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

Summary

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.

Abstract

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.

Introduction

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 50–60 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 bronchitis^2,3,4. 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’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.

Protocol

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

1. Assembling the air compressor with the valve system.
   1. 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.
   2. 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.
   3. Install the oxygen swivel barbed connector on each (4) flowmeter.
   4. Install a male adaptor on the air compressor’s top air outlet (part included with the medical air compressor instrument).
2. Assembling exposure chambers (make 4 units)
   1. Cut a 3/4 inch chlorinated polyvinyl chloride (CPVC) pipe into eight 4 inch segments.
   2. Insert each segment to one 3/4 inch 90° 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.
   3. Drill two holes (1 ¼ 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.
   4. Insert the threaded sides of the CPVC male adapter assembled before into each hole in the containers.
   5. 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).
   6. 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.
3. Assembling cigarette chambers (makes up to 4 murine exposure units)
   1. 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).
   2. 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).
   3. 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.
   4. Connect the two rubber stopper structures assembled before in steps 1.3.1–1.3.3 to an 8 inch x 1.75 inch glass cylinder from a laboratory glass drain tube.
4. Valve control system
   1. 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).
5. Assembling all components together (see Figure 1)
   1. Mount four solenoid valves to the sides of the valve control system using 1 inch fasteners.
   2. Connect the solenoid valves to the valve control system following the manufacturer’s instructions.
   3. Attach a 10–32 (M) threaded straight connector to the exhaust (“EXH”) connection on the solenoid valve and a threaded port adaptor on the “IN” and “OUT” connections of the same solenoid valve.
   4. Connect the flowmeter attached to the compressor to the solenoid valve through the “OUT” connection using a 7 ft medical grade vinyl tubing.
   5. Connect the 7 ft vinyl tube assembled with the rubber stopper in step 1.3.3. on the “IN” connector on the solenoid valve.
   6. Insert the small rubber stopper of the cigarette chamber on the chamber smoke input.
   7. Connect the solenoid valve to the second connection of the barbed Y connector on the cigarette pedestal assembled in step 1.3.1.

Figure 1: Schematic of the connections of our WBIS for exposure to CS. 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. Please click here to view a larger version of this figure.

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).

1. Setting pressure and airflow
   1. 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.
   2. Adjust the pressure of the air compressor to 40–50 psi by turning the knob on the pressure regulator.
   3. Adjust the airflow from the air compressor to 5 L using the flowmeter.
   4. Turn on the valve controller.
   5. Adjust the digital timer on the valve controller to the PULSE-C (shown in the display as “Pu-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 (i.e., Pu-C) as the working mode.
   6. Press the SET/LOCK to change timer 1 (shown in the display as “T1”).
   7. Press the UP or DOWN keys to set T1 to 20 s.
   8. Press the SET/LOCK to change timer 2 (shown in the display as “T2”).
   9. 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.
2. Cigarette smoke treatment
   NOTE: This system allows for the use of 1–4 murine exposure chambers at the same time.
   1. Turn on the air compressor and wait for the safety alarm to turn off on its own.
   2. Turn on the valve controller.
   3. 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.
   4. 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.
   5. 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.
      1. 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.
   6. 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.
   7. 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.
   8. 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.
   9. Remove the mice from the exposure chambers back into their corresponding cages.
   10. Turn off the valve controller and the air compressor.
   11. Remove the exposure and cigarette chambers and wash with water and soap to remove any residue of tar.
   12. Let the chambers fully dry before using them again.

Results

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’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

Discussion

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...

Materials

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