Name: Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure (Video) | JoVE Journal
Uploaded: 2017-08-25T14:00:00.000+00:00
Duration: 8 min 17 s
Description: Learn to generate a chronic obstructive pulmonary disease model in mice using repeated ozone exposure. This method recapitulates human COPD pathologies and enables drug screening in six to eight weeks.

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The authors would like to express gratitude to Mr. Boyin Qin (Shanghai Public Health Clinical Center) for the technical assistance with the &#181;CT evaluation in this 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
<ol>
	<li>House pathogen-free, 7- to 9-week-old female BALB/c mice in individual ventilated cages in an animal facility under controlled temperature (20 &#176;C) and humidity (40 - 60%). Provide a 12&#160;h light and 12&#160;h dark cycle in the facility. Provide food and water ad libitum.</li>
</ol>
2. Ozone or Air Exposure
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Micro-computed Tomography
<ol>
	<li>At the end of week 7, anesthetize the mice with an intraperitoneal injection of pelltobarbitalum natricum (1%, 0.6 - 0.8 ml/100 g)&#160;adjust the dose according to individual situations to see that the mouse does not respond to a toe pinch.&#160;Monitor and keep the mouse at a steady breathing frequency; and make sure no voluntary motions exist during the procedures.</li>
	<li>Place the anesthetized mice in the chamber of a micro-computed tomography (&#181;CT).</li>
	<li>Calibrate the &#181;CT using the standard protocol and the manufacturer&#39;s instructions. Set the X-ray tube at 50 kV and the current at 450 &#181;A. 
	NOTE: Both the X-ray and the detector rotate around the mice.</li>
	<li>Perform the &#181;CT 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.</li>
	<li>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.</li>
	<li>Calculate the percentage of LAA (LAA%) by dividing the LAA volume by the total lung volume.</li>
</ol>
4. Treadmill Test
<ol>
	<li>Give the mice an adaptation test at a speed of 10 m/min for 10 min on a running treadmill machine. &#160;Note: The electricity is always off when the procedure is being conducted.&#160;</li>
	<li>Administer a fatigue test to the mice.
	<ol>
		<li>Warm up the mice at a speed of 10 m/min for 5 min.</li>
		<li>Increase the speed to 15 m/min for 10 min.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Record the total running distance and running time as fatigue distance and fatigue time, respectively.</li>
</ol>
5. Pulmonary Function Measurements
<ol>
	<li>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&#160;and wait until the mice have maintained spontaneous breathing.&#160;Monitor and keep the mouse at a steady breathing frequency; and make sure no voluntary motions exist during the procedures.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Perform the quasi-static pressure-volume maneuver with the device by using the negative pressures generated in the plethysmograph.</li>
	<li>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.</li>
</ol>
6. BALF Collection
<ol>
	<li>Following terminal anesthesia with pelltobarbitalum natricum ((1%, 1.8-2.4 ml/100 g)&#160;adjust the dose according to individual situations to see that the mouse does not respond to a toe pinch and lose breath),&#160;lavage the mice with 2 mL of PBS via a 1 mm-diameter endotracheal tube and then retrieve the BALF10.</li>
	<li>Pool the retrieved lavage aliquots and centrifuge them at 4 &#176;C and 250 x g for 10 min.</li>
	<li>Collect the supernatant for immediate use and store the remainder at -80 &#176;C or liquid nitrogen.</li>
	<li>Count the total number of cells using a hemocytometer.</li>
	<li>Resuspend the cell pellet in PBS and then spin (1,400 x g, 6 min) 250 &#181;L of resuspended cells onto slides using a slide spinner centrifuge.</li>
	<li>Apply Wright staining to cells on the slides according to the manufacturer&#39;s protocol.</li>
	<li>Count 200 cells per mouse; identify the cells as macrophages or neutrophils, according to standard morphology, under 400X magnification; and count their numbers.</li>
</ol>
7. Cardiac Blood Sampling
<ol>
	<li>Collect blood via cardiac puncture, load it into 1.5-mL tubes, and keep it on ice for 30 min.</li>
	<li>Centrifuge the blood samples for 5 min at 2,000 x g and 4 &#176;C.</li>
	<li>Transfer the supernatant (serum) to a new tube and store it at -80 &#176;C or liquid nitrogen.</li>
	<li>Prepare the serum for IL-1&#946;, IL-10, and TNF-&#945; detection tests using the respective ELISA kits.</li>
</ol>
8. Lung Morphometric Analysis
<ol>
	<li>Dissect the lungs and tracheas from the mice.
	<ol>
		<li>Position each euthanized mouse onto a surgical board immediately after sacrifice.</li>
		<li>Dissect away the platysma and anterior tracheal muscles to visualize and access the tracheal rings.</li>
		<li>Open up the thoracic cavity. Dissect the lungs and the trachea, but do not separate the heart from the lungs.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Inflate the lung completely using 4% paraformaldehyde (10 drops, ~200 &#181;L) through the endotracheal catheter. Remove the heart after the completion of inflation.</li>
	<li>Maintain the lung in a 15&#160;mL tube containing 10 mL of 4% paraformaldehyde for at least 4 h.</li>
	<li>Embed the lung in paraffin. Obtain 5-&#181;m 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.</li>
	<li>For morphometric analysis, perform hematoxylin and eosin (H&#38;E) staining on the sections.</li>
	<li>Image the sections with a bright-field upright microscope (objective lens, 20X; exposure time, 1.667 ms).</li>
	<li>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:
	<ol>
		<li>Open the images of the sections in Photoshop and draw a reticule-grid on the image with five 550 &#181;m&#160;long lines.</li>
		<li>Count the number of alveoli across the grid line.</li>
		<li>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 &#177; 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 &#60;0.05 was considered significant.</li>
	</ol>
	</li>
</ol>

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.

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&#38;D period for new drugs. Compared...

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-&#945; and IL-1&#946;) 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.

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

generation of a chronic obstructive pulmonary disease model in mice by repeated ozone exposure

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

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.

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</su...

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.

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high ...

The overall goal of this experiment is to induce chronic obstructive pulmonary disease or C-O-P-D via ozone exposure, O-E in mice. This new OE model can happen in the characterization of the underlying mechanism of COPD and it can be used for the screening of new drugs. The main adavantages of this OE model are that it recapitulates the pathological processes of COPD patients.It can be generated in only six to eight weeks. Demonstrating the procedure with me will be Weijaian Sang, a research fellow in our group. To generate ozone, use an electric generator and a small air blower to blow the air out sealing Perspex box through an air vent pipe connected on the inside and outside of the box using an ozone probe to monitor the concentration of ozone in the box.When the ozone concentration reaches 2.5 parts per million place the mice into the box for 3 hours. At the end of week 7, image the ozone treated animals by micro-computed tomography according to standard protocols. To test the ozone induced changes and functional capacities such as lung fatigue, first warm up the mice at a speed of 10 meters per minute on a treadmill.After 5 minutes, increase the speed to 15 meters per minute for a period of 10 minutes. Continue to increase the speed by five meters per minute for every 30 minutes until the mice can no longer run. Recording the total running distance and running time for each animal as fatigue distance and fatigue time, respectively.To measure the pulmonary function of the ozone exposed animals, place an anesthetized mouse in a body plethysmograph connected to a computer controlled ventilator. Use pressure controlled ventilation to impose an average breathing frequency of a 150 breaths per minute to the anesthetized animal until a regular breathing pattern and a complete expiration at each breathing cycle is obtained. Then record the forced expiratory volume in the first 25, 50, and 75 milliseconds of exhalation rejecting any suboptimal maneuvers.Following terminal anesthesia, use a one millimeter diameter endotracheal tube to lavage the animals two times with one milliliter of PBS per lavage. Pool the retrieved lavage aloquats from each animal for centrifugation and collect the supernatants on liquid nitrogen. Re-suspend the pellets in fresh PPS for counting and equip two holders per mouse with a microscope slide, filter card, and funnel.Add approximately 100 to 125 microliters of the re-suspended cells to each funnel. Load the holders on to a cyto centrifuge and spin the cells on to the slides. Then apply right staining solution to the cells according to the manufacturer's instructions and count 200 cells per mouse under a 400 times magnification.For cardiac blood sampling, collect approximately 500 microliters of blood from the ozone exposed animals via cardiac puncture. And add the blood to 1.5 milliliter tubes on ice. After 30 minutes on ice, centrifuge the blood samples and transfer the serum supernatants into new tubes for storage on liquid nitrogen.The samples can later be thawed for the assessment of serum cytokine expression levels by ELISA according to standard protocols. For lung morphometric analysis, place the first mouse in the supine position after cardiac sampling. Then open up the thoracic cavity and dissect away the platysma and the anterior tracheal muscles to access the tracheal rings.Harvest the lungs and the trachea without separating the heart from the lungs. Next use a PE90 polyethylene tube to connect an endotracheal catheter to a syringe containing 4%power formaldehyde and completely inflate the lungs with approximately 200 microliters of paraformaldehyde. Then place the lungs in a 15 milliliter tube containing 10 milliliters of fresh four percent power formaldehyde for at least four hours.As assessed by microcomputed tomography, ozone exposed mice exhibit a significantly larger total lung volume and percentage of low attenuation area than ambient air exposed control animals. The emphysema phenotype is also evidence by the enlarged lung alveoli and increased mean linear intercepts. Lung function assessment by plethysmograph demonstrates that all the parameters that decrease in ozone exposed mice are consistent with the typical lung functional defects observed in chronic obstructive pulmonary disease patients.Further, ozone exposure significantly decreases the fatigue time and fatigue distance in an exercise tolerance tests. Ozone treated animals also display significant increase in inflammatory cells including macrophages and neutrophils as well as a significant decrease in IL-10 and an increase in IL-1 beta and TNF alpha demonstrating that the ozone exposure model recapitulates human chronic obstructive pulmonary disease like symptoms. Following this procedure on other strains of mice like the C57 BR6 mice can be used to generate this OE model After its development, this technique paves the way for researchers in the field of pulmonary disease to explore the underlying mechanisms of COPD.Don't forget that working with the paraformaldehyde can be hazardous and that precautions such as wearing gloves, safety glasses, and using the solution inside the fume hood should always be taken while working with this reagent.

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Learn to generate a chronic obstructive pulmonary disease model in mice using repeated ozone exposure. This method recapitulates human COPD pathologies and enables drug screening in six to eight weeks.

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

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