1) main inlet for aerosol closed by valve; (2) inlet for bias flow with 4‐way stopcock; (3) outlet for aerosol with 4‐way stopcock. Abbreviations: MCh, methacholine.
  •   FigureFigure 15.18.2 Changes in box flow waveform following methacholine challenge. Box flow waveform derived from a normal mouse following 3 min of nebulization with (A) aerosolized saline or (B) aerosolized methacholine (50 mg/ml in saline). f, respiratory rate (breaths/min); Penh, enhanced Pause; PIF, peak inspiratory flow; PEF, peak expiratory flow. Pause is defined as ( texpirationtrelaxation)/ trelaxation, where trelaxation is the time of pressure decay up to 36% of the total expiratory pressure signal.
  •   FigureFigure 15.18.3 Unrestrained whole‐body plethysmography system (from Buxco; http://www.buxco.com). (A) Bias Flow Supply PLY 1020 or 1040; (B) Whole‐Body Plethysmograph; (C) MAX II unit, which houses preamps and interface; (D) BioSystem XA software.
  •   FigureFigure 15.18.4 Invasive system (from Buxco; http://www.buxco.com). (A) BioSystem XA software with R/C analyzer; (B) MAX II unit, which houses preamps and interface; (C) ventilator; (D) PLY3111 or 3114 for anesthetized rodents.
  •   FigureFigure 15.18.5 Penh increases in allergen‐sensitized and challenged mice. Animals were sensitized with ovalbumin/alum intraperitoneally on days 1 and 14 and challenged with ovalbumin via the airways on days 28, 29, and 30. Airway responsiveness to aerosolized methacholine (MCh) was measured by whole‐body plethysmography (). Compared are nontreated (N, n = 8); nonsensitized, allergen‐challenged (Neb, n = 12); sensitized, nonchallenged (ip, n = 12); and sensitized, allergen‐challenged (ipNeb, n = 12) mice. Expressed are the means ± SEM of the increase in Penh values compared to Penh values after saline nebulization of three independent experiments.

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Literature Cited

Literature Cited
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   Beck, L. and Spiegelberg, H.L. 1989. The polyclonal and antigen‐specific IgE and IgG subclass response of mice injected with ovalbumin in alum or complete Freund's adjuvant. Cell Immunol. 123:1‐8.
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   Glaab, T., Daser, A., Braun, A., Neuhaus‐Steinmetz, U., Fabel, H., Alarie, Y., and Renz, H. 2001. Tidal midexpiratory flow as a measure of airway hyperresponsiveness in allergic mice. Am. J. Physiol. Lung Cell Mol. Physiol. 280:L565‐L573.
   Hamelmann, E., Oshiba, A., Loader, J., Larsen, G.L., Gleich, G., Lee, J., and Gelfand, E.W. 1997a. Antiinterleukin‐5 antibody prevents airway hyperresponsiveness in a murine model of airway sensitization. Am. J. Respir. Crit. Care Med. 155:819‐825.
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   Lofgren, J.L., Mazan, M.R., Ingenito, E.P., Lascola, K., Seavey, M., Walsh, A., and Hoffman, A.M. 2006. Restrained whole body plethysmography for measure of strain‐specific and allergen‐induced airway responsiveness in conscious mice. J. Appl. Physiol. 101:1495‐1505.
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