The overall goal of this video is to present a method for the construction and operation of e-vapor and cigarette smoke chambers that are easily adjustable and deliver e-vapor and tobacco smoke to rodents in order to study related health issues. These chambers help us perform experiments that enable us to determine the harmful effects of e-vapor and tobacco smoke on health, especially in the areas of pulmonary and cardiovascular diseases. These easily built chambers are inexpensive and readily customizable to various inhalation or exhalation times to simulate human smoking habits.
Using these chambers, we can expose rodents to e-vapor with and without nicotine to study the involvement of nicotine in diseases like lung cancer, COPD, allergies, and asthma. The tobacco smoke chamber will help us establish cigarette smoke induced disease models in rodents, such as abdominal aortic aneurysm. This will help us understand the biochemical changes that initiate this disease.
These chambers will help determine whether e-vapor use is safer than conventional tobacco smoke. Then cut some quarter inch inner diameter silicone tubing into two 15 centimeter segments and attach the ends to either side of a T-connector. Thread both of the tubes through pre-made holes near the front of the chamber lid so that the T-connector is inside the chamber.
Then use tape to secure the tubing to the lid. Next, connect the free ends of the silicone tubing to the output ends of two micro air pumps and use double-sided adhesive tape to mount the pumps to the lid of the chamber. Cut a four centimeter piece of silicone tubing and attach one end of it to the input side of one of the air pumps.
This is where the e-cigarette will be inserted during the chamber's use. Ensure that the diameter of the tube allows for a snug fit around the end of the e-cigarette. Next, attach one end of a new silicone tube to the input side of the other air pump.
This pump will introduce room air into the chamber. As such, the end of the tube should be placed outside of the fume hood. The length of the tube isn't critical, but it should be as short as possible to limit airflow resistance.
Inside the chamber, use double-sided adhesive tape to attach two small hooks which will hold the oxygen and carbon monoxide gas monitors. To construct the tobacco cigarette smoke chamber, begin by preparing a chamber similar to the e-cigarette chamber but with some modifications. First, construct a cigarette lighting device as described in the accompanying text protocol.
Attach one end of a new silicone tube to the cigarette lighting device and the other end to the input side of the air pump. Then place the cigarette lighting device inside the fume hood but outside the chamber during its use. Next, attach one end of a new silicone tube to the other air pump.
As this pump will introduce room air into the chamber, place the end of this tube outside the fume hood. Then cut several five millimeter wide vertical slits in the front wall of the chamber and mount a computer fan on the outside so that it covers this opening. Ensure that the front of the fan is facing towards the chamber, such that the fan blows air into the chamber through the opening.
Control the fan and the air pumps using a micro controller like the one shown here. Assemble the micro controller by following the detailed instructions in the accompanying text protocol and house it outside of the fume hood if possible. Use adult rats weighing 450 to 520 grams and group them according to the type of exposure they will receive.
Collect 500 microliters of blood from the tail vein and spin down at 20, 000 times g for 30 minutes at four degrees Celsius to isolate the serum. Ensure that the samples are chilled on ice throughout this process. Following the manufacturer's protocol, use a Cotinine ELISA Kit to measure the baseline serum cotinine concentration in each of the animals.
For daily operation of the e-cigarette chamber, start by cleaning the inside of the chamber with 70%ethanol and then with deionized water. Let it air dry until the chamber is completely dry, which should take about 30 minutes. Additionally, a clean absorbent pad should be placed in the bottom of the chamber after cleaning.
Next, calibrate the gas monitors and mount the entire monitor on the wall inside the chamber. Once cleaned and calibrated, place one group of rats inside the chamber. Ensure that the e-cigarette is fully charged and then insert the e-cigarette into the input tube.
Monitor the battery and e-liquid levels throughout the duration of the 90 minute exposure. When ready, turn the air pumps on and start the timer. During exposure, observe the gas monitors to ensure that the oxygen levels do not fall below 20%and zero ppm carbon monoxide.
Once the exposure time has reached 90 minutes, remove the e-cigarette and continue to run the gas pumps to vent the remaining vapor. When the vapor has cleared, remove the rats and clean the chamber. Collect 500 microliters of blood from the tail vein of each rat approximately one hour after exposure at the conclusion of the experimental protocol, and measure the levels of cotinine in each animal.
Before starting the tobacco cigarette exposure, clean the chamber as before, recalibrate the monitors, and place the proper animals inside the chamber, up to three at a time. Insert a cigarette into the cigarette lighting device and place it so that the end of the cigarette is against the heating element. Then turn on the cigarette lighting device until the cigarette butt begins to smolder.
Once the cigarette is lit, turn on the pumping system, start the timer, and observe the cigarette burn to completion. Then carefully remove the spent cigarette from the cigarette lighting device using a pair of forceps. Monitor the carbon monoxide and oxygen levels.
Ensure that the carbon monoxide levels do not rise above 1, 000 ppm and that the oxygen levels do not fall below 20%If needed, adjust the computer fan, as proper timing and run duration are critical for preventing carbon monoxide accumulation. After four minutes, turn off the pumping system and repeat the process for a full 90 minutes of tobacco smoke exposure. When the exposure is complete, continue to leave the pumping system on to ventilate residual smoke.
When the carbon monoxide falls below 100 ppm, remove the rats and clean the chamber. Collect 500 microliters of blood from the tail vein of the rats approximately one hour after exposure and measure the levels of cotinine in each animal. The carbon monoxide concentration in the chamber was recorded every 30 seconds while introducing smoke from 1R6F cigarettes.
The results shown here are the averages from three consecutive four minute cycles. During the test, carbon monoxide concentrations did not exceed 1, 000 parts per million. In contrast, in the electronic cigarette exposure system, carbon monoxide concentrations remained undetectable throughout the exposure.
Cotinine, a major metabolite of nicotine, is shown here measured from the serum of animals, both pre-exposure and one hour post-exposure. This study shows similar pre-exposure numbers, and animals subjected to the e-cigarette vapor and cigarette smoke had serum cotinine levels within a clinically relevant range. These chambers will help deliver consistent, clinically relevant e-vapor and cigarette smoke to rodents and aid the research effort in determining the adverse health effects of e-cigarettes and cigarette smoke.
Once mastered, this technique can be completed in about two hours if performed properly. One of the greatest advantages of these chambers is the versatility that allows for the use of any brand of e-cigarette or e-cigarette liquid. We can use these chambers to study whether nicotine-free e-vapors of different flavors have any adverse health concerns.
E-vapor and smoke induced rodent models of diseases will help us deliver better therapeutic agents for these diseases. Don't forget that operating the cigarette lighting device involves a heating element that can be extremely hazardous. You should check with your fire marshal to ensure the lab space has the appropriate safety requirements.
