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.
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.
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 for 6 months causes the development of emphysema and small airway remodeling (SAR) in mice, but the severity of the disease that is induced varies depending on the murine strain studied. For example, NZWLacZ mice are resistant to the development of CS-induced emphysema whereas AKR/J mice are extremely sensitive(10). Most investigators study C57BL/6 strain mice in the CS exposure model as many gene-targeted mice are available in this strain. After 6 months of CS exposure, emphysema and small airway fibrosis develop in wild type (WT) C57BL/6 mice, and both lesions are relatively mild in severity(5,10). Researchers use two types of CS exposure: nose-only and whole-body exposures. The major disadvantages of the nose-only exposure technique are that: 1) it is a more labor-intensive method; and 2) mice have to be restrained in small chambers which can induce a stress response and hyperthermia in the animals(11). The major disadvantage of whole-body exposure (described herein) is that the animals can ingest (as well as inhale) nicotine and tar products when they clean their fur. Mice exposed to whole-body CS also have lower carboxyhemoglobin levels and reduced loss of body weight when compared with animal exposed to nose-only CS(12).
Pulmonary function test (PFTs): Measures of lung compliance and elastance are usually similar in C57BL/6 wild type (WT) mice exposed to air or CS for 6 months due to the relatively mild emphysema that develops when this strain is exposed to CS(10). However, when emphysematous destruction is more severe, increases in lung compliance and left shifts in the pressure-volume (PV) flow loops can be detected. The latter can be observed, for example, in murine strains that are more susceptible to the effects of CS(10), in CS-exposed C57BL/6 strain gene-targeted mice that have a more severe emphysema type than C57BL/6 WT mice(13), or in CS-exposed mice subjected to environmental changes that render them more susceptible to the effects of CS(14). This protocol uses a small animal ventilator to measure reductions in the elastic recoil of the lung (increases in quasistatic lung compliance [Cst] and reductions in tissue elastance [H]), PV flow loops, and changes in airway and tissue resistance in anesthetized mice(15,16).
Measures of pulmonary emphysema: Analysis of emphysema development in CS-exposed C56BL/6 strain mice is challenging because its distribution is spatially heterogeneous. Several different methods quantify airspace enlargement in mice. The first method used was the mean linear intercept (Lm)(17). However, the Lm method is a slow, manual process which may not capture the heterogeneity of the disease (unless all sections of the lung are randomly sampled) and its use may therefore introduce observer bias into the analysis. The destructive index [DI,(18)] also quantifies airspace enlargement using a transparent sheet with 50 equally distributed points placed over a printed digitized image of a hematoxylin and eosin-stained lung section. The PI method scores the area surrounding each point according to the extent to which the alveolar ducts and alveolar walls within this area are destroyed. The main disadvantage of the DI method is that it is time-consuming and not more accurate than other methods(19,20).
This protocol measures mean alveolar chord length and alveolar area on paraffin-embedded lung sections stained with Gill’s stain. Morphometry software converts images of lung sections to binary images (in which tissue is white and airspace is black), and then superimposes a uniform grid of horizontal and vertical lines (chords) and the software then quantifies the length of each chord within areas identified by software as airspace. Using this method, it is possible to measure the size of the alveoli in all parts of the lung in a standardized and relatively automated manner(21).
Small airway remodeling (SAR): The increased deposition of ECM proteins (especially interstitial collagens) around small airways occurs in CS-exposed animals and contributes to airflow obstruction. Researchers do not study SAR in animal models of COPD as frequently as emphysema development(22). To quantify SAR in CS-exposed mice, this protocol uses image analysis software to measure the thickness of the layer of ECM proteins that is deposited around the small airways (airways having a mean diameter between 300 and 899 m) in paraffin-embedded lung sections stained with Masson’s trichrome stain.
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/Harvard Medical School.
1. Whole-Body Cigarette Smoke Exposure
2. Pulmonary Function Test (PFTS) And Lung Inflation
3. Emphysema
4. Morphometry To Measure Emphysema
The protocol uses Scion Image and customized macros to analyze airspace enlargement. Scion Image is a Windows-compatible version of the original NIH Image application which runs under the Macintosh operating system. Scion Image runs under Windows XP and is still available online through Wikiversity.org, where a search for 'Scion Image' will direct the user to links to the manual and beta 4.0.2 release of Scion Image. Installation and software operation is detailed in the online supplement manual and summarized below. The alveolar chord length macro was adapted from the macro available in NIH Image.
5. Small Airway Remodeling
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). It is important that the researcher practices the lung inflation technique using the inflation device (Figure 2) and carefully removes the lung after inflation in order to obtain well inflated lung sections for accurate analysis of airspace enlargement. Figure 3A shows a well inflated lung whereas Figure 3B shows a poorly inflated lung. Figure 3C shows an image of an inflated lung section prepared for the thresholding step (macrophages in the alveolar spaces are painted white, and vessels and bronchi are painted black to generate. The thresholding step creates a binary image in which all pixels in the alveolar space are white and all pixels in areas of the lung which are not alveoli are black (Figure 3D). Figure 3E and 3F show the vertical and horizontal alveolar chord lengths that the macros generate, respectively.
Pulmonary function tests show modest (and not statistically significant) left shifts in the pressure volume (PV) loops reflecting modest loss of elastic recoil of the lung consistent with the mild emphysema that develops in C57BL/6 WT mice exposed to CS for 6 months (Figure 4A). Significant left shifts in the PV loops are observed only in murine strains that are very sensitive to the effects of CS or in CS-exposed gene-targeted mice having a more severe emphysema phenotype than CS-exposed C57BL/6 WT mice.
Figure 5 shows representative images Masson’s trichrome-stained lung sections of C57BL/6 WT mice exposed to air (Figure 5A) or CS (Figure 5B) for 6 months illustrating increases in ECM protein deposition around small airways in the CS-exposed animals. Figure 5C illustrates how an image analysis software program quantifies ECM protein deposition around the airways having the desired internal diameter. Figure 5D shows the analysis of ECM protein deposition around airways with a diameter of 300-899 μm in CS-exposed C57BL/6 WT mice.
Figure 1. A cartoon of the whole-body cigarette exposure system. A smoke exposure device is connected to a smoke exposure chamber. Smoke is pulled from the side-stream collection chamber and smoke is pulled from the cigarettes by the pump, and both smoke samples are mixed and diluted with ambient air in the mixing and dilution chamber (left), and then the smoke flows into the exposure chamber. The researcher places mice in their cages in the exposure chamber (right); mice are able to move freely in their cages, and have access to food and water for the duration of the smoke exposure.
Figure 2. Inflation of murine lungs. The researcher fills a flask with sterile PBS, seals it with a rubber stopper, and inverts it and secures it a distance of 25 cm above the heart of the animal using a ring stand. An intravenous giving set delivers the PBS to the lungs via the tracheal cannula. A cut-down serologic pipette is inserted through the rubber stopper and this allows air into the flask to replace the volume of PBS that drains into the lungs of the mice by gravity.
Figure 3. Emphysema analysis. (A) shows a representative image of Gill’s-stain inflated lung sections from mice exposed to air or CS for 6 months, the black arrows indicate a vessel and alveolar macrophages. (B) shows a representative image of an under-inflated lung that is not suitable for analysis. (A) shows the “before” and (C) shows the “after” image of a representative lung section that the researcher prepares for generation of a binary image. The black arrows in (A) and (C) indicate either a vessel (which the researcher paints black in (C)) or alveolar macrophages (which the researcher paints white in (C)). (D) shows the binary image after the researcher performs the threshold step. (E) and (F) show the horizontal and vertical alveolar chord lengths that the researcher generates, respectively. Magnification of all images is x 200. Scale bar representing 400 μm is shown in (A).
Figure 4. Alveolar chord length and pressure-volume curves. (A) shows a typical analysis of alveolar chord lengths in C57BL/6 WT mice exposed to air (n = 13) or CS (n = 24) for 6 months. The asterisk indicates p<0.001. (B) shows typical PV loops performed on C57BL/6 WT mice exposed to air (n = 13) or CS (n = 14) for 6 months. Data are expressed as mean + SEM.
Figure 5. Small airway remodeling (SAR) assessment. (A) and (B) show representative images of Masson’s trichrome-stained lung sections from C57BL/6 WT mice exposed to air (A) or CS (B) for 6 months. (C) shows how image analysis software analyzes SAR in CS-exposed mice. (D) shows typical measurements of the thickness of the extracellular matrix protein layer deposited around small airways having a diameter of 300-899 m in C57BL/6 WT mice exposed to air (n = 11) or CS (n = 16) for 6 months. Data are expressed as mean + SEM and asterisk indicates p<0.05.
Scale bars are shown on the images of each lung section.
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, during the cigarette smoke exposure, it is essential to clean the machine at least once-a-week because tar products deposit in the device which can result in CS exposures that are not sufficient to induce lung disease. Second, it is essential to inflate lungs well to accurately measure airspace enlargement. If lungs are nicked during their removal from the thorax, this deflates lobes and results in an under-estimation of airspace size. Thus, to protect the lungs, only cut tissue when the tips of the scissor are visible. Third, for accurate analysis of emphysema development stain the alveolar walls of the lung section darkly with Gill’s stain, and optimize the exposure time for image acquisition to ensure ideal thresholding of the image to generate an accurate binary image for subsequent analysis. Fourth, capture images in a randomized fashion to avoid observer bias, and acquire and analyze at least 20 images per animal to ensure accurate analysis of airspace enlargement which is heterogeneously distributed in CS-exposed C57BL/6 WT mice. Fifth, in the SAR analysis do not make measurements in areas of adventitia that are shared by adjacent bronchi and vessels in broncho-vascular bundles which will lead to an over-estimation of ECM protein deposition around small airways.
There are several limitations of this protocol. First, the relatively long time needed to induce emphysema and SAR in CS-exposed mice (6 months). Second, there are well documented differences in the susceptibility of different strains of mice to develop emphysema in response to CS (10). Third, much less is known about the susceptibility of murine strains other than the C57BL/6 strain to CS-induced SAR. Fourth, the average researcher needs substantial practice in the lung inflation technique in order to consistently obtain optimally inflated lungs for analysis of both emphysema and SAR.
An advantage of this protocol is that whole–body cigarette smoke exposure minimizes distress to the mice, and is not labor-intensive to perform (unlike the nose-only exposure method). Compared with other methods, this protocol measures alveolar chord length on randomly-acquired images (as a readout of emphysema), is less time-consuming to complete, less susceptible to observer bias, and more effective at capturing the heterogeneous nature of the pathology in mice as it can analyze the disease in entire lobes and/or both lungs. The protocol also measures both contributors to airflow obstruction (emphysema and SAR) on lung sections from the same mice.
The protocol enables the researcher to assess the effects of CS on SAR in airways having different sizes. It can be adapted to measure individual ECM protein components deposited around airways in CS-exposed lungs to provide additional information about the nature of the small airway disease in CS-exposed animals. Overall, this protocol can quantify major chronic pulmonary pathologies in the lungs of mice exposed to CS in a relatively automated, accurate, and unbiased manner.
The authors have nothing to disclose.
We wish to thank Francesca Polverino MD, a Research Fellow at Brigham and Women’s Hospital for her contribution to this article, and also Monica Yao, BS, and Kate Rydell, BS for their assistance with murine husbandry and exposing the mice to cigarette smoke.
This work was supported by Public Health Service, National Heart, Lung, and Blood Institute Grants HL111835, HL105339, HL114501, Flight Attendants Medical Research Institute Grant #CIA123046, the Brigham and Women’s Hospital-Lovelace Respiratory Research Institute Consortium, and the Cambridge NIHR Biomedical Research Centre.
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 |