The overall goal of this procedure is to demonstrate how to measure and analyze the lung pressure volume curve in mice. This is accomplished by first setting up a syringe pump to generate a constant rate of mouse lung inflation and deflation. In the second step, the pump is adapted to enable measurement of the air volume delivered to the animal, and then the system is corrected for the gas compression.
Finally, the airway pressure is measured and the pressure volume curve is digitally recorded. Ultimately, assessment of the pressure volume curve allows the evaluation of multiple variables that can reflect the structural changes that occur in lung disease. The main advantage of this method, which has never been standardized in a mouse model, is that the lung diss extensibility reflects the pathologic changes in lung structure associated with different lung diseases, especially emphysema and fibrosis.
This method is easy to perform and it can help answer key questions related to the changes associated that with lung disease. For measurement of the lung volume, use a syringe pump with a switch that allows a quick pump reversal after the pressure limit is reached to generate a constant rate of inflation and deflation of the lung. For mouse pressure volume curves use a five milliliter glass syringe with the prior to inflation volume set.
At three milliliters of air, attach a linear differential transformer to the pump housing with a small sensor rod connected to the moving syringe plunger, and measure the volume delivered by the pump. For measurement of the lung pressure, use a standard inexpensive pressure gauge with a range of zero to 60 centimeters.H2O. To record the pressure volume curve, use any digital recorder with XY capabilities.
Setting one channel to record the corrected volume signal and another, to record the trans pulmonary pressure, use a bridge pre amplifier that connects to the recorder to measure the pressure. Calibrate the pressure channel from zero to 30 centimeters H2O and calibrate the volume channel from zero to one milliliter. This figure shows a step change in pressure of 30 centimeters H2O, and a volume infusion from the syringe pump of one milliliter.
The raw and corrected volume channels show the same calibration since the system is open to air during this calibration. Correcting the gas compression is a critical initial step in the setup since as the pressure increases, the gas volume decreases and thus the volume air delivered to the mouse will become increasingly lower than the dis displacement of the syringe barrier. Before correcting for the gas compression, close the stop cock that will connect the pressure volume system to the lungs so that no gas can leave the system.
Then start the infusion and check if the corrected volume channel on the recorder indicates any measurable changes as the pressure increases to about 40 centimeters. H2O use a pump speed of one milliliter per minute for this volume correction. This tracing shows a properly corrected system where the raw volumes shows a change as the pressure increase compresses the air, but the corrected volume channel shows no changes in volume.
This means that when the system is connected to a mouse with a Degas lung, the changes in lung volume will be accurate. Note if the starting syringe volume and tubing is always the same, this correction for system gas compression never needs to be redone To measure a pressure volume curve in the mouse, after confirming the complete sedation of a six to 12 week old anesthetized mouse by lack of a response to a toe pinch, make a small incision in the skin overlying the trachea of the animal, then locate the trachea and make a small slit in the tissue. Insert an 18 gauge stub needle cannula into the slit, and then secure the cannula with thread.
Allow the mouse to breathe 100%oxygen for at least five minutes with a ventilator nominally set with a tidal volume of 0.2 milliliters at 150 breaths per minute. Then close the tracheal cannula to allow the mouse to absorb all of the oxygen for three to four minutes. Over the course of several minutes, the oxygen in the lungs will be absorbed as the lung volume goes to zero.
During this period, the heart rate will cease and the mouse will die. This can be confirmed by measuring the heart rate with an ECG recording showing that by three or four minutes the mouse's heart has stopped. After confirming cessation of the heartbeat with ECG electrodes, the lung volume will be at zero, and then the lung PV curve inflation can begin at a rate of three milliliters per minute.
Monitor the pressure trace on the digital recorder. When the pressure reaches 35 centimeters, H2O reverse the pump. Follow the deflation curve until the pressure reaches negative 10 centimeters.
H2O, by which time the airways will have collapsed, immediately reverse the pump again, allowing the lung to reinflate as the collapsed airways open, which is apparent by the noisy looking inflation limb at the initial part of this second inflation. When the pressure returns to 35 centimeters H2O, reverse the pump direction again and continue to deflate the lung until this second deflation limb reaches zero centimeters H2O. Then stop the pump.
Finally analyze the chart record of the pressure and flow and the pressure volume curve to evaluate any phenotypic changes in the lung parenchyma that have occurred within the different experimental lung pathologies. The variables obtained from the pressure volume curve are shown here. However, the raw data needed to determine these variables can be more easily obtained directly from the chart record.
As shown in this figure, the RV is measured as the volume at the end of the first deflation, and the TLC is measured as the volume at the end of the second inflation. We also measure the V 10 as the volume at 10 centimeters H2O on the second deflation. The deflation lung compliance is also measured on the second deflation limb by measuring the lung volumes at three and eight centimeters, H2O PV curves are routinely measured in control mice and mice with experimentally induced lung fibrosis caused by blio, mycin and emphysema caused by elastase.
This figure shows typical PV curves obtained under these three conditions. The results for the variables measured from the pressure volume curves in the fibrosis model are shown in this figure. There were highly significant decreases in all the measured variables.
The results for the variables measured from the pressure volume curves in the emphysematous mice are shown in this. Figure two doses of elastase were used to create two different degrees of lung damage. There were significant dose dependent increases in TLC, RV percent, V 10, and compliance and significant dose dependent decreases in specific compliance.
The table summarizes the changes in these variables in both lung disease models taken together these data confirm that measuring lung pressure volume curves is useful in detecting changes in the mechanical properties of diseased mouse lung. Once master, this procedure can be D complete in about 10 minutes per mouse, but since much of the time is leaded to the d ventilation and com oxygen gassing, severe mouse can be run in parallel. After watching this video, you should have a good understanding of how to measure the lung PV curve in mice.
Such curves can be used to phenotype the extent of the different lung pathologies, and these standardized procedures can generate data that can be compared with results from other labs.
