Measurement of the Pressure-volume Curve in Mouse Lungs

Summary

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung’s structural changes with developing pathology.

Abstract

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Introduction

The mouse is now the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measuring the changes in lung elasticity. Although there are many ways to measure elasticity, the classical method is that of the total pressure-volume (PV) curve measured from residual volume (RV) to total lung capacity (TLC). This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years^1-3. Such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development^4-7. Despite the PV curve’s importance as a measurement of the lung’s phenotype, there has been no standardized way to perform this measurement. It has been done simply by inflating and deflating the lung with discrete steps (waiting a variable time for equilibration after each) or with pumps that can continuously inflate and deflate the lung. The PV curve is often done over a volume range between zero and some user-define lung capacity, but the time duration of each pressure volume loop reported by different labs has been extremely variable, varying from a few sec⁸ to hr². Some investigators refer to this total lung PV curve as static or quasistatic, but these are qualitative terms that offer little insight, and they are not used here. In addition, the PV curve has not been widely reported in the mouse, despite the fact that it can provide useful information on the macroscopic effects of structural changes in the lung.

Several issues have resulted in variability in PV curve acquisition including: 1) the rate of inflation and deflation; 2) the pressure excursions for inflation and deflation; and 3) the means to determine an absolute lung volume measurement. In the method present here, a rate of 3 ml/min was chosen as a compromise, being not too short as to reflect the dynamic elasticity associated with normal ventilation and not too slow as to make the measurement impractical, particularly when studying large cohorts. Since a nominal total lung capacity in a C57BL/6 healthy mouse is on the order of 1.2 ml⁹, this rate typically allows two complete closed PV loops to be done in about 1.5 min.

In the extended literature where PV curves have been reported, the peak inflation pressure used has been extremely variable, varying from as low as 20 to over 40 cm H₂O. Part of this variability may be related to species, but a primary goal of setting the upper pressure limit for PV curves is to inflate the lung to total lung capacity (TLC), or maximal lung volume. The TLC in humans is defined by the maximal voluntary effort an individual can make, but unfortunately this can never be duplicated in any animal model. Thus, the maximal volume in experimental PV curves is determined by a maximal pressure arbitrarily set by the investigator. The goal is to set a pressure where the PV curve is flat, but unfortunately the inflation limb of a mammalian lung PV curve is never flat. So most investigators set a pressure where the inflation curve begins to flatten substantially, typically 30 cm H₂O. In the mouse, however, the PV curve is even more complex with a double hump on the inflation limb, and where this inflation limb is often still rising steeply at 30 cm H₂O¹⁰, so 30 is not a good end point for the PV curve. For this reason, we use 35 cm H₂O as the pressure limit for the mouse PV curve, which is a pressure at which the inflation limbs of all strains we have examined begin to flatten.

Since the PV curve itself is very nonlinear, the appearance of a PV loop will depend on the volume from where the curve starts. Some commercial ventilators allow users to do large PV loops, starting from FRC, but if the FRC volume is unknown then it is impossible to interpret changes in such PV curve with any pathology, since these changes could simply result from a change in starting volume, and not structural alterations in the lung. Thus without an absolute volume measurement, PV curves are almost impossible to interpret and thus have little utility. Although, there are several ways to measure lung volumes, these are often cumbersome and require special equipment. In the simple approach described here, the PV curve starts at zero volume after an in vivo degassing procedure.

In summary, this paper demonstrates a straightforward method to standardize lung PV curve measurement in the mouse lung, and defines several metrics that can be calculated from this curve that are linked to lung structure. The PV curve thus provides a pulmonary function test that has direct application in being able to detect phenotypic structural changes in mice with common lung pathologies such as emphysema and fibrosis.

Protocol

The Johns Hopkins University Animal Care and Use Committee approved all animal protocols.

1. Equipment

The composite system set up, ready to measure the PV curve is shown in Figure 1.

1. Volume measurement:
   1. Generate a constant rate of inflation and deflation by using a syringe pump with a switch that allows the user to quickly reverse the pump after reaching the pressure limits. For mouse PV curves, use a very lightly greased 5 ml glass syringe with the initial volume (prior to inflation) set at 3 ml of air. 3 ml is sufficiently large to measure volumes in nearly all mouse PV curves.
   2. Measure the volume delivered by the pump by attaching a linear differential transformer to the pump housing, with a small sensor rod connected to the moving syringe plunger.
      Note: An empirical means to correct for gas compression in the system is described under the PV curve recording section.
2. Pressure measurement:
   1. Use a standard inexpensive pressure gauge with a range of 0-60 cm H₂O (0-1 PSI).
3. Recording measurement:
   1. To record the PV curve use any digital recorder with XY capabilities (e.g., PowerLab). Set one channel to record the corrected volume signal and another channel to record the transpulmonary pressure (Ptp), in order to graph the PV curve. Use a bridge preamplifier that connects to the main Powerlab to measure the pressure. Calibrate the pressure channel from 0-40 cm H₂O, and calibrate the volume channel from 0-3 ml.

2. Correction for Gas Compression

Note: This is a critical initial step in the set up, since as the pressure increases, the gas volume decreases, and thus the volume of air delivered to the mouse will be increasingly less than the displacement of the syringe barrel.

1. Close the stopcock that will connect the PV system to the lungs, so no gas can leave the system. Start the infusion and observe if the corrected Volume channel on the recorder shows any measurable changes as the pressure increases to about 40 cm H₂O. If so, then correct as in the next steps.
   1. Correct for gas compression empirically by subtracting from the plunger displacement measurement (i.e., the uncorrected volume) a term proportional to the inflation pressure. Do this on a Powerlab channel (called Vc) to show the volume signal minus a coefficient times the pressure.
   2. Determine the coefficient in the equation. First, make an initial guess, turn the chart recording on, and start the pump. Since the inflation tubing is sealed, adjust the pressure coefficient multiplier to make the Vc channel read zero as the pressure rises from 0-40 cm H₂O. If it goes up or down, simply adjust the correction factor until it stays flat over this pressure range. This correction factor will always be the same, if the same 3 ml starting volume in the syringe is not changed.

3. Experimental Tests in Mice

1. Procedure for measurement of the PV curve in mice. All animal protocols were approved by the Johns Hopkins University Animal Care and Use Committee.
   1. Anesthetize mice (C57BL/6 mice at 6-12 weeks of age) with ketamine (90 mg/kg) and xylazine (15 mg/kg), and confirm anesthesia by the absence of reflex motion.
      Note: The PV curve can be completed in anesthetized mice in less than 10 min and is a terminal procedure. 
   2. Tracheostomize the mice with an 18 G stub needle cannula. Do this by making a small incision in the skin overlying the trachea, locating the trachea, then making a small slit in the trachea, where the stub needle can be inserted. Secure the cannula by tying with thread.
   3. Allow the mice to breath 100% oxygen for at least 4 min. This can be via spontaneous breathing from a bag or with a ventilator nominally set with a tidal volume of 0.2 ml at 150 breaths/min.
   4. Close the tracheal cannula and allow 3-4 min for the mouse to absorb all the oxygen. This oxygen absorption procedure results the death of the animals and in a nearly complete degassing of the lung¹¹. Confirm death of the mouse by measuring the cessation of the heartbeat with ECG electrodes or direct observation.
   5. Once the degassing of the lung is complete and the lung volume is zero, begin inflating the lung with room air in the syringe pump at a rate of 3 ml/min. Monitor the pressure trace on the digital recorder, and when it reaches 35 cm H₂O, reverse the pump.
   6. Follow the deflation curve until pressure reaches negative 10 cm H₂O, by which time the airways have collapsed, trapping air in alveoli preventing further volume reduction. Immediately reverse the pump again, allowing the lung to reinflate as the collapsed airways open. This heterogeneous opening is normally apparent by the noisy looking inflation limb at the initial part of this 2nd inflation.
   7. When the pressure again reaches 35 cm H₂O, reverse the pump direction, and continue to deflate the lung until this 2nd deflation limb reaches 0 cm H₂O. Then stop the pump.
   8. View the PowerLab chart record of pressure and flow and the PV curve. Then analyze the PV curve to detect phenotypic changes in lung parenchyma that occur with different lung pathologies.

Results

Although the procedure for the PV curves is demonstrated in the video only for control healthy mice, we examined the ability of the PV curve to detect functional and pathologic changes in mice with two different common pathologies, emphysema and fibrosis. Details of these traditional models described elsewhere^12,13. Very briefly, after anesthesia with 3% isoflurane the emphysema was caused by 3 or 6 U porcine pancreatic elastase instilled into the trachea and studied 3 weeks later, and the fibrosis was caused ...

Discussion

In this paper a straightforward reproducible method has been described to measure in mice a classical method of phenotyping lung elasticity, the total lung PV curve. Such curves were instrumental in the discovery of pulmonary surfactant and its importance in providing lung stability. Here it is shown how the PV curve is also useful in providing a means to measure several variables related to lung elasticity in adult mouse lungs. There were highly significant changes in all variables in two commonly used mouse models to g...

Materials

Name	Company	Catalog Number	Comments
Name of Material/ Equipment	Company	Catalog Number	Comments/Description
Syringe Pump	Harvard Apparatus	55-2226	Infuse/Withdraw syringe pump
Pump 22 Reversing Switch	Harvard Apparatus	552217	included with pump
Linear displacement transformer	Trans-Tek, Inc.	0244-0000
5 mL glass syringe	Becton Dickenson		Several other possible vendors
Digital recorder	ADInstruments	PL3504	Several other possible vendors
Bridge Amp Signal Conditioner	ADInstruments	FE221
Gas tank,100% oxygen	Airgas, Inc		Any supplier or hospital source will work
Pressure Transducer - 0-1psi  millivolt output	Omega Engineering	PX-137	Range: ≈0-60 cmH2O