Name: measurement of the pressure-volume curve in mouse lungs
Uploaded: 2015-01-27T15:00:00
Description: Watch this Scientific Journal Video about Measurement of the Pressure-volume Curve in Mouse Lungs at JoVE.com

This work has been supported by NIH HL-1034.

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.
<ol>
	<li>Volume measurement:
	<ol>
		<li>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 ...

<ol>
	<li>Neergaard, K. v. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neue+Auffasungen+&#252;ber+einn+Grundbergriff+der+Atemtechnik.+Die+Retraktionskraft+der+unge,+abhangig+von+den+Oberflachenspannung+in+den+Alveolen.+(New+interpretations+of+basic+concepts+of+respiratory+mechanics.+Correlation+of+pulmonary+recoil+force+with+surface+tension+in+the+alveoli.).">Neue Auffasungen &#252;ber einn Grundbergriff der Atemtechnik. Die Retraktionskraft der unge, abhangig von den Oberflachenspannung in den Alveolen. (New interpretations of basic concepts of respiratory mechanics. Correlation of pulmonary recoil force with surface tension in the alveoli.).</a> Zeitschrift Fur Gesamte Experi Medizin. 66, 373-394 (1929).</li><li>Hildebrandt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure-volume+data+of+cat+lung+interpreted+by+a+plastoelastic,+linear+viscoelastic+model.">Pressure-volume data of cat lung interpreted by a plastoelastic, linear viscoelastic model.</a> J. Appl. Physiol. 28, 365-372 (1970).</li><li>Hoppin, F. G., Hildebrandt, J., West, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioengineering Aspects of the Lung. , 83-162 (1977).</li><li>Avery, M. E., Mead, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+properties+in+relation+to+atelectasis+and+hyaline+membrane+disease).">Surface properties in relation to atelectasis and hyaline membrane disease).</a> AMA. J. Dis. Child. 97, 517-523 (1959).</li><li>Clements, J. A., Hustead, R. F., Johnson, R. P., Gribetz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+surface+tension+and+alveolar+stability.">Pulmonary surface tension and alveolar stability.</a> Tech Rep CRDLR US Army Chem. Res. Dev. Lab. 3052, 1-24 (1961).</li><li>Radford, E. P., Remington, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Tissue Elasticity. , 177-190 (1957).</li><li>Mitzner, W., Johnson, J. W. C., Scott, R., London, W. T., Palmer, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+betamethasone+on+the+pressure-volume+relationship+of+fetal+rhesus+monkey+lung.">Effect of betamethasone on the pressure-volume relationship of fetal rhesus monkey lung.</a> Journal of Applied Physiology. 47, 377-382 (1979).</li><li>Smaldone, G. C., Mitzner, W., Itoh, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+alveolar+recruitment+in+lung+inflation:+Influence+on+pressure-volume+hysteresis.">The role of alveolar recruitment in lung inflation: Influence on pressure-volume hysteresis.</a> Journal of Applied Physiology. 55, 1321-1332 (1983).</li><li>Tankersley, C. G., Rabold, R., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+lung+mechanics+are+genetically+determined+in+inbred+murine+strains.">Differential lung mechanics are genetically determined in inbred murine strains.</a> Journal of Applied Physiology. 86, 1764-1769 (1999).</li><li>Soutiere, S. E., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+defining+total+lung+capacity+in+the+mouse.">On defining total lung capacity in the mouse.</a> J. Appl. Physiol. 96, 1658-1664 (2004).</li><li>Stengel, P. W., Frazer, D. G., Weber, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+degassing:+an+evaluation+of+two+methods.">Lung degassing: an evaluation of two methods.</a> Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology. 48, 370-375 (1980).</li><li>Limjunyawong, N., Mitzner, W., Horton, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+chronic+idiopathic+pulmonary+fibrosis.">A mouse model of chronic idiopathic pulmonary fibrosis.</a> Physiol Rep. 2, e00249 (2014).</li><li>Fallica, J., Das, S., Horton, M. R., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+Carbon+Monoxide+Diffusing+Capacity+in+the+Mouse+Lung.">Application of Carbon Monoxide Diffusing Capacity in the Mouse Lung.</a> J. Appl. Physiol. 110, 1455-1459 (2011).</li><li>Brown, R. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structural+basis+of+airways+hyperresponsiveness+in+asthma.">The structural basis of airways hyperresponsiveness in asthma.</a> J. Appl. Physiol. 101 (1), 30-39 (2006).</li><li>Smargiassi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasonographic+Assessment+of+the+Diaphragm+in+Chronic+Obstructive+Pulmonary+Disease+Patients:+Relationships+with+Pulmonary+Function+and+the+Influence+of+Body+Composition+-+A+Pilot+Study.">Ultrasonographic Assessment of the Diaphragm in Chronic Obstructive Pulmonary Disease Patients: Relationships with Pulmonary Function and the Influence of Body Composition - A Pilot Study.</a> Respiration: International Review of Thoracic Diseases. 87 (5), 364-371 (2014).</li><li>Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airway-parenchymal+interdependence.">Airway-parenchymal interdependence.</a> Comprehensive Physiol. 2, 1921-1935 (2012).</li><li>Johnson, J. W., Permutt, S., Sipple, J. H., Salem, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Intra-Alveolar+Fluid+on+Pulmonary+Surface+Tension+Properties.">Effect of Intra-Alveolar Fluid on Pulmonary Surface Tension Properties.</a> J. Appl. Physiol. 19, 769-777 (1964).</li><li>Palmer, S., Morgan, T. E., Prueitt, J. L., Murphy, J. H., Hodson, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+development+in+the+fetal+primate,+Macaca+nemestrina.+II.+Pressure-volume+and+phospholipid+changes.">Lung development in the fetal primate, Macaca nemestrina. II. Pressure-volume and phospholipid changes.</a> Pediatr. Res. 11, 1057-1063 (1977).</li><li>Lum, H., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+species+comparisonof+alveolar+size+and+surface+forces.">A species comparisonof alveolar size and surface forces.</a> Journal of Applied Physiology. 62, 1865-1871 (1987).</li><li>Faridy, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+distension+on+release+of+surfactant+in+excised+dogs'+lungs.">Effect of distension on release of surfactant in excised dogs' lungs.</a> Respir. Physiol. 27, 99-114 (1976).</li><li>Faridy, E. E., Permutt, S., Riley, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ventilation+on+surface+forces+in+excised+dogs'+lungs.">Effect of ventilation on surface forces in excised dogs' lungs.</a> J. Appl. Physiol. 21, 1453-1462 (1966).</li><li>Comroe, J. H., Forster, R. E., Dubois, A. B., Briscoe, W. A., Carlsen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Lung: Clinical Physiology and Pulmonary Function Tests. , (1962).</li><li>Martinez, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+course+of+patients+with+idiopathic+pulmonary+fibrosis.">The clinical course of patients with idiopathic pulmonary fibrosis.</a> Ann. Intern. Med. 142, 963-967 (2005).</li></ol>

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...

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 years1-3. Such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development4-7. Despite the PV curve&#8217;s importance as a measurement of the lung&#8217;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&#160;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.

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			<td height="21" style="height:21px;width:244px;">Name of Material/ Equipment</td>
			<td style="width:127px;">Company</td>
			<td style="width:155px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
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			<td height="21" style="height:21px;width:244px;"></td>
			<td style="width:127px;"></td>
			<td style="width:155px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:244px;"></td>
			<td style="width:127px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">&#160;Syringe Pump</td>
			<td style="width:127px;">Harvard Apparatus</td>
			<td>55-2226</td>
			<td>Infuse/Withdraw syringe pump</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Pump 22 Reversing Switch&#160;</td>
			<td style="width:127px;">Harvard Apparatus</td>
			<td>552217</td>
			<td>&#160;included with pump</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:127px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Linear displacement transformer</td>
			<td style="width:127px;">Trans-Tek, Inc.</td>
			<td style="width:155px;">0244-0000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;"></td>
			<td style="width:127px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">5 mL glass syringe</td>
			<td style="width:127px;">Becton Dickenson</td>
			<td style="width:155px;"></td>
			<td>Several other possible vendors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;"></td>
			<td style="width:127px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Digital recorder</td>
			<td style="width:127px;">ADInstruments</td>
			<td>PL3504</td>
			<td>Several other possible vendors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bridge Amp Signal Conditioner</td>
			<td style="width:127px;">ADInstruments</td>
			<td>FE221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:127px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Gas tank,100% oxygen</td>
			<td style="width:127px;">Airgas, Inc</td>
			<td style="width:155px;"></td>
			<td>Any supplier or hospital source will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;"></td>
			<td style="width:127px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pressure Transducer - 0-1psi&#160; millivolt output</td>
			<td>Omega Engineering</td>
			<td>PX-137</td>
			<td>Range: &#8776;0-60 cmH2O</td>
		</tr>
	</tbody>
</table>

measurement of the pressure-volume curve in mouse lungs

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.

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 elsewhere12,13. 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 ...

Watch this Scientific Journal Video about Measurement of the Pressure-volume Curve in Mouse Lungs at JoVE.com

Measurement of the Pressure-volume Curve in Mouse Lungs

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&#8217;s structural changes with developing pathology.

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 ...

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