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JoVE Journal Medicine

Medicine

Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure

Published: August 25, 2017 doi: 10.3791/56095
Automatic Translation
1,2, 3, 3, 1,2
1Cellular Biomedicine Group, Shanghai, 2Cellular Biomedicine Group, Cupertino, 3Department of Respiratory Medicine, Shanghai General Hospital, Shanghai Jiaotong University

Summary

This study describes the successful generation of a new chronic obstructive pulmonary disease (COPD) animal model by repeatedly exposing mice to high concentrations of ozone.

Abstract

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high incidence in aging populations. The current conventional therapies for COPD focus mainly on symptom-modifying drugs; thus, the development of new therapies is urgently needed. Qualified animal models of COPD could help to characterize the underlying mechanisms and can be used for new drug screening. Current COPD models, such as lipopolysaccharide (LPS) or the porcine pancreatic elastase (PPE)-induced emphysema model, generate COPD-like lesions in the lungs and airways but do not otherwise resemble the pathogenesis of human COPD. A cigarette smoke (CS)-induced model remains one of the most popular because it not only simulates COPD-like lesions in the respiratory system, but it is also based on one of the main hazardous materials that causes COPD in humans. However, the time-consuming and labor-intensive aspects of the CS-induced model dramatically limit its application in new drug screening. In this study, we successfully generated a new COPD model by exposing mice to high levels of ozone. This model demonstrated the following: 1) decreased forced expiratory volume 25, 50, and 75/forced vital capacity (FEV25/FVC, FEV50/FVC, and FEV75/FVC), indicating the deterioration of lung function; 2) enlarged lung alveoli, with lung parenchymal destruction; 3) reduced fatigue time and distance; and 4) increased inflammation. Taken together, these data demonstrate that the ozone exposure (OE) model is a reliable animal model that is similar to humans because ozone overexposure is one of the etiological factors of COPD. Additionally, it only took 6 - 8 weeks, based on our previous work, to create an OE model, whereas it requires 3 - 12 months to induce the cigarette smoke model, indicating that the OE model might be a good choice for COPD research.

Introduction

It has been estimated that COPD, including emphysema and chronic bronchitis, might be the third leading cause of death in the world in 20201,2. The potential incidence of COPD in a population over 40 years old is estimated to be 12.7% in males and 8.3% in females within the next 40 years3. No medications are currently available to reverse the progressive deterioration in COPD patients4. Reliable animal models of COPD not only demand the imitation of the disease pathological process but also require a short generation period. Current COPD models, including the LPS or a PPE-induced model, can induce emphysema-like symptoms5,6. A single administration or a week-long challenge of LPS or PPE to mice or rats results in marked neutrophilia in the bronchoalveolar lavage fluid (BALF), increases pro-inflammatory mediators (e.g., TNF-α and IL-1β) in the BALF or serum, produces lung parenchymal destruction-enlarged air spaces, and limits airflow5,6,7,8,9,10. However, LPS or PPE are not causes of human COPD and thus do not mimic the pathological process11. A CS-induced model produced a persistent airflow limitation, lung parenchymal destruction, and reduced functional exercise capacity. However, a traditional CS protocol requires at least 3 months to generate a COPD model12,13,14,15. Thus, it is important to generate a new, more efficient animal model that meets the two requirements.

Recently, in addition to cigarette smoking, air pollution and occupational exposure have become more common causes of COPD16,17,18. Ozone, as one of the major pollutants (though not the major component of air pollution), can directly react with the respiratory tract and damage the lung tissue of both children and young adults19,20,21,22,23,24,25. Ozone, as well as other stimulators including LPS, PPE, and CS, are involved in a serious of biochemical pathways of pulmonary oxidative stress and DNA damage and are linked to the initiation and promotion of COPD26,27. Another factor is that the symptoms of some COPD patients deteriorate after being exposed to ozone, indicating that ozone can disrupt lung function18,28,29. Therefore, we generated a new COPD model by repeatedly exposing mice to high concentrations of ozone for 7 weeks; this resulted in airflow defects and lung parenchymal damage similar to those of previous investigations30,31,32. We extended the OE protocol to female mice in this study and successfully reproduced the emphysema observed in male mice in our previous studies30,31,32. Because COPD mortality has decreased in men but increased in women in many countries33, a COPD model in females is needed to study the mechanisms and to develop therapeutic methods for female COPD patients. The applicability of the OE model to all genders lends further support to its use as a COPD model.

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Protocol

NOTE: The OE model has been generated and used in previously reported research30,31,32. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Jiaotong University.

1. Mice

  1. House pathogen-free, 7- to 9-week-old female BALB/c mice in individual ventilated cages in an animal facility under controlled temperature (20 °C) and humidity (40 - 60%). Provide a 12 h light and 12 h dark cycle in the facility. Provide food and water ad libitum.

2. Ozone or Air Exposure

  1. Generate ozone with an electric generator in a sealing acrylic (e.g. Perspex) box. Blow the air out of the box using a small air blower through an air vent pipe that is connected on the inside and outside of the box. Monitor the concentration of ozone in the box using an ozone probe. Wait until the concentration of ozone in the box reaches 2.5 parts per million (ppm).
    NOTE: The ozone probe can automatically switch the ozone generator on or off and can maintain the ozone level in the box at 2.5 ppm.
  2. Place the mice into the box when the level of ozone reaches 2.5 ppm. Keep the mice in the box for 3 h each time to expose them to ozone.
    NOTE: The box can maintain an ozone level of 2.5 ppm during the 3 h by automatically switching the ozone generator on or off and blowing the CO2 that is produced by the mice out of the box.
  3. Give two ozone exposures (each exposure lasting 3 h) per week (once every 3 days) for 7 weeks; expose the control mice to air at the same time and for the same period.

3. Micro-computed Tomography

  1. At the end of week 7, anesthetize the mice with an intraperitoneal injection of pelltobarbitalum natricum (1%, 0.6 - 0.8 ml/100 g) adjust the dose according to individual situations to see that the mouse does not respond to a toe pinch. Monitor and keep the mouse at a steady breathing frequency; and make sure no voluntary motions exist during the procedures.
  2. Place the anesthetized mice in the chamber of a micro-computed tomography (µCT).
  3. Calibrate the µCT using the standard protocol and the manufacturer's instructions. Set the X-ray tube at 50 kV and the current at 450 µA.
    NOTE: Both the X-ray and the detector rotate around the mice.
  4. Perform the µCT analysis by acquiring 515 projections with an effective pixel size of 0.092 mm, an exposure time of 300 ms for one slice, and a slice thickness of 0.093 mm.
  5. Reconstruct the lung with the acquired images using a software. Adjust the grayscale image brightness by setting the minimum and maximum of the grayscale at -750 and -550 Hounsfield units, respectively.
    NOTE: The software will automatically calculate the volumes of the lung parenchyma and the low-attenuation area (LAA)34,35.
  6. Calculate the percentage of LAA (LAA%) by dividing the LAA volume by the total lung volume.

4. Treadmill Test

  1. Give the mice an adaptation test at a speed of 10 m/min for 10 min on a running treadmill machine.  Note: The electricity is always off when the procedure is being conducted. 
  2. Administer a fatigue test to the mice.
    1. Warm up the mice at a speed of 10 m/min for 5 min.
    2. Increase the speed to 15 m/min for 10 min.
    3. Increase the exercise intensity: increasing the speed by 5 m/min, starting at 20 m/min, every 30 min until mice cannot continue to run36.
  3. Record the total running distance and running time as fatigue distance and fatigue time, respectively.

5. Pulmonary Function Measurements

  1. Anesthetize the mice with an intraperitoneal injection of pelltobarbitalum natricum (1%, 0.6 - 0.8 ml/100 g) adjust the dose according to individual situations to see that the mouse does not respond to a toe pinch and wait until the mice have maintained spontaneous breathing. Monitor and keep the mouse at a steady breathing frequency; and make sure no voluntary motions exist during the procedures.
  2. Carefully tracheostomize the mice and place them in a body plethysmograph that is connected to a computer-controlled ventilator.
    NOTE: Ventilation is controlled via a valve located proximally to the endotracheal tube. The setup provides different semi-automatic maneuvers, including the maneuver of the quasi-static pressure volume and the maneuver of the fast flow volume.
  3. Impose an average breathing frequency of 150 breaths/min to the anesthetized mouse via pressure-controlled ventilation until a regular breathing pattern and complete expiration at each breathing cycle is obtained.
  4. Perform the quasi-static pressure-volume maneuver with the device by using the negative pressures generated in the plethysmograph.
  5. Perform the fast flow volume maneuver within the quasi-static pressure-volume loops to record the FVC and the FEV. Inflate the lung to +30 cm H2O and immediately afterwards connect it to a highly negative pressure to enforce expiration until the residual volume is at -30 cm H2O. Record the FEV in the first 25, 50, and 75 ms of exhalation (FEV25, FEV50, and FEV75, respectively). Reject the suboptimal maneuvers. For each test with every single mouse, conduct a minimum of three acceptable maneuvers to obtain a reliable mean for all numerical parameters.

6. BALF Collection

  1. Following terminal anesthesia with pelltobarbitalum natricum ((1%, 1.8-2.4 ml/100 g) adjust the dose according to individual situations to see that the mouse does not respond to a toe pinch and lose breath), lavage the mice with 2 mL of PBS via a 1 mm-diameter endotracheal tube and then retrieve the BALF10.
  2. Pool the retrieved lavage aliquots and centrifuge them at 4 °C and 250 x g for 10 min.
  3. Collect the supernatant for immediate use and store the remainder at -80 °C or liquid nitrogen.
  4. Count the total number of cells using a hemocytometer.
  5. Resuspend the cell pellet in PBS and then spin (1,400 x g, 6 min) 250 µL of resuspended cells onto slides using a slide spinner centrifuge.
  6. Apply Wright staining to cells on the slides according to the manufacturer's protocol.
  7. Count 200 cells per mouse; identify the cells as macrophages or neutrophils, according to standard morphology, under 400X magnification; and count their numbers.

7. Cardiac Blood Sampling

  1. Collect blood via cardiac puncture, load it into 1.5-mL tubes, and keep it on ice for 30 min.
  2. Centrifuge the blood samples for 5 min at 2,000 x g and 4 °C.
  3. Transfer the supernatant (serum) to a new tube and store it at -80 °C or liquid nitrogen.
  4. Prepare the serum for IL-1β, IL-10, and TNF-α detection tests using the respective ELISA kits.

8. Lung Morphometric Analysis

  1. Dissect the lungs and tracheas from the mice.
    1. Position each euthanized mouse onto a surgical board immediately after sacrifice.
    2. Dissect away the platysma and anterior tracheal muscles to visualize and access the tracheal rings.
    3. Open up the thoracic cavity. Dissect the lungs and the trachea, but do not separate the heart from the lungs.
  2. Connect the endotracheal catheter to a syringe containing 4% paraformaldehyde through a PE90 polyethylene tube.
    Caution: Paraformaldehyde is toxic. Wear gloves and safety glasses and use the solution inside a fume hood.
  3. Inflate the lung completely using 4% paraformaldehyde (10 drops, ~200 µL) through the endotracheal catheter. Remove the heart after the completion of inflation.
  4. Maintain the lung in a 15 mL tube containing 10 mL of 4% paraformaldehyde for at least 4 h.
  5. Embed the lung in paraffin. Obtain 5-µm sections by paraffin block sectioning with a rotary microtome. During the sectioning, expose the maximum surface area of lung tissue within the bronchial tree area.
  6. For morphometric analysis, perform hematoxylin and eosin (H&E) staining on the sections.
  7. Image the sections with a bright-field upright microscope (objective lens, 20X; exposure time, 1.667 ms).
  8. Have two investigators blinded to the treatment protocol independently count the histological sections. Use the mean linear intercept (Lm) as a parameter for measuring the distance of the inter-alveolar septal wall. Determine the Lm using the following steps:
    1. Open the images of the sections in Photoshop and draw a reticule-grid on the image with five 550 µm long lines.
    2. Count the number of alveoli across the grid line.
    3. Calculate the Lm by dividing the length of the grid line by the number of alveoli. For quantification, image five sections per mouse. Acquire ten images of each section (one image per field) and assess randomly. During field selection, avoid fields of airways and vessels by moving one field ahead or in another direction.
      Note: The data are presented as the mean ± S.E.M. An un-paired t-test was carried out for comparison between air-exposed mice and ozone-exposed mice. Three animals of each group were used to calculate the significant difference. A p-value of <0.05 was considered significant.

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Representative Results

Examples of 3D µCT images of each group are displayed in Figure 1a. The ozone-exposed mice had a significantly larger total lung volume (Figure 1a and b) and LAA% (Figure 1c) than did the air-exposed control mice. The lung volume and LAA% remained elevated after six weeks of ozone exposure31,32. Increased lung volume and LAA% represent the emphysema phenotype. Examples of lung alveolar enlargement in Figure 2a illustrate the emphysema formation. An increase in the mean linear intercept (Lm) was observed in ozone-exposed mice (Figure 2b), which confirmed that lung parenchymal destruction occurred after ozone exposure.

Lung function was measured by the airflow rate parameters, shown as FEV25/FVC, FEV50/FVC, and FEV75/FVC. The results showed that all of the parameters that decreased in ozone-exposed mice (Figure 3a-c) were consistent with the typical lung functional defects in COPD patients4,37. To further evaluate the OE model, we carried out an exercise tolerance test that substituted for the 6-min walk test (6MWT)38,39, which is commonly used to assess changes in functional exercise capacity in COPD patients. The ozone exposure significantly decreased the fatigue time and fatigue distance (Figure 4a and b).

To address the pathogenesis of COPD in the OE model, the macrophages and neutrophils were counted from the BALFs of the mice; pro-inflammatory cytokines (IL-1β and TNF-α) and anti-inflammatory cytokines (IL-10) were detected in the mouse sera. The ozone-exposed mice showed a significant increase in inflammatory cells, including macrophages and neutrophils (Figure 5a-c), as well as a significant decrease in IL-10 and an increase in IL-1β and TNF-α (Figure 6a-c). All of the data demonstrated that the OE model recapitulated human COPD-like symptoms.

Figure 1
Figure 1.Ozone exposure increased the lung volume and LAA% detected by µCT.(a) Representative 3D images showing the lungs of air- or ozone-exposed mice at 7 weeks after the respective exposure. (b) Individual total lung volumes of the two groups were extracted from the 3D images for statistics. Ozone-exposed mice showed a significant increase in total lung volume. (c) Individual and mean LAA% of the two groups. Ozone-exposed mice showed a significant increase in LAA%. The red color indicates LAA (voxels with densities of 2,550-2,700 Hounsfield units). The trachea and bronchi shown in red in this figure were removed to calculate the lung LAA. The data are presented as the mean ± S.E.M. **P<0.01, ***P<0.001. Please click here to view a larger version of this figure.

Figure 2
Figure 2. Ozone exposure increased the Lm. (a) Representative micrographs of lung alveolar spaces in HE-stained sections of air-exposed or ozone-exposed mice. (b) Individual values of Lm were quantified from the lung sections of the two groups for statistics. An increase in Lm was observed in ozone-exposed mice. The data are presented as the mean ± S.E.M. **P<0.01. Please click here to view a larger version of this figure.

Figure 3
Figure 3. Ozone exposure decreased pulmonary function. (a-c) For both the air-exposed and the ozone-exposed mice, the individual FEV in the first 25, 50, and 75 ms of fast expiration (FEV25, FEV50, and FEV75, respectively), as well as the FVC, were all recorded. The percentages of FEV25, FEV50, and FEV75 to FVC were calculated separately. FEV25/FVC, FEV50/FVC, and FEV75/FVC all decreased significantly in ozone-exposed mice. The data are presented as the mean ± S.E.M. *P<0.05, **P<0.01. Please click here to view a larger version of this figure.

Figure 4
Figure 4. Ozone exposure decreased fatigue time and fatigue distance. (a) Individual running times for air- or ozone-exposed mice were recorded. Ozone-exposed mice showed a significant decrease in fatigue time. (b) Individual running distances of the two groups were recorded. Ozone-exposed mice exhibited a significant decrease in fatigue distance. The data are presented as the mean ± S.E.M. *P<0.05. Please click here to view a larger version of this figure.

Figure 5
Figure 5. Inflammatory cells counted from BALF. (a) Individual and mean numbers of total cells (TOTAL) in air- or ozone-exposed mice. (b) Individual and mean numbers of macrophages (MAC) in the two groups. (c) Individual and mean numbers of neutrophils (NEU) in the two groups. The data are presented as the mean ± S.E.M. *P<0.05, **P<0.01. Please click here to view a larger version of this figure.

Figure 6
Figure 6. Inflammatory and anti-inflammatory cytokine detection in serum. (a) Individual and mean values of the amount of IL-10 in air- or ozone-exposed mice. (b) Individual and mean values of the amount of IL-1β in the two groups. (c) Individual and mean values of the amount of TNF-α in the two groups. The data are presented as the mean ± S.E.M. *P<0.05. Please click here to view a larger version of this figure.

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Discussion

In this study, we present a reliable method for generating a new COPD model. Compared to other models (i.e., LPS or PPE models), this OE model recapitulates the pathological process of COPD patients. Because cigarette smoke is the main hazardous material that causes COPD in human patients40, the CS model remains the most popular COPD model41,42. However, the CS model requires a 3- to 12-month R&D period for new drugs. Compared to the CS model, the current OE model reduces the generation period to 6-8 weeks. We have observed emphysema in male mice after 6 weeks of exposure to ozone in our previous studies30,31,32. In this study, we applied the OE protocol to female mice for 7 weeks and successfully generated a female OE model. Because it has been reported that COPD mortality has decreased in men but increased in women in some countries33, it is necessary to study the pathogenic mechanisms and to develop cure approaches for female COPD patients using a female COPD model. We know that the abovementioned COPD models (i.e., LPS, PPE, and CS) can work in both male and female animals43. The aim of this work was to propose an additional COPD model that both recapitulates the pathological process of COPD patients and requires a very short generation-period.

The key step for generating this model was exposing the mice to ozone (at a level of 2.5 ppm) two times per week (once every 3 days) for 6-8 weeks (in this study, we used 7 weeks, although we did not try dose escalation). By controlling the critical parameters of exposure frequency and ozone concentration, we successfully reproduced emphysema in male C57BL/6 mice31,32, male BALB/c mice30, and female BALB/c mice (the current study). The emphysema phenotype resulted from ozone exposure, with the airflow limitation and lung parenchymal destruction similar to the changes seen in COPD patients44,45.

There are still limitations to this study. For example, because the pathogenesis of human COPD is related to the anatomy of the lungs, an ideal COPD model should be generated in animals that have a pulmonary anatomical structure similar to that of humans.Compared to large animals, small animals possess even less extensive airway branching than humans46. We must admit that it would be more meaningful to generate a COPD model in large animals. However, it is very difficult to establish large-scale models in animals. Another limitation of this model is its clinical relevance. Although it is certain that ozone can react with the respiratory tract and damage lung tissue19,22, and although there is evidence that the symptoms of some COPD patients deteriorate after exposure to ozone28, ozone is not the main cause of COPD in patients. However, we are still proposing and using this OE model because both ozone and CS cause damage to the respiratory system by inducing inflammation and lead to oxidative stress26,27. Thus, a new drug that can cure COPD in the OE model can assumably also work in the CS model and therefore potentially be developed for COPD patients.

Applications of the OE model are not restricted to deciphering the molecular and cellular mechanisms of COPD. Our two recent papers also investigated the efficacy of N-acetylcysteine (NAC)31 and NaHS32 (an exogenous donor of H2S) at treating COPD via the exogenous administration of the two drugs to the OE model. In the first study, we found a reversal of airway hyper-responsiveness and a reduction of airway smooth muscle mass after the administration of NAC. These effects might indicate the basis for potential clinical benefits of NAC in COPD patients31. In the second study of the OE model, we found that the administration of exogenous NaHS reversed lung inflammation and partially reversed the features of emphysema. Thus, with this OE model, we demonstrated in a preliminary study that NaHS could be developed as a potential drug candidate for COPD patients32. Therefore, the OE model has potential applications for both mechanism research and drug screening for COPD.

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Disclosures

Z.W.S. and W.W. are current employees and stock option holders of the Cellular Biomedicine Group (NASDAQ: CBMG). The other authors declare that they have no competing interests.

Acknowledgments

The authors would like to express gratitude to Mr. Boyin Qin (Shanghai Public Health Clinical Center) for the technical assistance with the µCT evaluation in this protocol.

Materials

Name Company Catalog Number Comments
BALB/c mice Slac Laboratory Animal,Shanghai, China N/A 7-to-9-week-old female BALB/c mice were used in this study.
Individual ventilated cages Suhang, Shanghai, China Model Number: MU64S7 The cages were used for housing mice in the animal facility.
Sealing perspex-box Suhang, Shanghai, China N/A The box was used  to contain the ozone generator. Mice were exposed to ozone within the box.
Electric generator Sander Ozoniser, Uetze-Eltze, Germany Model 500  The device was used for generating ozone.
Ozone probe ATi Technologies, Ashton-U-Lyne, Greater Manchester, UK Ozone 300 The device was used for monitoring and controlling the generation of ozone.
Pelltobarbitalum natricum Sigma, St. Louis, MO, USA P3761 Mice were anesthetized by intraperitoneal injection of pelltobarbitalum natricum.
Micro-Computed Tomography GE Healthcare, London, ON, Canada RS0800639-0075 This device was used for acquiring images of the lung.
Micro-view 2.01 ABA software GE Healthcare, London, ON, Canada Micro-view 2.01  This device was used for reconstruct the lung and analyze volume, LAA of the lung.
Treadmill machine  Duanshi, Hangzhou, Zhejiang, China DSPT-208 This machine was usd for fatigue test.
Body plethysmograph eSpira™ Forced Manoeuvres System, EMMS, Edinburgh, UK Forced Manoeuvres System This device was used to test spirometry pulmonary function.
Ventilator eSpira™ Forced Manoeuvres System, EMMS, Edinburgh, UK Forced Manoeuvres System This device was used to test spirometry pulmonary function.
Slide spinner centrifuge Denville Scientific, Holliston, MA, USA C1183  It was used to spin BALF cells onto slides.
Wright Staining Hanhong, Shanghai, China RE04000054  It was used to staining macrophages, neutrophils in the suspended BALF.
Hemocytometer Hausser Scientific, Horsham, PA, USA 4000 It was used to count cells.
IL-1β Abcam, Cambridge, MA, USA ab100704 They were used to test the respective factors in serum.
IL-10 Abcam, Cambridge, MA, USA ab46103 They were used to test the respective factors in serum.
TNF-α Abcam, Cambridge, MA, USA ab100747 They were used to test the respective factors in serum.
Paraformaldehyde  Sigma, St. Louis, MO, USA P6148 The lung was inflated by 4% paraformaldehyde.
Paraffin Hualing, Shanghai, China 56# It was used to embed the lung.
Rotary Microtome Leica, Wetzlar,  Hesse, Germany RM2255 It was used for sectioning the lung.
Hgaematoxylin and Eosin (H&E) staining solution Solarbio, Beijing, China G1120 H&E staining was done for morphometric analysis.
Upright bright field microscope Olympus, Center Valley, PA, USA CX41 It was used to image the H&E staining slides.
Adobe Photoshop 12 Adobe, San Jose, CA, USA Adobe Photoshop 12 It was used to count the number of alveoli on the H&E stained images.
GraphPad prism 5 Graphpad Software Inc., San Diego, CA GraphPad prism 5 It was used for data analysis and production of figures.

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Chronic Obstructive Pulmonary Disease COPD Ozone Exposure OE Model Mice Underlying Mechanism Screening Of New Drugs Pathological Processes Ozone Concentration Perspex Box Air Vent Pipe Ozone Probe Micro-computed Tomography Lung Fatigue Treadmill Running Distance Running Time
Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure
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Sun, Z., Li, F., Zhou, X., Wang, W.… More

Sun, Z., Li, F., Zhou, X., Wang, W. Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure. J. Vis. Exp. (126), e56095, doi:10.3791/56095 (2017).

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