AR, XH, and PE were supported by NIH grant R01HL140398 and a Gilson Longenbaugh Foundation grant. DEMM and KK were supported by the NIH grants R01HL136333 and R01HL134880 (KYK), and a grant from the Helis Medical Research Foundation. DEMM is also supported by the Howard Hughes Medical Institute (HHMI) Gilliam Fellowship for Advanced Study. PE is also supported by Training in Precision Environmental Health Sciences NIEHS T32 ES027801 Fellowship Program. JC and MF are supported by Tobacco Research Funds from the Department of Epigenetics and Molecular Carcinogenesis and by the Center for Epigenetics (Scholar Award to MF) at MD Anderson. FK and YZ are supported by NIH grants R01 ES029442-01 and R01 AI135803-01 as well as VA Merit grant CX000104. This project was supported by the Cytometry and Cell Sorting Core at Baylor College of Medicine with funding from the CPRIT Core Facility Support Award (CPRIT-RP180672), the NIH (CA125123 and RR024574), and the assistance of Joel M. Sederstrom.

All the animals involved in the experiments and the development of this technique have been under our animal use protocol approved by the Institutional Animal Care and Use Committee (IACUC) and under Baylor College of Medicine and MD Anderson institutions that are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).
1. Building the apparatus
<ol>
	<li>Assembling the air compressor with the valve system.
	<ol>
		<li>Connect the fl...

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+implications+of+clonal+hematopoiesis.">Clinical implications of clonal hematopoiesis.</a> Mayo Clinic Proceedings. 93, 1122-1130 (2018).</li><li>Tobacco. Federal Trade Comission Available from: <a target="_blank" href="https://www.ftc.gov/tips-advice/business-center/selected-industries/tobacco">https://www.ftc.gov/tips-advice/business-center/selected-industries/tobacco</a> (2020)</li><li>3R4F Cigarettes. University of Kentucky - College of Agriculture Food and Environment Available from: <a target="_blank" href="https://ctrp.uky.edu/products/gallery/Reference">https://ctrp.uky.edu/products/gallery/Reference</a> (2020)</li><li>Shan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoke+induction+of+osteopontin+(SPP1)+mediates+T+H+17+inflammation+in+human+and+experimental+emphysema.">Cigarette smoke induction of osteopontin (SPP1) mediates T H 17 inflammation in human and experimental emphysema.</a> Science Translational Medicine. 4, 1-10 (2012).</li><li>Yuan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+C3a+receptor+is+required+in+cigarette+smoke-mediated+emphysema.">Activation of C3a receptor is required in cigarette smoke-mediated emphysema.</a> Nature Mucosal Immunology. 8, 874-885 (2014).</li><li>Yuan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoke+-+induced+reduction+of+C1q+promotes+emphysema.">Cigarette smoke - induced reduction of C1q promotes emphysema.</a> JCI Insight. 4, 1-17 (2019).</li><li>Shan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agonistic+induction+of+PPAR+g+reverses+cigarette+smoke+-+induced+emphysema+Find+the+latest+version:+Agonistic+induction+of+PPAR+&#947;+reverses+cigarette+smoke+-+induced+emphysema.">Agonistic induction of PPAR g reverses cigarette smoke - induced emphysema Find the latest version: Agonistic induction of PPAR &#947; reverses cigarette smoke - induced emphysema.</a> Journal of Clinical Investigation. 124, 1371-1381 (2014).</li><li>Hong, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+role+of+gd+T+cells+in+cigarette+smoke+and+influenza+infection.">Protective role of gd T cells in cigarette smoke and influenza infection.</a> Nature Mucosal Immunology. 11, 834-908 (2018).</li><li>Kim, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoke+induces+intestinal+inflammation+via+a+Th17+cell-neutrophil+axis.">Cigarette smoke induces intestinal inflammation via a Th17 cell-neutrophil axis.</a> Frontiers in Immunology. 10, 1-11 (2019).</li><li>Lu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microRNA+miR-22+inhibits+the+histone+deacetylase+HDAC4+to+promote+T+H+17+cell+-+dependent+emphysema.">The microRNA miR-22 inhibits the histone deacetylase HDAC4 to promote T H 17 cell - dependent emphysema.</a> Nature Immunology. 16, 1185-1194 (2015).</li><li>Hendrix, A. Y., Kheradmand, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Role of Matrix Metalloproteinases in Development, Repair, and Destruction of the Lungs. Progress in Molecular Biology and Translational Science. 148, (2017).</li><li>Siggins, R. W., Hossain, F., Rehman, T., Melvan, J. N., Welsh, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoke+alters+the+hematopoietic+stem+cell+niche.">Cigarette smoke alters the hematopoietic stem cell niche.</a> Med Sci. 2, 37-50 (2014).</li><li>Kheradmand, F., You, R., Gu, B. H., Corry, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoke+and+DNA+cleavage+promote+lung+inflammation+and+emphysema.">Cigarette smoke and DNA cleavage promote lung inflammation and emphysema.</a> Transactions of the American Clinical and Climatological Association. 128, 222-233 (2017).</li><li>Ha, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Menthol+attenuates+respiratory+irritation+and+elevates+blood+cotinine+in+cigarette+smoke+exposed+mice.">Menthol attenuates respiratory irritation and elevates blood cotinine in cigarette smoke exposed mice.</a> PLoS ONE. , 1-16 (2015).</li><li>Moreno-Gonzalez, I., Estrada, L. 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Here we provide the information required for the construction of an apparatus for WBIS of mice to CS. After installation of the system, it is critically important that investigators calibrate the system based on the delivered dose of nicotine or cotinine in animals. The apparatus contains a timer and pressure gauges that can be used to adjust cigarette puff volume, puff frequency, combined smoke exposure period, and rest intervals that animals receive between each cigarette. Furthermore, the actual number of cigarettes a...

Cigarette smoking remains one of the major causes of preventable diseases in the US despite the steady decline in the number of cigarette-smoking adults in the past 50&#8211;60 years1. It is widely known that smoking is linked to multiple diseases of the lungs and blood including chronic obstructive pulmonary disease (COPD), a group of diseases that includes emphysema and chronic bronchitis2,3,4. According to the Center for Disease Control (CDC), in 2014, COPD was the third leading cause of death in the United States with over 15 million Americans suffering from this disease5.
CS has also recently been associated with a higher risk of developing clonal hematopoiesis (CH)6,7, a condition in which a single hematopoietic stem cell disproportionately produces a large percentage of a person&#8217;s peripheral blood. This finding indicates a potential connection between smoking and bone marrow function. Given the widespread and highly significant health implications of CS and given that murine models of diseases are a cornerstone of progress in biomedical research, it is useful to develop efficient and affordable systems to model CS in mice.
Here, we provide a step-by-step guide for building an affordable system for treating and studying the in vivo effects of CS on lung emphysema and bone marrow homeostasis. The assembly of this equipment does not require the user to have specialized knowledge and thus allows for DIY assembly.

<table>
	<tbody>
		<tr>
			<td>1 in fastener</td>
			<td>Lowes</td>
			<td>756990</td>
			<td></td>
		</tr>
		<tr>
			<td>1/4 in Barbed Y connector</td>
			<td>VWR</td>
			<td>89093-282</td>
			<td></td>
		</tr>
		<tr>
			<td>1/4 in straight tubing connector</td>
			<td>VWR</td>
			<td>62866-378</td>
			<td></td>
		</tr>
		<tr>
			<td>1/8 hex nipple</td>
			<td>Lowes</td>
			<td>877221</td>
			<td></td>
		</tr>
		<tr>
			<td>1/8 in threaded coupling fitting</td>
			<td>Lowes</td>
			<td>877208</td>
			<td></td>
		</tr>
		<tr>
			<td>1/8 in threaded male adapter nipple fitting</td>
			<td>Lowes</td>
			<td>877243</td>
			<td></td>
		</tr>
		<tr>
			<td>10/32 (M) threaded straight connector</td>
			<td>Bimba</td>
			<td>EB60</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4 in 90-degree elbow CPVC fitting</td>
			<td>Lowes</td>
			<td>22643</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4 in chlorinated polyvinyl chloride (CPVC) pipe</td>
			<td>Lowes</td>
			<td>23814</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4 in CPVC cap</td>
			<td>Lowes</td>
			<td>23773</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4 in CPVC Drip irrigation female adapter</td>
			<td>Lowes</td>
			<td>194629</td>
			<td></td>
		</tr>
		<tr>
			<td>3/4 in diameter CPVC male adapter</td>
			<td>Lowes</td>
			<td>23766</td>
			<td></td>
		</tr>
		<tr>
			<td>8.5 L airtight container with lid (11.25in x 7.75in x 6 in)</td>
			<td>Komax</td>
			<td>N/A</td>
			<td>Listed as &#34;Komax Biokips Large Bread Box | (280-oz) Large Storage Container&#34;</td>
		</tr>
		<tr>
			<td>Glass drain tube (1.75 in diameter x 8 in length)</td>
			<td>KIMAX</td>
			<td>6500</td>
			<td></td>
		</tr>
		<tr>
			<td>Isonic Solenoid Valves</td>
			<td>Bimba</td>
			<td>V2A02-AW1</td>
			<td></td>
		</tr>
		<tr>
			<td>Marlboro Red 100&#39;s</td>
			<td>Marlboro</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen swivel barbed connector</td>
			<td>Global Medical Solutions</td>
			<td>RES002</td>
			<td></td>
		</tr>
		<tr>
			<td>Panasonic Timer LT4H-W</td>
			<td>Panasonic</td>
			<td>LT4HW</td>
			<td>Item was built-in the valve controller by Shepherd Controls &#38; Associates</td>
		</tr>
		<tr>
			<td>Pressure regulator</td>
			<td>Allied Electronics and Automation</td>
			<td>70600552</td>
			<td>Also listed as &#34;Norgren R07-100-RGKA&#34;</td>
		</tr>
		<tr>
			<td>Rubber stopper # 1 (one hole)</td>
			<td>VWR</td>
			<td>59581-163</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber stopper # 8.5 (one hole)</td>
			<td>VWR</td>
			<td>59581-389</td>
			<td></td>
		</tr>
		<tr>
			<td>Scireq inExpose system</td>
			<td>Scireq and Emka Technologies</td>
			<td>N/A</td>
			<td>Commercial system used for comparison with our DIY WBIS</td>
		</tr>
		<tr>
			<td>Straight barbed fitting (8mm opening)</td>
			<td>VWR</td>
			<td>10028-872</td>
			<td></td>
		</tr>
		<tr>
			<td>Thread Sealant tape</td>
			<td>Lowes</td>
			<td>1184243</td>
			<td></td>
		</tr>
		<tr>
			<td>Threaded port adaptor</td>
			<td>Bimba</td>
			<td>P1SA1</td>
			<td></td>
		</tr>
		<tr>
			<td>Timeter Aridyne 2000 Medical Air Compressor</td>
			<td>MFI Medical</td>
			<td>AHC-TE20</td>
			<td></td>
		</tr>
		<tr>
			<td>Timeter flowmeter</td>
			<td>Allied Healthcare Products</td>
			<td>15006-03YP2</td>
			<td>Also listed as &#34;Puritan Air Meter&#34;</td>
		</tr>
		<tr>
			<td>Valve Control system</td>
			<td>Shepherd Controls and Associates</td>
			<td>N/A</td>
			<td>Company custom designed the valve control system for this model.</td>
		</tr>
		<tr>
			<td>Vinyl pipes</td>
			<td>Vitality Medical</td>
			<td>RES3007</td>
			<td></td>
		</tr>
	</tbody>
</table>

cigarette smoke exposure in mice using a whole-body inhalation system

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular diseases have been widely studied, however, the impact of CS in other tissues and organs such as blood and bone marrow remain incompletely defined. Finding the appropriate system to study the effects of CS in rodents can be prohibitively expensive and require the purchase of commercially available systems. Thus, we set out to build an affordable, reliable, and versatile system to study the pathologic effects of CS in mice. This whole-body inhalation exposure system (WBIS) set-up mimics the breathing and puffing of cigarettes by alternating exposure to CS and clean air. Here we show that this do-it-yourself (DIY) system induces airway inflammation and lung emphysema in mice after 4-months of cigarette smoke exposure. The effects of whole-body inhalation (WBI) of CS on hematopoietic stem and progenitor cells (HSPCs) in the bone marrow using this apparatus are also shown.

One of the main hallmarks of CS exposure is emphysema that is characterized by the damage and destruction of air sacs (alveoli) in the lung. Thus, initial experiments focused on the DIY system&#8217;s ability to provoke emphysematous changes in the lungs of female mice upon repeated whole-body exposure to CS. The CS dosing regimen was chosen based on our prior publications in which we utilized the DIY system described here to treat mice with CS and study the molecular pathophysiology of emphysema10</sup...

Watch this Scientific Journal Video about Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System at JoVE.com

Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System

This protocol demonstrates the study of the pathophysiologic effects of cigarette smoke (CS) with a whole-body inhalation (WBI) exposure system (WBIS) built in-house. This system can expose animals to CS under controlled repeatable conditions for research of CS-mediated effects on lung emphysema and hematopoiesis.

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular ...

This technique can be used to study the physiologic effects of cigarette smoke in chronic airway inflammation and emphysema, as well as bone marrow immune cell development in mice. The low cost and the straightforward building procedure of this equipment makes it accessible to many labs around the world. After assembling all components of the whole body inhalation exposure system, turn on the air compressor and wait for the safety alarm to turn off on its own.Adjust the pressure of the air compressor to 40 to 50 PSI by turning the knob on the pressure regulator. Adjust the airflow from the air compressor to five liters using the flow meter. Then turn on the valve controller.Adjust the digital timer on the valve controller to the pulse C operating mode by pressing the set lock key while holding down the up key at the first digit of the timer. Then press the up key until the PU-C mode is reached. Press the reset key to set the displayed operating mode as the working mode.Press set lock to change timer one shown in the display as T1, then press the up or down keys to set it to 20 seconds. Repeat this process to set timer two or T2 to three seconds. Turn on the air compressor and wait for the safety alarm to turn off on its own.Then turn on the valve controller. Transfer five mice into each of the four 8.5 liter exposure chambers with airtight removable lids. Place the exposure chambers inside a Class II type B2 laminar flow biological safety cabinet.Inside the biological safety cabinet, light a cigarette and insert it into the cigarette chamber. Switch on the valves on the valve controller that correspond to the chambers that are currently in use. Clean air will be pumped into the exposure chamber for 20 seconds causing the cigarette to burn and smoke will be pumped into the exposure chamber for three seconds.Allow the cigarette to burn out completely until it reaches the filter. Adjust the timer settings to perform an average of 10 puffs per cigarette over a four-minute period. Remove the cigarette filter and dispose of it by placing the cigarette butt in a glass beaker with water to extinguish the flame and dampen the odor.Close the cigarette chamber and let the machine pump clean air for 10 minutes. Repeat the cigarette smoke exposure for a total of four cigarettes per chamber per day. When finished, remove the mice from the exposure chambers and place them back into their corresponding cages.Turn off the valve controller and the air compressor. Remove the exposure and cigarette chambers and wash them with water and soap to remove any residue of tar. Let the chambers fully dry before using them again.Mice were exposed to the smoke of four commercial cigarettes daily with smoke-free intervals of 10 minutes in between each cigarette five days a week for a duration of four months. Hematoxylin and Eosin stained lung histology showed the destruction of the alveoli in mice exposed to cigarette smoke. Blinded histomorphometric analysis of lung sections confirmed that the mean linear intercept was significantly higher in mice exposed to cigarette smoke compared to air controls.As expected, whole body exposure to cigarette smoke provokes a decrease in body weight. The exposed mice also showed enhanced airway infiltration of immune cells, as well as induction of matrix metalloproteinases 9 and 12 gene expression, which are responsible for tissue damage. Cotinine, a metabolite of nicotine, was significantly elevated in the serum of mice exposed to four months of cigarette smoke, but undetectable in air-exposed mice.After exposure, bone marrow populations were analyzed using flow cytometry. A significant increase in hematopoietic stem and progenitor populations after four months of cigarette smoke exposure was detected. Whole body exposure to cigarette smoke of mice utilizing a commercially available system also showed an alteration in HSPC populations.It is important to ensure that all the tools delivering air or cigarette smoke to the mouse chambers are unobstructed and free of kinks that could disrupt the airflow. Furthermore, mice need to be monitored at all times during the exposure to cigarette smoke. The procedure can also be used to study the physiologic effects of cigarette smoke on other organ system such as the heart and the GI tract.With a few modifications, this machine can also be adaptive for a whole body inhalation exposure of mice to electronic cigarettes. This could allow researchers to study the potential hazards associated with vaping in a murine animal model.

cigarette-smoke-exposure-in-mice-using-a-whole-body-inhalation-system

Research

JoVE Journal

Immunology and Infection

6.0K visualizações.  Baylor College of Medicine. This technique can be used to study the physiologic effects of cigarette smoke in chronic airway inflammation and emphysema, as well as bone marrow immune cell development in mice. The low cost and the straightforward building procedure of this equipment makes it accessible to many labs around the world. After assembling all components of the whole body inhalation exposure system, turn on the air compressor and wait for the safety alarm to turn off on its own.Adjust the pressure of the...