Mouse Models of Asthma

Nincy Debeuf1, Eline Haspeslagh1, Mary van Helden1, Hamida Hammad1, Bart N. Lambrecht1

1 Department of Internal Medicine, Ghent University, Ghent, Belgium
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/cpmo.4
Online Posting Date:  June, 2016
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Abstract

Allergic asthma is a chronic inflammatory disease of the conducting airways characterized by the presence of allergen‐specific IgE, Th2 cytokine production, eosinophilic airway inflammation, bronchial hyperreactivity, mucus overproduction, and structural changes in the airways. Investigators have tried to mimic these features of human allergic asthma in murine models. Whereas the surrogate allergen ovalbumin has been extremely valuable for unravelling underlying mechanisms of the disease, murine asthma models depend nowadays on naturally occurring allergens, such as house dust mite (HDM), cockroach, and Alternaria alternata. Here we describe a physiologically relevant model of acute allergic asthma based on sensitization and challenge with HDM extracts, and compare it with the ovalbumin/alum‐induced asthma model. Moreover, we propose a detailed readout of the asthma phenotype, determining the degree of eosinophilia in bronchoalveolar lavage fluids by flow cytometry, visualizing goblet cell metaplasia, and measuring Th cytokine production by lung‐draining mediastinal lymph node cells restimulated with HDM. © 2016 by John Wiley & Sons, Inc.

Keywords: allergic asthma; bronchoalveolar lavage (BAL); goblet cell hyperplasia; house dust mite (HDM); mediastinal lymph nodes (MLNs); murine model; ovalbumin (OVA)

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: House Dust Mite (HDM)‐Induced Asthma Model
  • Basic Protocol 2: OVA‐Induced Asthma Model
  • Support Protocol 1: Bronchoalveolar Lavage and Harvesting Organs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: House Dust Mite (HDM)‐Induced Asthma Model

  Materials
  • House dust mite (lyophilized Dermatophagoides pteronyssinus extracts; Greer Laboratories)
  • Phosphate‐buffered saline (PBS; Life Technologies, cat. no. 10010023), sterile
  • 6‐ to 12‐week‐old mice, specific‐pathogen free, of either sex (the protocol has been optimized for C57Bl/6 mice, but also works well in BALB/c mice)
  • Isoflurane
  • 70% (v/v) ethanol
  • Isoflurane vaporizer
  • Intubating platform allowing hyperextension of the mouse neck to facilitate access to the larynx (Fig.  )
  • Repeat pipettor and 100‐μl tips
  • Curved forceps
  • Curved forceps with serrated tips

Basic Protocol 2: OVA‐Induced Asthma Model

  Materials
  • Ovalbumin grade V (Sigma‐Aldrich) or ovalbumin (Worthington); these have a low endotoxin content to assure that immunity is solely raised against ovalbumin
  • Phosphate‐buffered saline (PBS; Life Technologies, cat. no. 10010023), sterile
  • Aluminum hydroxide [Al(OH) 3; alum; Sigma‐Aldrich]
  • 6‐ to 12‐week‐old mice, specific‐pathogen‐free, of either sex (C57Bl/6 and BALB/c backgrounds work well)
  • 70% (v/v) ethanol
  • Ovalbumin grade III (Sigma‐Aldrich; this ovalbumin is less pure—the higher endotoxin content boosts the challenges)
  • End‐over‐end rotator
  • Syringe and 26‐G needle
  • Small animal aerosol chamber with nebulizer
  • Additional reagents and equipment for intraperitoneal injection of mice (Donovan and Brown, )

Support Protocol 1: Bronchoalveolar Lavage and Harvesting Organs

  Materials
  • Optimum Cutting Temperature (OCT) compound (Tissue‐Tek, cat. no. 4853)
  • Phosphate‐buffered saline (PBS; Life Technologies, cat no. 10010023), sterile
  • Antibodies for staining mix (Table 15.2.2800)
  • FITC‐labeled counting beads (CaliBRITE beads; BD Biosciences, cat. no. 349502; optional)
  • Mice, 3 to 4 days after last HDM challenge ( protocol 1 or 2) or 1 day after last OVA challenge ( protocol 2)
  • Pentobarbital (Nembutal) for euthanasia
  • PBS‐EDTA: 0.1 mM disodium EDTA in PBS
  • Hanks’ balanced salt solution (HBSS) or serum‐free RPMI medium
  • Liquid nitrogen
  • 4% (w/v) paraformaldehyde (PFA; optional)
  • FACS buffer: PBS containing 0.5% (w/v) bovine serum albumin (BSA) and 0.05% (w/v) NaN 3
  • 0.4% (w/v) trypan blue
  • Compensation beads (UltraComp eBeads, eBioscience, cat. 01‐2222‐42)
  • Viability dye, e.g., eFluor506 (eBioscience, cat. no. 65‐0866)
  • Total cell medium (see recipe)
  • House dust mite (lyophilized Dermatophagoides pteronyssinus extracts; Greer Laboratories)
  • ELISA kit for Th cytokines (IL‐5, IL‐10, IL‐13, IL‐17 A, IFN‐γ)
  • Xylene
  • 70%, 96% and 100% ethanol
  • 0.5% periodic acid (Sigma‐Aldrich, cat. no. P7875)
  • Schiff's reagent (Sigma‐Aldrich, cat. no. S5133)
  • Gills’ hematoxylin (Polysciences, cat. no. 24244)
  • Entallan mounting medium
  • Bürker counting chamber
  • Dissection instruments including scissor and forceps
  • 500‐μl microcentrifuge tubes (for blood samples)
  • Small strings (5 cm length)
  • Cannulas, made of a 23‐G needle with a small piece of polyethylene tubing (Becton Dickinson) around the sharp needle point (the end of the tubing should be cut slightly oblique, to facilitate entry in the tracheal incision)
  • 15‐ml conical centrifuge tubes
  • 24‐well plate
  • Benchtop centrifuge and microcentrifuge
  • 96‐well round‐bottom microtiter plates
  • Refrigerated centrifuge with microtiter plate adapter
  • FACS tubes
  • 3‐laser flow cytometer (see Robinson et al., )
  • 100‐μm or 70‐μm filters (as many as you have groups)
  • Plungers from syringes
  • Small petri dishes (as many as you have groups)
  • Microscope slides and coverslips
  • Additional reagents and equipment for injection of rodents (Donovan and Brown, ), ELISA (Hornbeck, ), and flow cytometry (Robinson et al., )
Table 5.2.1   MaterialsAntibody Panel for Staining Bronchoalveolar Lavage Cells. Prepare the Staining Mix in PBS. Make 50 μl Staining Mix per Sample

Antigen Clone Fluorochrome Laser Dilution
SiglecF E50‐2440 PE 488 or 561 nm 1/1000
CD3 145‐2C11 PE‐Cy5 488 or 561 nm 1/200
CD19 eBio1D3(1D3) PE‐Cy5 488 or 561 nm 1/400
CD11c N418 PE‐Cy7 488 or 561 nm 1/800
CD11b M1/70 V450 405 nm 1/800
Ly‐6 G 1A8 AF700 633 nm 1/500
MHCII M5/114.15.2 APC‐eFluor780 633 nm 1/800
Fc Block 2.4G2 1/400
Viability dye eFluor506 405 nm 1/400

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Figures

Videos

Literature Cited

Literature Cited
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