Preparation of Specific Compartments of the Lungs for Pathologic and Biochemical Analysis of Toxicologic Responses

Laura S. Van Winkle1, Jacklyn S. Kelty1, Charles G. Plopper2

1 University of California, Davis, California, 2 School of Veterinary Medicine, University of California, Davis, California
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 24.5
DOI:  10.1002/cptx.18
Online Posting Date:  February, 2017
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Abstract

This unit focuses on protocols for assessing microenvironment‐specific responses in the thoracic lung tissues. Aspects of the entire respiratory system serve as potential targets for candidate toxicants, but each candidate toxicant may impact distinct sites due to differential distribution of either the toxicant or the target cells. Within the conducting airways, the composition of resident cell populations and the metabolic capabilities of the cell populations vary greatly. Thus, studies of this region of the lung require unique, site‐selective methods to clearly define the toxic response. Without site‐specific sampling, as described in this chapter, the experimental limit of detection for toxicant effects in conducting airways is weakened because differences unrelated to treatment, but related to location, may dominate the response. The protocols included here allow assessment of toxicological responses in the tracheobronchial airways and the gas exchange area of the lung, with specific application to laboratory mammals. © 2017 by John Wiley & Sons, Inc.

Keywords: respiratory; microenvironment; microdissection; lungs; toxicant; environmental pollutants; toxicology

     
 
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Table of Contents

  • Introduction: Rationale for Site‐Selective Sampling of Lung
  • Basic Protocol 1: Constant‐Pressure Fixation of Lung Within the Thorax
  • Alternate Protocol 1: Constant Pressure Fixation of Lung Outside of the Thorax
  • Basic Protocol 2: Lung Volume Measurement by Fluid Immersion
  • Basic Protocol 3: Cryopreservation of Lung
  • Basic Protocol 4: Lung Microdissection for Imaging
  • Basic Protocol 5: Site‐Specific Sampling of Lung by Microdissection
  • Basic Protocol 6: Isolation of Live Airway Tissues
  • Basic Protocol 7: Lysis Lavage of Lung Epithelial Tissue
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Constant‐Pressure Fixation of Lung Within the Thorax

  Materials
  • Fixative of choice (see recipes in Reagents and Solutions):
    • 2.5% (w/v) glutaraldehyde
    • Dilute Karnovsky's fixative
    • 1% (w/v) paraformaldehyde
  • Test animal: rat or mouse
  • Sodium pentobarbital anesthetic (see Donovan and Brown, , for dosage information)
  • 70% ethanol
  • Manometer: Apparatus for maintaining constant fluid pressure during instillation; a vessel for holding fixative with a sealable cover containing an opening that reaches through the fixative to the bottom of the vessel and that also needs an extension at the base for fluid outflow to tubing connected to a cannula with a valve (Fig. )
  • Stand for holding manometer a fixed distance (20 to 30 cm) above the level of the heart (maintaining the same height above the thorax is critical for consistent inflation)
  • Dissecting board with pins or other restraining capabilities for anesthetized animals
  • Dissection instruments: forceps, scissors, hemostats, scalpels with blades, single‐edge razor blades
  • Sutures
  • Cannula whose external diameter approximates the internal diameter of the trachea, scored on the end: 18‐G, 2‐in. feeding tubes are appropriate for rat tracheas and 22‐G, 1.25–in. tubes are used for mice
  • 1‐ml syringe
  • Container to store fixed tissue immersed in fixative
  • Additional reagents and equipment for injection (Donovan and Brown, ) and anesthesia (Donovan and Brown, ) of rodents
CAUTION: All aldehyde fixatives are carcinogens. Appropriate ventilation and personal protective equipment is required. Fixatives are hazardous waste when disposed.CAUTION: Phenobarbital is a controlled substance. Choice of anesthetic and necropsy method must be approved by Institutional Animal Care and Use Committee.

Alternate Protocol 1: Constant Pressure Fixation of Lung Outside of the Thorax

  Materials
  • Test animal: rat or mouse
  • Phosphate‐buffered saline [PBS ( appendix 2A); alternatively, fixative can be used instead of PBS; fixative should be consistent with the original fixative used for tissue fixation and requires ventilation of the workspace]
  • Suture
  • Beaker or container (about 100 ml capacity)
  • Balance
  • Hemostat or other clamp to hold fixed lungs immersed in fluid
  • Dissection instruments to trim tissue: forceps, scissors, hemostats, scalpels with blades, single‐edge razor blades
  • Absorbent towel or tissue
  • Additional reagents and equipment for fixation of rodent lung ( protocol 1)

Basic Protocol 2: Lung Volume Measurement by Fluid Immersion

  Materials
  • Liquid nitrogen
  • 2‐methylbutane (Fisher, cat. no. O3551‐4)
  • Lung (unfixed, cannulated; obtained at protocol 2, step 12a)
  • 50:50 (v/v) solution of OCT (Fisher, cat. no. 23‐730) and RNase‐free phosphate buffered saline (PBS; Fisher, cat. no. AM9624)
  • OCT cryoprotectant (Fisher, cat. no. 23‐730)
  • Cryogenic storage Dewar with a wide‐mouth opening
  • 600‐ml glass beaker attached to a glass rod by tape that is resistant to approximately −200°C
  • Ring stand and clamp
  • Syringe (3‐ml or greater)
  • Cryomolds labeled with marking pen
  • Strips of cork board backing cut to fit cryomolds
  • Teflon‐coated razor blade
  • 9‐in. or longer forceps
  • Styrofoam container of dry ice
  • Airtight storage bags

Basic Protocol 3: Cryopreservation of Lung

  Materials
  • Rodent lung: either obtained fresh protocol 2 (step 12a) or fixed as described in protocol 1 (for fixed tissues, rinsing in PBS and use of an aldehyde‐vapor‐scavenging device is recommended)
  • Phosphate buffered saline (PBS; appendix 2A)
  • 3 M Vetbond Tissue Adhesive
  • Fine forceps and scissors
  • Culture dishes
  • 22 × 22‐mm glass cover slips, No. 1 ½
  • Transfer pipets
  • Modeling clay
  • High‐resolution dissecting microscope with wide field of view and an ocular micrometer and protractor mounted in the eyepieces
  • High‐intensity light source with flexible mounting for lights

Basic Protocol 4: Lung Microdissection for Imaging

  Materials
  • Rodent lung: either obtained fresh ( protocol 2, step 12a) or fixed as described in protocol 1 (for fixed tissues, rinsing in PBS and use of an aldehyde‐vapor‐scavenging device is recommended)
  • Phosphate‐buffered saline [PBS ( appendix 2A); alternatively, fixative can be used instead of PBS; fixative should be consistent with the original fixative used for tissue fixation and requires ventilation of the workspace]
  • High‐resolution dissecting microscope with a wide field of view and an ocular micrometer and protractor mounted in the eyepieces to measure length and diameter
  • High‐intensity light source with flexible mounting for lights
  • Hemostat
  • Dissection instruments: forceps, scissors of various sizes, hemostats, scalpels with blades, single‐edge razor blades
  • Labeled containers for holding sampled tissues
  • Petri dish or other low‐sided container for holding lung in fixative or buffer during dissection
  • Bronchial cast (Fig. A), binary numbering system (Fig. B,D), or airway map (Fig. C,E)

Basic Protocol 5: Site‐Specific Sampling of Lung by Microdissection

  Materials
  • F12 medium, plain (see recipe)
  • 2× Waymouth's medium (see recipe)
  • 2× agarose (see recipe)
  • Lung (unfixed, cannulated; obtained at protocol 2, step 12a)
  • F12 medium, supplemented (see recipe)
  • Small beaker
  • Water bath at 40°C
  • 3‐ to 6‐ml syringes
  • High‐resolution dissecting microscope
  • Dissecting instruments including forceps
  • High‐intensity light source with flexible mounting for lights
  • Sterile hood for making medium, otherwise protocol does not require sterile conditions

Basic Protocol 6: Isolation of Live Airway Tissues

  Materials
  • Incubation solution (see recipe)
  • 0.75% (w/v) agarose (see recipe)
  • 1% (w/v) dextrose (prepare ∼2.0 ml per mouse lung; good for 1 week when stored at 4°C)
  • Lung (unfixed, cannulated; obtained at protocol 2, step 12a)
  • 40°C water bath
  • Plastic 1‐ml syringes
  • 3‐valve stopcock
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Figures

Videos

Literature Cited

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