The overall goal of the following experiment is to learn how to measure the diffusing capacity of the lungs of a mouse using a calibrated gas chromatograph. This is accomplished by inflating the lungs of an anesthetized mouse with gas containing neon and carbon monoxide, holding the gas in the lungs for nine seconds and collecting the gas. The gas withdrawn from the lungs is then diluted to two milliliters and injected into the gas chromatograph.
The differential uptake of neon and carbon monoxide in the lung is used to calculate the diffusing capacity. This measurement can be used to assess the loss of lung function in a wide spectrum of lung disease models. The main advantage of this technique over all other existing methods to measure pulmonary function in the mouse is that this is a very simple reproducible measurement that can be directly compared with similar measurements in humans.
This method can be used to follow the changes in lung structure that occur with a variety of lung pathologies. This protocol begins with setting up the gas chromatograph module supplied with the machine to measure peaks for nitrogen, oxygen, neon, and carbon monoxide. For this application, only the neon and carbon monoxide data is needed.
The instrument uses a molecular sieve column with helium as the carrier gas. This particular gas chromatograph has a 0.8 milliliter column and a two milliliter sample is used to ensure adequate clearing of this column before starting. A series of measurements always calibrate the machine using two milliliters of gas directly from the gas sample bag.
The first peak to appear is the neon, then oxygen and nitrogen, which we do not measure for this procedure. Lastly, the carbon monoxide peak appears the time for the gas chromatograph. To measure all of these peaks is just under one minute.
To begin, anesthetize the mice with ketamine and xylazine. Confirm the anesthesia by the absence of reflex motion in response to a toe pinch. Next to protect the eyes, apply veterinary ointment, then tracheostomies the mice with a studen cannula.
Use an 18 gauge cannula for adults or a 20 gauge cannula for very young mice. Next, start the metronome with audible clicks every one second. For mice older than six weeks, use a three milliliter syringe to withdraw 0.8 milliliters of gas from the gas mixture bag.
If the mouse is less than four weeks old, use 0.4 milliliters of gas, then connect the syringe to the tracheal cannula and quickly inflate the lungs. Start counting mentally from one to 10 in synchrony with the metronome clicks. When the count reaches 10, quickly withdraw the 0.8 milliliter volume.
Dilute this exhaled gas to two milliliters by adding room, air and weight at least 15 seconds. It is very important to inflate and deflate the lungs as quickly as possible. A rapid inflation is very easy, but it takes a little practice to precisely withdraw the 0.8 mls quickly, Then inject the whole sample into the gas chromatograph.
For analysis, It's important to avoid contamination of the sample when transferring to and from the animal and to the gas chromatograph. This can be done careful by capping the sign with a finger While the GC is measuring the sample. Inflate the mouse lung with another 0.8 milliliters of gas mixture from the bag, and then process this sample identically as the first.
Using the average values from the two measurements, calculate the DFCO. The C subscript refers to the calibration gasses, and the nine subscript refers to the samples collected from the animal. After the nine second breath hold.
A nominal control DFCO value taken from C 57 black six mice is about 0.77. In a study using a PR eight influenza model, a progressive loss of function was seen at day six and eight in a model of emphysema 21 days after elastases installation. The DFCO was moderately decreased, but there were much larger changes in the model of fibrosis induced by Blio Mycin installation.
The fibrotic pathway caused by bleomycin resulted in the largest change in DFCO observed in any of the pathologic models. This is important since the diffusing capacity is also a reliable marker of fibrosis in humans. The CFTR gene plays a crucial role in cystic fibrosis and in a study of mice lacking this gene, there was a significant decrease in DFCO.
The DFCO was further decreased with fungal infection in these knockout mice. A commonly used model for lung injury is caused by LPS. In mice exposed to LPS, there was progressive time dependent loss of lung function from day one to four as assessed by DFCO measurements, the measurement of DFCO can be successfully made in mice as young as two weeks of age.
The smaller size of the lung requires a smaller inflation volume of 0.4 milliliters. The DFCO measured with this smaller volume is able to show the expected increase as the lung matures Once mastered. This technique can be done in just a few minutes per mouse prior to doing any other pulmonary function measurements or assessments.
While attempting this procedure is important to make sure to perform the inflation and deflation step quickly and to be careful when transferring the sample so contamination is prevented.
