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 flowmeters (two 15 L/min with Y bar and 2 power takeoffs) to the miniature pressure regulator using a 1/8 inch threaded male adapter nipple fitting. Make sure to use the thread seal tape in all threaded ends.</li>
		<li>Connect the assembled pressure regulator with the flowmeter to the medical air compressor instrument using the following: a 1/8 inch hex nipple on the compressor air outlet, a 1/8 inch threaded coupling fitting, and a 1/8 inch threaded male adapter nipple fitting that connects to the pressure regulator.</li>
		<li>Install the oxygen swivel barbed connector on each (4) flowmeter.</li>
		<li>Install a male adaptor on the air compressor&#8217;s top air outlet (part included with the medical air compressor instrument).</li>
	</ol>
	</li>
	<li>Assembling exposure chambers (make 4 units)
	<ol>
		<li>Cut a 3/4 inch chlorinated polyvinyl chloride (CPVC) pipe into eight 4 inch segments.</li>
		<li>Insert each segment to one 3/4 inch 90&#176; elbow CPVC fitting and attach the fitting side of the elbow to a 3/4 inch diameter CPVC male adapter. There should be eight CPVC segments, each attached to one CPVC elbow fitting and one CPVC male adapter.</li>
		<li>Drill two holes (1 &#188; inch diameter) on opposite sides most distant from each other of an 8.5 L airtight container (11.25 x 7.75 x 6 inch) with a lid (see Figure 1 exposure chamber). The positioning of the holes is to be centered top to bottom and left to right.</li>
		<li>Insert the threaded sides of the CPVC male adapter assembled before into each hole in the containers.</li>
		<li>From the inside of the container, attach a 3/4 inch CPVC cap on the other side (Chamber smoke input) and a 3/4 inch CPVC Drip irrigation female adapter on one side (Chamber smoke output).</li>
		<li>Drill five 3 mm holes on the top of the CPVC cap of the chamber smoke output in a quincunx (shower head) pattern. This will allow the cigarette smoke to enter the chamber with higher velocity and ensures that it spreads evenly inside the chamber in all directions.</li>
	</ol>
	</li>
	<li>Assembling cigarette chambers (makes up to 4 murine exposure units)
	<ol>
		<li>Take a one-hole rubber stopper (manufacturer size 8.5) and insert a 1/4 inch barbed Y connector on the wider side and a straight barbed fitting (8 mm opening) on the narrower side. The cigarette will be placed here during the smoking procedure (cigarette pedestal).</li>
		<li>Connect one end of a 12 inch long medical grade vinyl pipe to one of the barbed connectors on the Y connector attached to the rubber stopper and the other end to a 1/4 inch fitting and insert the opposite side of this fitting on a one-hole rubber stopper (manufacturer size 1).</li>
		<li>On another rubber stopper (manufacturer size 8.5), insert one 1/4 inch straight tubing connector on the wider side of the stopper and connect the outer end of the fitting to a 7 ft medical grade vinyl pipe.</li>
		<li>Connect the two rubber stopper structures assembled before in steps 1.3.1&#8211;1.3.3 to an 8 inch x 1.75 inch glass cylinder from a laboratory glass drain tube.</li>
	</ol>
	</li>
	<li>Valve control system
	<ol>
		<li>The system is controlled by a rhythmic opening and closing of solenoid valves that simulate inhalation (puffing) of cigarette smoke and clean air. The system that controls the solenoid valves was commercially designed (see Table of Materials).</li>
	</ol>
	</li>
	<li>Assembling all components together (see Figure 1)
	<ol>
		<li>Mount four solenoid valves to the sides of the valve control system using 1 inch fasteners.</li>
		<li>Connect the solenoid valves to the valve control system following the manufacturer&#8217;s instructions.</li>
		<li>Attach a 10&#8211;32 (M) threaded straight connector to the exhaust (&#8220;EXH&#8221;) connection on the solenoid valve and a threaded port adaptor on the &#8220;IN&#8221; and &#8220;OUT&#8221; connections of the same solenoid valve.</li>
		<li>Connect the flowmeter attached to the compressor to the solenoid valve through the &#8220;OUT&#8221; connection using a 7 ft medical grade vinyl tubing.</li>
		<li>Connect the 7 ft vinyl tube assembled with the rubber stopper in step 1.3.3. on the &#8220;IN&#8221; connector on the solenoid valve.</li>
		<li>Insert the small rubber stopper of the cigarette chamber on the chamber smoke input.</li>
		<li>Connect the solenoid valve to the second connection of the barbed Y connector on the cigarette pedestal assembled in step 1.3.1.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61793/61793fig01.jpg" /> 
Figure 1: Schematic of the connections of our WBIS for exposure to CS.&#160;This figure demonstrates how all components are assembled to form a working apparatus. The figure shows only one assembled smoking chamber of the four that the machine is capable of operating. <a href="https://www.jove.com/files/ftp_upload/61793/61793fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Cigarette smoke exposure
CAUTION: Avoid second- and third-hand exposure to cigarette smoke. Cigarette and exposure chambers should be used within a Class II Type B2 Laminar Flow Biological Safety Cabinets. Proper PPE should be worn while conducting the smoke exposure experiments (i.e., masks, gloves, hairnet, gown).
<ol>
	<li>Setting pressure and airflow
	<ol>
		<li>Once all the components are assembled as shown in Figure 1, turn on the air compressor and wait for the safety alarm to turn off on its own.</li>
		<li>Adjust the pressure of the air compressor to 40&#8211;50 psi by turning the knob on the pressure regulator.</li>
		<li>Adjust the airflow from the air compressor to 5 L using the flowmeter.</li>
		<li>Turn on the valve controller.</li>
		<li>Adjust the digital timer on the valve controller to the PULSE-C (shown in the display as &#8220;Pu-c&#8221;) 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 (i.e., Pu-C) as the working mode.</li>
		<li>Press the SET/LOCK to change timer 1 (shown in the display as &#8220;T1&#8221;).</li>
		<li>Press the UP or DOWN keys to set T1 to 20 s.</li>
		<li>Press the SET/LOCK to change timer 2 (shown in the display as &#8220;T2&#8221;).</li>
		<li>Press the UP or DOWN keys to set T2 to 3 s. 
		NOTE: Steps 2.1.5 through 2.1.9 are tailored to be used with the specific timer (see Table of Materials). For further instructions on other uses for this product, see its corresponding user manual.</li>
	</ol>
	</li>
	<li>Cigarette smoke treatment 
	NOTE: This system allows for the use of 1&#8211;4 murine exposure chambers at the same time.
	<ol>
		<li>Turn on the air compressor and wait for the safety alarm to turn off on its own.</li>
		<li>Turn on the valve controller.</li>
		<li>Transfer 5 mice into each of the four exposure chambers with airtight removable lids with a volume of 8.5 L. Place the four exposure chambers with mice within a Class II Type B2 Laminar Flow Biological Safety Cabinets.</li>
		<li>Inside the laminar flow biological safety cabinet, light up a cigarette and insert the cigarette inside of the cigarette chamber. Use commercially available cigarettes which contain 15 mg/cig tar and 1.1 mg/cig nicotine8 as compared to Kentucky 3RF4 research cigarettes (9.5 mg/cig tar and 0.73 mg/cig nicotine)9.</li>
		<li>Switch ON the valves on the valve controller that correspond to the chambers that are currently in use. The exposure is divided into 2 phases: (T1) clean air is pumped into the exposure chamber for 20 s and (T2) airflow causes the cigarette to burn and smoke from the cigarette chamber is pumped into the exposure chamber for 3 s. Allow the cigarette to burn out completely until it reaches the filter.
		<ol>
			<li>Adjust the timer settings to perform an average of ~10 puffs/cigarette over an ~4-min period. Note that the timer and system are easily customizable for enhancing or lowering CS dosing regimen according to the research needs of the investigators.</li>
		</ol>
		</li>
		<li>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.</li>
		<li>Make sure the cigarette chamber is closed again and without a cigarette. Let the machine pump clean air for 10 min. It is of utmost importance to maintain constant monitoring of the vertebrate animals that are exposed to CS. This exposing regimen is optimized for 5 female mice over 9-weeks old per exposure chamber.</li>
		<li>Repeat steps 2.3.4 through 2.3.7 three times for a total of 4 cigarettes per chamber a day. This procedure is repeated 5 days a week for as long as the researcher needs for their experiments.</li>
		<li>Remove the mice from the exposure chambers back into their corresponding cages.</li>
		<li>Turn off the valve controller and the air compressor.</li>
		<li>Remove the exposure and cigarette chambers and wash with water and soap to remove any residue of tar.</li>
		<li>Let the chambers fully dry before using them again.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Current Cigarette Smoking Among Adults in the United States. Center for Disease Control and Prevention Available from: <a target="_blank" href="https://www.cdc.gov/tobacco/data_statistics/fact_sheets/adult_data/cig_smoking/index.htm">https://www.cdc.gov/tobacco/data_statistics/fact_sheets/adult_data/cig_smoking/index.htm</a> (2018)</li><li>Salvi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+smoking+and+evironmental+risk+factors+for+chronic+obstructive+pulmonary+disease.">Tobacco smoking and evironmental risk factors for chronic obstructive pulmonary disease.</a> Clinics in Chest Medicine. 35, 17-27 (2014).</li><li>Sunyer, 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=Longitudinal+relation+between+smoking+and+white+blood+cells.">Longitudinal relation between smoking and white blood cells.</a> American Journal of Epidemiology. 144, 734-741 (1996).</li><li>Freedman, D. S., Flanders, D., Barboriak, J. J., Malarcher, A. M., Gates, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoking+and+leukocyte+subpopulations+in+men.">Cigarette smoking and leukocyte subpopulations in men.</a> Annals of Epidemiology. 6, 299-306 (1996).</li><li>Chronic Obstructive Pulmonary Disease (COPD). Center for Disease Control and Prevention Available from: <a target="_blank" href="https://www.cdc.gov/copd/basics-about.html">https://www.cdc.gov/copd/basics-about.html</a> (2019)</li><li>Genovese, G., 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=Clonal+hematopoiesis+and+blood-cancer+risk+inferred+from+blood+DNA+sequence.">Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence.</a> New England Journal of Medicine. , (2014).</li><li>Steensma, D. 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. D., Sanchez-Mejias, E., Soto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smoking+exacerbates+amyloid+pathology+in+a+mouse+model+of+Alzheimer's+disease.">Smoking exacerbates amyloid pathology in a mouse model of Alzheimer's disease.</a> Nature Communications. 4, 1-10 (2013).</li><li>Madison, M. C., 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=Electronic+cigarettes+disrupt+lung+lipid+homeostasis+and+innate+immunity+independent+of+nicotine.">Electronic cigarettes disrupt lung lipid homeostasis and innate immunity independent of nicotine.</a> Journal of Clinical Investigation. 129, 4290-4304 (2019).</li></ol>

The authors have nothing to disclose.

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>
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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 ...

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

Research

JoVE Journal

Immunology and Infection

6.1K Views.  Baylor College of Medicine. 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.

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

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Humanized NOG Mice for Intravaginal HIV Exposure and Treatment of HIV Infection

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes the establishment of a human immune system in adult NOG mice. Here, we explain all the practical steps from isolation of umbilical cord blood derived human CD34+ cells and their subsequent intravenous transplantation into the mice, to the manipulation of the model through HIV infection, combination antiretroviral therapy (cART), and blood sampling. Approximately 75,000 hCD34+ cells are injected intravenously into the mice and the level of human chimerism, also known as humanization, in the peripheral blood is estimated longitudinally for months by flow cytometry. A total of 75,000 hCD34+ cells yields 20%&#8211;50% human CD45+ cells in the peripheral blood. The mice are susceptible to intravaginal infection with HIV and blood can be sampled once weekly for analysis, and twice monthly for extended periods. This protocol describes an assay for quantification of plasma viral load using droplet digital PCR (ddPCR). We show how the mice can be effectively treated with a standard-of-care cART regimen in the diet. The delivery of cART in the form of regular mouse chow is a significant refinement of the experimental model. This model can be used for preclinical analysis of both systemic and topical pre-exposure prophylaxis compounds as well as for testing of novel treatments and HIV cure strategies.

We have developed a protocol for the generation and evaluation of a humanized and human immunodeficiency virus-infected NOG mouse model based on stem cell transplant, intravaginal human immunodeficiency virus exposure, and droplet digital PCR RNA quantification.

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes ...

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes susceptibility to bacterial infections in the respiratory system. However, the effects of cigarette smoking on bacterial infections in human lung epithelial cells have yet to be thoroughly studied. Described here is a detailed protocol for the preparation of cigarette smoking extracts (CSE), treatment of human lung epithelial cells with CSE, and bacterial infection and infection determination. CSE was prepared with a conventional method. Lung epithelial cells were treated with 4% CSE for 3 h. CSE-treated cells were, then, infected with Pseudomonas at a multiplicity of infection (MOI) of 10. Bacterial loads of the cells were determined by three different methods. The results showed that CSE increased Pseudomonas load in lung epithelial cells. This protocol, therefore, provides a simple and reproducible approach to study the effect of cigarette smoke on bacterial infections in lung epithelial cells.

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Described here is a protocol to study how cigarette smoke extract affects bacterial colonization in lung epithelial cells.

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...