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

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 2020^1,2. 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 symptoms^5,6. 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 airflow^5,6,7,8,9,10. 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 model^12,13,14,15. 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 COPD^16,17,18. 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 adults^{19,20,21,22,23,24,25}. 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 COPD^26,27. Another factor is that the symptoms of some COPD patients deteriorate after being exposed to ozone, indicating that ozone can disrupt lung function^18,28,29. 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 investigations^30,31,32. We extended the OE protocol to female mice in this study and successfully reproduced the emphysema observed in male mice in our previous studies^30,31,32. 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.

Protocol

NOTE: The OE model has been generated and used in previously reported research^30,31,32. 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 (L_m) as a parameter for measuring the distance of the inter-alveolar septal wall. Determine the L_m 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 L_m 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.

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 exposure^31,3...

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 model^41,42. However, the CS model requires a 3- to 12-month R&D period for new drugs. Compared...

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.