Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

Summary

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

Abstract

COPD is projected to be the third most common cause of mortality world-wide by 2020⁽¹⁾. Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs⁽²⁾. However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD^(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients⁽⁶⁾, but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill’s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson’s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

Introduction

The use of animal models to study COPD is challenging because no model can perfectly replicate all features of the human disease⁽²⁾. Most investigators use mice to model COPD because of the similarities between mice and humans in their pulmonary physiology, pathology, genetics, and metabolites. Also, mice are relatively inexpensive to study, and both emphysema and small airway remodeling develop within 6 months of CS exposure^(5,7-9).

Cigarette smoke-induced COPD: Several methods can induce COPD in mice. Most researchers expose mice to CS, which is the main etiologic factor for human COPD. CS exposure f....

Protocol

The protocol takes ~25 weeks to complete. The protocol exposed mice to air or smoke for 24 weeks. At the end of the smoke exposures, the protocol measures pulmonary function in the mice, and lungs are inflated to a fixed pressure, fixed, and removed on the same day. Additional time is needed for the researcher to embed, cut, and stain the lung sections (2-3 days), and capture and analyze the images (2-4 days depending on the number of animals studied). This protocol can also be used to measure age-related airspace enlargement in mice.

All procedures described in this protocol have been approved by the Institutional Animal Care and Use Committee at Brigham and Women's Hospital/H....

Results

This protocol begins with whole-body exposure of mice for CS. Adequate oversight and maintenance of the device and monitoring of TPM counts ensure consistent smoke exposures (Figure 1). It is important that the researcher practices the lung inflation technique using the inflation device

This protocol begins with whole-body exposure of mice for CS. Adequate oversight and maintenance of the device and monitoring of TPM counts ensure consistent smoke exposures (Figure 1

Discussion

Most researcher use mice to model the main chronic lung pathologies and abnormal lung physiology in COPD (airspace enlargement, SAR, and increases in lung compliance) present in the human disease. A comprehensive approach to assess the effect of molecules of interest on both emphysema development and SAR is needed in mice in order to comprehensively assess the activities of molecules of interest in these chronic disease processes.

There are several critical steps in this protocol. First, dur.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Whole-body smoke exposure device	Teague Enterprise	TE-10z	Chronic Smoke exposures to induce chronic lung disease in mice
Research Cigarette	University of Kentucky	3R4F reference cigarettes
Pallflex® Air Monitoring Filters, Emfab Filters TX40HI20WW, 25 mm	Pall Corporation	7219	For measurement of TPMs
25 mm filter holder	Pall Corporation
Filter sampler	Intermatic	Metal T100
Gas meter	AEM	Gas meters G1.6; G2.5; G4
Tracheal Cannula for mouse 18 gauge	Labinvention		Analysis of pulmonary function
Mechanical ventilator	Scireq	FlexiVent
Gill's hematoxylin solution	Sigma-Aldrich	GSH316	For Gill staining, work under a fume hood
Hematoxylin solution, Harris modified	Sigma-Aldrich	HHS16
Cytoseal-60	Thermo Scientific	8310-16
Micro-Slide-Field-Finder	Andwin Scientific INC	50-949-582	For analysis of emphysema
Scion Image Program	Scion Corporation
Mason's trichrome stain	Sigma-Aldrich	HT15	For analysis of small airway fibrosis
MetaMorp Offline version 7.0	Molecullar Devices LLC	31032