Name: generation of a chronic obstructive pulmonary disease model in mice by repeated ozone exposure
Uploaded: 2017-08-25T14:00:00
Description: Watch this Scientific Journal Video about Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure at JoVE.com

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 (4...

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.

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	<tbody>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">BALB/c mice</td>
			<td style="width:217px;">Slac Laboratory Animal,Shanghai, China</td>
			<td style="width:143px;">N/A</td>
			<td style="width:455px;">7-to-9-week-old female BALB/c mice were used in this study.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">Individual ventilated cages</td>
			<td style="width:217px;">Suhang, Shanghai, China</td>
			<td style="width:143px;">Model Number: MU64S7</td>
			<td>The cages were used for housing mice in the animal facility.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">Sealing perspex-box</td>
			<td style="width:217px;">Suhang, Shanghai, China</td>
			<td style="width:143px;">N/A</td>
			<td style="width:455px;">The box was used&#160; to contain the ozone generator. Mice were exposed to ozone within the box.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">Electric generator</td>
			<td style="width:217px;">Sander Ozoniser, Uetze-Eltze, Germany</td>
			<td style="width:143px;">Model 500&#160;</td>
			<td>The device was used for generating ozone.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">Ozone probe</td>
			<td style="width:217px;">ATi Technologies, Ashton-U-Lyne, Greater Manchester, UK</td>
			<td style="width:143px;">Ozone 300</td>
			<td>The device was used for monitoring and controlling the generation of ozone.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">Pelltobarbitalum natricum</td>
			<td style="width:217px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:143px;">P3761</td>
			<td style="width:455px;">Mice were anesthetized by intraperitoneal injection of pelltobarbitalum natricum.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">Micro-Computed Tomography</td>
			<td style="width:217px;">GE Healthcare, London, ON, Canada</td>
			<td style="width:143px;">RS0800639-0075</td>
			<td>This device was used for acquiring images of the lung.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">Micro-view 2.01 ABA software</td>
			<td style="width:217px;">GE Healthcare, London, ON, Canada</td>
			<td style="width:143px;">Micro-view 2.01&#160;</td>
			<td>This device was used for reconstruct the lung and analyze volume, LAA of the lung.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">Treadmill machine&#160;</td>
			<td style="width:217px;">Duanshi, Hangzhou, Zhejiang, China</td>
			<td style="width:143px;">DSPT-208</td>
			<td>This machine was usd for fatigue test.</td>
		</tr>
		<tr height="38">
			<td height="38" style="width:211px;height:38px;">Body plethysmograph</td>
			<td style="width:217px;">eSpira&#8482; Forced Manoeuvres System, EMMS, Edinburgh, UK</td>
			<td style="width:143px;">Forced Manoeuvres System</td>
			<td height="80">This device was used to test spirometry pulmonary function.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">Ventilator</td>
			<td style="width:217px;">eSpira&#8482; Forced Manoeuvres System, EMMS, Edinburgh, UK</td>
			<td style="width:143px;">Forced Manoeuvres System</td>
			<td height="80">This device was used to test spirometry pulmonary function.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">Slide spinner centrifuge</td>
			<td style="width:217px;">Denville Scientific, Holliston, MA, USA</td>
			<td>C1183&#160;</td>
			<td>It was used to spin BALF cells onto slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:211px;height:21px;">Wright Staining</td>
			<td style="width:217px;">Hanhong, Shanghai, China</td>
			<td style="width:143px;">RE04000054</td>
			<td>&#160;It was used to staining macrophages, neutrophils in the suspended BALF.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">Hemocytometer</td>
			<td style="width:217px;">Hausser Scientific, Horsham, PA, USA</td>
			<td style="width:143px;">4000</td>
			<td>It was used to count cells.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:211px;height:21px;">IL-1&#946;</td>
			<td style="width:217px;">Abcam, Cambridge, MA, USA</td>
			<td style="width:143px;">ab100704</td>
			<td height="63">They were used to test the respective factors in serum.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:211px;height:21px;">IL-10</td>
			<td style="width:217px;">Abcam, Cambridge, MA, USA</td>
			<td style="width:143px;">ab46103</td>
			<td height="63">They were used to test the respective factors in serum.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:211px;height:21px;">TNF-&#945;</td>
			<td style="width:217px;">Abcam, Cambridge, MA, USA</td>
			<td style="width:143px;">ab100747</td>
			<td height="63">They were used to test the respective factors in serum.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:211px;height:21px;">Paraformaldehyde&#160;</td>
			<td style="width:217px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:143px;">P6148</td>
			<td>The lung was inflated by 4% paraformaldehyde.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:211px;height:21px;">Paraffin</td>
			<td style="width:217px;">Hualing, Shanghai, China</td>
			<td style="width:143px;">56#</td>
			<td>It was used to embed the lung.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:211px;height:21px;">Rotary Microtome</td>
			<td style="width:217px;">Leica, Wetzlar,&#160; Hesse, Germany</td>
			<td style="width:143px;">RM2255</td>
			<td>It was used for sectioning the lung.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">Hgaematoxylin and Eosin (H&#38;E) staining solution</td>
			<td style="width:217px;">Solarbio, Beijing, China</td>
			<td style="width:143px;">G1120</td>
			<td>H&#38;E staining was done for morphometric analysis.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">Upright bright field microscope</td>
			<td style="width:217px;">Olympus, Center Valley, PA, USA</td>
			<td style="width:143px;">CX41</td>
			<td>It was used to image the H&#38;E staining slides.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:211px;height:21px;">Adobe Photoshop 12</td>
			<td style="width:217px;">Adobe, San Jose, CA, USA</td>
			<td style="width:143px;">Adobe Photoshop 12</td>
			<td>It was used to count the number of alveoli on the H&#38;E stained images.</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:211px;height:42px;">GraphPad prism 5</td>
			<td style="width:217px;">Graphpad Software Inc., San Diego, CA</td>
			<td style="width:143px;">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

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 &#181;CT 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 exposure31,3...

Watch this Scientific Journal Video about Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure at JoVE.com

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

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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