Guinea Pig Models of Asthma

Alice E. McGovern1, Stuart B. Mazzone1

1 School of Biomedical Sciences, University of Queensland, St Lucia
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 5.26
DOI:  10.1002/0471141755.ph0526s67
Online Posting Date:  December, 2014
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Abstract

Described in this unit are methods for establishing guinea pig models of asthma. Sufficient detail is provided to enable investigators to study bronchoconstriction, cough, airway hyperresponsiveness, inflammation, and remodeling. © 2014 by John Wiley & Sons, Inc.

Keywords: bronchospasm; hyperresponsiveness; cough; inflammation; remodeling

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Measurements of Pulmonary Insufflation Pressure in Anesthetized, Paralyzed, Mechanically Ventilated Guinea Pigs
  • Alternate Protocol 1: Measurement of Reflex Tracheal Contractions in Anesthetized, Paralyzed, Mechanically Ventilated Guinea Pigs
  • Basic Protocol 2: Induction of Airways Hyperresponsiveness in Guinea Pigs Through Active Sensitization and Allergen Challenge
  • Alternate Protocol 2: Chronic Allergen Challenge for Assessing Airways Remodeling
  • Basic Protocol 3: Measurement of Cough Reflexes in Conscious Guinea Pigs
  • Alternate Protocol 3: Measurement of Cough Reflexes in Anesthetized Guinea Pigs
  • Basic Protocol 4: Measurement of Inflammatory Cell Influx into the Airways by Bronchoalveolar Lavage (BAL)
  • Basic Protocol 5: Quantification of Plasma Extravasation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurements of Pulmonary Insufflation Pressure in Anesthetized, Paralyzed, Mechanically Ventilated Guinea Pigs

  Materials
  • Guinea pig (Harlan Bioproducts for Science; also see Strategic Planning)
  • 25% (w/v) urethane (ethyl carbamate; Sigma) in sterile distilled water
  • 2 mg/ml succinylcholine chloride (Sigma) in 0.9% NaCl
  • 750 U/ml heparin (Sigma) in 0.9% NaCl
  • 5 mg/ml propranolol (Sigma) in 0.9% NaCl (not needed for every experimental design)
  • Test compounds (provocative and therapeutic agents; see recipe)
  • 100 mM KCl, 100% CO 2 source, or sodium pentobarbital for euthanasia of guinea pigs
  • Absolute ethanol or 0.1 N HCl
  • Flexible plastic tubing (4 mm i.d. × 7 mm o.d.) and Y connectors (VWR Scientific)
  • Constant‐volume ventilator (e.g., Harvard Apparatus rodent ventilator, Model 683)
  • Air humidifier constructed from a water‐filled glass flask equipped with a two‐holed rubber stopper (Fig.  A)
  • Ultrasonic nebulizer (DeVilbiss, Model 25; optional)
  • Amplifier and pressure transducers (e.g., , Grass Instruments polygraph with Isotec pressure transducers from Harvard Apparatus); this equipment can be as simple as the older‐style paper chart recorders (e.g., the model 70D polygraph) or integrated into a more sophisticated computer‐based recording system via an analog‐to‐digital (A‐D) converter and compatible software
  • 1‐ml disposable syringes for intravenous, subcutaneous, and intraperitoneal injections (use a 27‐G intradermal needle for subcutaneous and intraperitoneal injections)
  • 27‐G needles (e.g., Becton Dickinson)
  • Heated pad with limb and head restraints (Harvard Apparatus)
  • Fiber‐optic light (e.g., FiberLite, Model 9745‐00; World Precision Instruments)
  • Animal clippers for shaving fur (optional; similar results can be obtained with a disposable razor or a small battery‐powered hair trimmer available from the men's grooming section of most department stores)
  • Surgical instruments including:
    • Scalpel and blades
    • Fine scissors and forceps
    • 30‐mm bulldog clamp (World Precision Instruments)
    • Silk surgical suture (sizes 4–0, 2–0; George Tiemann and Co.)
  • Surgical retractors: these can be easily assembled in‐house using a 27‐G needle that has been bent into a ‘U’ shape with a 15‐cm length of string or suture secured to the plastic luer of the needle
  • Surgical gauze or cotton balls
  • Surgical cauterizer (optional)
  • Tracheal cannula: 15‐G luer stub adapter 10 to 20 mm in length (commercially prepared cannulas also available from Harvard Apparatus)
  • 25‐G needles
  • Venous and arterial cannulas made from PE‐60 tubing, 22‐G luer stub adapters, 3‐way stopcocks, and 5‐ml disposable syringes
  • 5‐ml syringes
  • Additional reagents and equipment for injections (Donovan and Brown, )

Alternate Protocol 1: Measurement of Reflex Tracheal Contractions in Anesthetized, Paralyzed, Mechanically Ventilated Guinea Pigs

  Additional Materials ( protocol 1)
  • Trachea perfusion buffer (see recipe), 37°C, continuously oxygenated with 95% O 2/5% CO 2
  • 10 mM atropine stock solution in H 2O (to make 1 μM final buffer concentration)
  • 1 M barium chloride stock solution in H 2O (to make 300 mM final buffer concentration)
  • Fiber optic light (e.g., FiberLite, Model 9745‐00; World Precision Instruments)
  • Two small stainless steel hooks (e.g., Mustad size 14 dry fly hooks; available from fishing supply stores)
  • Small laboratory scaffold components (for holding manipulators, heating coil, clamps)
  • 10‐cm length of PE‐60 tubing
  • Vacuum source
  • Isometric force transducer (Grass Model FT03) and polygraph
  • Coarse manipulators (for moving force transducers, fixed bar; Harvard Apparatus)
  • 18‐G luer stub adapter
  • Small water‐jacketed heating coil (Radnoti; available from Harvard Apparatus)
  • Heating water circulator with tubing
  • Peristaltic pump with tubing (e.g., Cole‐Parmer variable speed L/S pump with standard pump head designed for L/S 16 tubing)
  • Gauze
  • Blue surgical mats (Fisher Scientific)

Basic Protocol 2: Induction of Airways Hyperresponsiveness in Guinea Pigs Through Active Sensitization and Allergen Challenge

  Materials
  • Guinea pig (Harlan Bioproducts for Science; also see Strategic Planning)
  • 10 mg/ml chicken egg ovalbumin prepared in sterile (0.9% NaCl) saline (to inject 10 mg/kg intraperitoneally)
  • Sterile saline (0.9% NaCl) for vehicle control experiments
  • 10 mg/ml bovine serum albumin (irrelevant allergen; as control)
  • Test compounds (see recipe)
  • 1‐ml syringes with 27‐G needles
  • Exposure chamber (Fig.  ) consisting of:
    • Transparent Perspex chamber (∼200 × 100 × 100 mm; easily constructed from Perspex sheeting or can be purchased from Harvard Apparatus)
    • Ultrasonic nebulizer (DeVilbiss, Model 25) and flexible plastic tubing (7 mm i.d. × 10 mm o.d.; VWR Scientific)
    • Chart recorder with flow meter or pressure transducer (Harvard Apparatus)
    • Microphone and audio tape recorder
    • Compressed air cylinder with regulator
  • Additional reagents and equipment for injections (Donovan and Brown, )

Alternate Protocol 2: Chronic Allergen Challenge for Assessing Airways Remodeling

  Additional Materials ( protocol 3)
  • Reference and experimental compounds, e.g.,
    • Corticosteroid therapy: dexamethasone, 5 mg/kg i.p. once daily for the duration of the challenge phase
    • Anti‐leukotriene therapy: Montelukast, 10 mg/kg i.p. once daily for the duration of the challenge phase;
    • Anticholinergic therapy: tiotropium bromide, 0.1 mM, inhaled for 3mins prior to each ovalbumin challenge
  • 0.1 M phosphate‐buffered saline (PBS), pH 7.4 (e.g., unit 6.2; 150 ml per animal), room temperature.
  • 4% (w/v) paraformaldehyde (PFA), pH 7.4 (made in 0.1 M PBS; 150 ml per animal), room temperature
  • Pentobarbital sodium (Lethabarb 100 mg/kg)
  • 18‐G needle with 6 × 50 ml syringes
  • Large and fine scissors
  • Rat's tooth forceps
  • Hemostat
  • 27‐G needle with 1‐ml syringe
  • Additional reagents and equipment for histological processing (see appendix 3D & )

Basic Protocol 3: Measurement of Cough Reflexes in Conscious Guinea Pigs

  Materials
  • Guinea pig (Harlan Bioproducts for Science; also see Strategic Planning)
  • Tussigenic agents, e.g.:
    • Citric acid: 0.04 to 0.4 M solution prepared in saline (0.9% NaCl)
    • Capsaicin: 100 mM stock solution prepared in absolute ethanol, diluted to 1 to 100 μM in saline for challenges (be sure to perform appropriate vehicle controls when using capsaicin)
    • 2 to 10 mg/ml bradykinin in saline
  • Test compound(s) (see recipe)
  • Exposure chamber (Fig.  ) consisting of:
    • Transparent Perspex chamber (∼200 × 100 × 100 mm; easily constructed from Perspex sheeting or purchased from Harvard Apparatus) or a commercial plethysmograph system (e.g., Buxco guinea pig chamber).
    • Ultrasonic nebulizer (DeVilbiss, Model 25) and flexible plastic tubing (7 mm i.d. × 10 mm o.d.; VWR Scientific)
    • Chart recorder with flow meter or pressure transducer (Harvard Apparatus); alternatively, several validated, commercial automated cough detection systems are now available (e.g., Buxco automated cough detection analyzer).
    • Microphone and audio tape recorder (optional)
    • Compressed air cylinder with regulator or small air pump (e.g., an aquarium pump)
NOTE: Results from studies of cough in conscious animals may vary widely among subjects. This makes it necessary to design experiments in which at least 8 to 10 animals are assigned per experimental group to assure sufficient statistical power for data analysis. A variety of agents are used to induce cough (Table 5.26.4). Perform vehicle control experiments when studying test agents, such as capsaicin and cinnamaldehyde, that are not soluble in saline.
Table 5.6.4   MaterialsInhaled Doses of Commonly Used Agents for Studying Cough in Conscious Guinea Pigs a

Tussive agent Receptor Dose
Capsaicin TRPV1 1‐60 mM
Citric Acid TRPV1/ASIC 100‐400 mM
AITC TRPA1 1‐30 mM
Cinnamaldehyde TRPA1 10‐30 mM
Bradykinin Bradykinin B2 0.3‐10 mM

 aThe values for the tussive agent doses listed above represent data extracted from the literature (Medline/PubMed) using common search terms (including guinea pig, cough, and agent name). Most drugs listed above are available from Sigma or Tocris. Many other compounds may sensitize cough without inducing cough per se. Many of the same agents listed in Tables 5.26.2 and 5.26.3 have been tested for antitussive activity against the tussive agents listed above. Abbreviations: TRPV1, Transient Receptor Potential Vanilloid 1; TRPA1, Transient Receptor Potential Ankyrin 1; ASIC, Acid Sensitive Ion Channels.

Alternate Protocol 3: Measurement of Cough Reflexes in Anesthetized Guinea Pigs

  Additional Materials ( protocol 11 and protocol 53 and protocol 2)
  • Krebs buffer (see recipe), continuously oxygenated with 95% O 2/5% CO 2
  • Stimuli: chemical tussive agents, mechanical/electrical stimulation, etc.
  • Test compounds (see recipe)
  • Dissecting equipment and sutures (see protocol 1)
  • Tracheal cannula (also see protocol 1): 15‐G luer stub adapter approximately the length of the bypassed airways, including the nasal passages
  • Pressure transducer or differential flow transducer and head (available from Harvard Apparatus)
  • Chart recorder or digital data acquisition system
  • 10‐ml water jacketed organ bath (available from Harvard Apparatus)
  • Circulating water bath with flexible tubing
  • Gauze
  • Von Frey filaments (optional; available from Stoelting). An alternative is to use short lengths of various sized PE tubing.
  • Platinum bipolar electrode (constructed in‐house from 0.4 mm platinum wire or available from WPI Inc.; note that a concentric stimulating electrode works equally well)
  • Electrical pulse generator (e.g., S48 square pulse stimulator from Grass Technologies Inc.)

Basic Protocol 4: Measurement of Inflammatory Cell Influx into the Airways by Bronchoalveolar Lavage (BAL)

  Materials
  • Guinea pig (Harlan Bioproducts for Science; also see Strategic Planning)
  • Pentobarbital
  • Lavage solution (see recipe), 37°C
  • Phosphate‐buffered saline, pH 7.4 (PBS; e.g., unit 6.2)
  • 75% methanol
  • Differential stain: e.g., Diff‐Quik (Baxter) or 10% (v/v) Giemsa stain
  • Immunoassay kits for inflammatory mediators (e.g., Cayman Chemical)
  • Tracheal cannula: 15‐G luer stub adapter 10 to 20 mm in length (commercially prepared cannulas also available from Harvard Apparatus)
  • 5‐ml syringe
  • Refrigerated centrifuge and 15‐ or 50‐ml tubes
  • Hemacytometer or automated cell counter
  • Glass microscope slides
  • Cytospin cytocentrifuge (Shandon) and tubes

Basic Protocol 5: Quantification of Plasma Extravasation

  Materials
  • Guinea pig (Harlan Bioproducts for Science; also see Strategic Planning)
  • 25% (w/v) urethane (ethyl carbamate) in 0.9% NaCl
  • 5 mg/ml atropine in 0.9% NaCl
  • 5 mg/ml propranolol in 0.9% NaCl
  • 30 mg/ml Evans blue dye (Sigma) prepared in 0.9% NaCl
  • Inflammatory insult: e.g., histamine, bradykinin, or allergen
  • Formamide
  • Phosphate‐buffered saline, pH 7.4 (PBS; e.g., unit 6.2)
  • Evans blue dye standards in formamide, 0.5 to 10 μg/ml
  • Transcardial perfusion system consisting of:
  • 500‐ml aspirator bottle and tubing
  • Blunt 14‐G needle, 2 cm in length
  • Syringes for injections
  • Large tray or dish for collecting excess perfusate
  • Surgical instruments (see protocol 1)
  • Large hemostat
  • Small artery clamp
  • Spectrophotometer
  • Additional reagents and equipment for intravenous or nebulized administration of inflammatory compounds and allergen (see other protocols in this unit)
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Figures

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Literature Cited

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