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

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.