Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

Summary

We describe a means to quickly and simply measure the lung diffusing capacity in mice and show that it is sufficiently sensitive to phenotype changes in multiple common lung pathologies. This metric thus brings direct translational relevance to the mouse models, since diffusing capacity is also easily measured in humans.

Abstract

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods are needed that can quantify the pathologic changes. Furthermore, to provide translational relevance to the mouse models, such measurements should be tests that can easily be done in both humans and mice. Unfortunately, in the present literature few phenotypic measurements of lung function have direct application to humans. One exception is the diffusing capacity for carbon monoxide, which is a measurement that is routinely done in humans. In the present report, we describe a means to quickly and simply measure this diffusing capacity in mice. The procedure involves brief lung inflation with tracer gases in an anesthetized mouse, followed by a 1 min gas analysis time. We have tested the ability of this method to detect several lung pathologies, including emphysema, fibrosis, acute lung injury, and influenza and fungal lung infections, as well as monitoring lung maturation in young pups. Results show significant decreases in all the lung pathologies, as well as an increase in the diffusing capacity with lung maturation. This measurement of lung diffusing capacity thus provides a pulmonary function test that has broad application with its ability to detect phenotypic structural changes with most of the existing pathologic lung models.

Introduction

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underly such pathologies, phenotypic methods are needed that can quantify the it the pathologic changes. Although there are many mouse studies where ventilation mechanics are measured, these measurements are generally unrelated to the standard assessments of pulmonary function normally done in humans. This is unfortunate, since the ability to perform equivalent measurements in mice and human subjects may facilitate the translation of results in mouse models to human disease.

One of the most common and easily made measurements in human subjects is the diffusing capacity for carbon monoxide (DLCO)^1,2, but this measurement has only rarely been done in mouse models. In those studies where it has been reported^3-7, there have been no follow-up studies, in part because the procedures are often cumbersome or may require complex equipment. Another approach is to use a CO rebreathing method in a steady state system, which has the advantage of being able to measure CO diffusion in conscious mice. However this method is very cumbersome, and results can vary with the level of the mouse’s ventilation as well as O₂ and CO₂ concentrations^8,9. These difficulties seem to have precluded routine use of diffusing capacity to detect lung pathologies in mice, despite its several advantages.

To circumvent the problems with measurement of diffusing capacity in mice, details of a simple means to measure it in mice have been recently reported¹⁰. The procedure eliminates the difficult problem of sampling uncontaminated alveolar gas by quickly sampling a volume equal to the entire inspired gas. This procedure results in a very reproducible measurement, termed the diffusion factor for carbon monoxide (DFCO), that is sensitive to a host of pathologic changes in the lung phenotype. The DFCO is thus calculated as 1 – (CO₉ / CO_c) / (Ne₉ / Ne_c), where the c and 9 subscripts refer to concentrations of the calibration gases injected and the gases removed after a 9 sec breath hold time, respectively. DFCO is a dimensionless variable, which varies between 0 and 1, with 1 reflecting complete uptake of all CO, and 0 reflecting no uptake of CO.

In this presentation we show how to make this diffusing capacity measurement, and how it can be used to document changes in nearly all of the existing mouse lung disease models, including emphysema, fibrosis, acute lung injury, and viral and fungal infections.

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Protocol

NOTE: All animal protocols were approved by the Johns Hopkins University Animal Care and Use Committee.

1. Animal Preparation

1. Prepare 6 C57BL/6 control mice for the DFCO measurement, by anesthetizing them with ketamine and xylazine as outlined in step 2.3 below.
2. Prepare all of the other mice with the different lung pathologies shown in Table 1 by using the same procedure as for the controls. Specific details needed to establish each of these models are found in the relevant references. Control mice and those in the other pathologic cohorts are all 6-12 weeks of age.

2. Measurement of Diffusion Factor for Carbon Monoxide (DFCO)

1. Set up the gas chromatograph module supplied with the machine to measure peaks for nitrogen, oxygen, neon, and carbon monoxide. For this application use only the neon and CO data.
   NOTE: This instrument uses a molecular sieve column with helium as carrier gas, with a 12.00 µm film, 320.00 µm ID and 10 m length. The chromatograph column has a volume of 0.8 ml, but we used 2 ml to ensure adequate clearing of the connecting tubing with the sample.
2. At the start of each experimental day, prior to making measurements of the samples from the mice, take a 2 ml sample directly from a gas mixture bag containing approximately 0.5% Ne, 0.5% CO, and balance air, and use this sample to calibrate the gas chromatograph.
3. Anesthetize mice with ketamine (90 mg/kg) and xylazine (15 mg/kg), and confirm anesthesia by the absence of reflex motion. Apply veterinary ointment on the eyes to prevent dryness. Tracheostomize the mice with a stub needle cannula (18 G in adults or 20 G in very young mice).
   NOTE: The DFCO is completed in less than 10 min after anesthesia and prior to any mechanical ventilation or other procedures.
4. In mice greater than 6 weeks of age, use a 3 ml syringe to withdraw 0.8 ml of gas from the gas mixture bag. Connect the syringe to the tracheal cannula and quickly inflate the lung. Using a metronome, count 9 sec, and then quickly withdraw the 0.8 ml (exhaled air).
5. Dilute this withdrawn 0.8 ml exhaled air to 2 ml with room air, allow it to rest for at least 15 sec. Then inject the whole sample into the gas chromatograph for analysis.
6. While analyzing this first DFCO sample, inflate the mouse lung with a second 0.8 ml from the gas mixture bag, and then process this sample identical to the first sample. Average the two DFCO measurements.
   NOTE: For measurements in mice as young as 2 weeks of age, use a volume of 0.4 ml, since 0.8 ml is too large a volume to make measurements in lungs of very young mice. It is better to use the 0.8 ml volume for mice older than 6 weeks, and that if the 0.4 ml volume is needed for some mice, it should be used consistently for all mice in the cohort being studied.
7. Calculate DFCO as 1 – (CO₉ / CO_c) / (Ne₉ / Ne_c), where c and 9 subscripts refer to concentrations of the calibration gases injected and the gases removed after a 9 sec breath hold time, respectively.
8. Analyze and compare differences with a one-way ANOVA and assess the significance level with Tukey’s correction for multiple comparisons in all the cohort mice. Consider p <0.05 as significant value.
   NOTE: All of the mice used here were part of experimental studies involving several subsequent measurements of lung ventilation, mechanics, lung lavage, or histology, which are not reported here. In addition, since the method is the same in all the experimental models as was done above in the control mice, only the results from the various pathologic models are presented. Further information on these models is presented in the supplemental table.
9. Euthanize the animals by deep anesthetic overdose followed by cervical dislocation or decapitation. Where needed, remove the lung cells and/or tissues from the dead mice for further biologic or histologic processing and analysis.

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Results

Figure 1 shows the DFCO measurements from the adult mice in groups A, B, C, D, E, and F. There were significant decreases with both the Aspergillus and influenza infections, as well as significant decreases in the fibrotic, emphysematous, and acute lung injury models. Figure 2 shows the Group G developmental changes in DFCO over time as the mice age from 2-6 weeks. There was a slight but significant increase with lung development over this time course. The effect of using a smaller infla...

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Discussion

In the present work, we defined a new metric to quantify the gas exchanging ability of the mouse lung. This metric is analogous to the diffusing capacity, a common clinical measurement that measures the primary function of the lung, that is, its ability to exchange gas. The diffusing capacity is the only lung functional measurement that can be easily and quickly done in both mice and humans. For the detection of lung disease in mice, a major objective is to quantify changes in lung function between control and experiment...

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Materials

Name	Company	Catalog Number	Comments
Gas Chromatograph	Inficon	Micro GC Model 3000A	Agilent makes a comparable model
18 G Luer stub needle	Becton Dickenson		Several other possible vendors
3 ml plastic syringe	Becton Dickenson		Several other possible vendors
Polypropylene gas sample bags	SKC	1 or 2 L capacity works well	Other gas tight bags will work well
Gas tank, 0.3% Ne, 0.3% CO, balance air; (size ME)	Airgas, Inc	Z04 NI785ME3012	This is the standard mixture used for DLCO in humans
25 TCID50/mouse of influenza virus A/PR8 diluted in phosphate buffered saline.
Porcine pancreatic elastase	Elastin Products, Owensville, MO		5.4 U
Bleomycin	APP Pharmaceuticals, Schaumburg, IL		0.25 U
Escherichia coli LPS	Sigma L2880		3 μg/g body weight; O55:B5
Aspergillus fumigatus (isolate Af293) conidia were collected from mature colonies grown on potato dextrose agar.