Mouse Models of Bleomycin‐Induced Pulmonary Fibrosis

Dianne M. Walters1, Steven R. Kleeberger1

1 Laboratory of Respiratory Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 5.46
DOI:  10.1002/0471141755.ph0546s40
Online Posting Date:  March, 2008
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Abstract

Pulmonary fibrosis is a component of many interstitial lung diseases, including idiopathic pulmonary fibrosis, a chronic, progressive disease for which there is currently no effective therapy. Bleomycin has been widely used in rodents to model pulmonary fibrosis for the study of mechanisms involved in fibrogenesis and for evaluation of potential therapies. Bleomycin induces DNA strand breaks, resulting in pulmonary inflammation, injury, and subsequent interstitial fibrosis. This unit describes methods for delivering bleomycin, either directly into the lung or systemically, to create models of pulmonary fibrosis in rodents. Also described is a rapid and easy procedure for measuring lung collagen content to quantify the severity of fibrosis. Curr. Protoc. Pharmacol. 40:5.46.1‐5.46.17. © 2008 by John Wiley & Sons, Inc.

Keywords: bleomycin; pulmonary fibrosis; aspiration; animal models; mice

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Direct Aspiration of Bleomycin into the Lungs of Mice or Rats
  • Basic Protocol 2: Systemic Delivery of Bleomycin in Mice to Induce Pulmonary Fibrosis
  • Basic Protocol 3: Measurement of Lung Collagen Content Using the Sircol Soluble Collagen Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Direct Aspiration of Bleomycin into the Lungs of Mice or Rats

  Materials
  • 10 to 15 units (U)/ml bleomycin stock solution: prepared by dissolving lyophilized bleomycin sulfate (e.g., Blenoxane, Sigma‐Aldrich) in sterile phosphate‐buffered saline (PBS; appendix 2A) or 0.9% saline, mixing by inversion, and storing in small aliquots up to 1 year at −70 °C
  • Phosphate‐buffered saline (PBS; see appendix 3D)
  • Animals (mice or rats; see )
  • Isoflurane or ketamine and xylazine
  • Gas anesthesia setup, including:
    • Anesthesia induction chamber
    • Gas anesthesia machine
    • Flexible tubing
    • Oxygen tank
  • 1‐cc syringe and 25‐G, 5/8‐inch needle (alternative for ketamine/xylazine anesthesia)
  • 60° incline board with pegs (constructed in‐house, e.g., of Plexiglas or a piece of Styrofoam with push pins; see Fig. )
  • Rubber bands
  • Padded forceps
  • Warming pad or heat lamp

Basic Protocol 2: Systemic Delivery of Bleomycin in Mice to Induce Pulmonary Fibrosis

  Materials
  • Mice
  • Phosphate‐buffered saline (see appendix 3D)
  • 0.9% saline, sterile
  • Bleomycin (e.g., Blenoxane; Sigma‐Aldrich)
  • Betadine
  • 70% (v/v) ethanol
  • Isoflurane and oxygen for anesthesia
  • Osmotic minipumps (Alzet 2001‐7 day), with filling tubes and flow modulators
  • 1‐cc syringes, sterile
  • 37°C incubator
  • Electric hair clipper
  • Nose cone (for anesthesia)
  • Surgical equipment: scissors, scalpel blade, hemostat (sterile)
  • Wound clips, sterile

Basic Protocol 3: Measurement of Lung Collagen Content Using the Sircol Soluble Collagen Assay

  Materials
  • RIPA lysis buffer with protease inhibitors (see recipe)
  • Frozen lung samples: removed from the animal, weighed (if collagen content is to be expressed per unit of tissue weight), and quickly frozen in liquid nitrogen (to prevent protease degradation of collagen)
  • Dry ice
  • DC protein assay kit (Pierce)
  • Sircol collagen assay kit (Biocolor, Ltd.; available from Accurate Chemical and Scientific), including:
    • Sircol dye reagent
    • 1.0 mg/ml collagen standard
    • Alkali reagent
  • 13‐ml round bottom polypropylene homogenization tubes (Sarstedt, or equivalent)
  • 1.5‐ml and 2‐ml microcentrifuge tubes
  • Probe homogenizer and sterilized probe (e.g., T‐25 Ultra‐Turrax high‐speed homogenizer, IKA)
  • Refrigerated microcentrifuge, precooled to 4°C
  • Platform shaker
  • Cotton swabs
  • 1.5‐ml disposable cuvettes (e.g., Plastibrand, Sigma‐Aldrich)
  • Visible light spectrophotometer (450 nm)
  • Graphing software, optional
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Figures

Videos

Literature Cited

Literature Cited
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