Name: phenotyping mouse pulmonary function in vivo with the lung diffusing capacity
Uploaded: 2015-01-06T10:00:00.000+00:00
Duration: 7 min 13 s
Description: Watch this Scientific Journal Video about Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity at JoVE.com

This work was supported by NIH HL-10342

NOTE: All animal protocols were approved by the Johns Hopkins University Animal Care and Use Committee.
1. Animal Preparation
<ol>
	<li>Prepare 6 C57BL/6 control mice for the DFCO measurement, by anesthetizing them with ketamine and xylazine as outlined in step 2.3 below.</li>
	<li>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.</li>
</ol>
2. Measurement of Diffusion Factor for Carbon Monoxide (DFCO)
<ol>
	<li>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 &#181;m film, 320.00 &#181;m 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>Dilute this withdrawn 0.8 ml exhaled air to 2 ml with room air, allow it to rest for at least 15 sec.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.</li>
	<li>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.</li>
	<li>Calculate DFCO as 1&#160;&#8211;&#160;(CO9&#160;/&#160;COc)&#160;/&#160;(Ne9&#160;/&#160;Nec), 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.</li>
	<li>Analyze and compare differences with a one-way ANOVA and assess the significance level with Tukey&#8217;s correction for multiple comparisons in all the cohort mice. Consider p &#60;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.</li>
	<li>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.</li>
</ol>

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No conflicts of interest, and nothing to disclose.

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 experimental cohorts. To achieve this goal, we have defined and utilized the DFCO to demonstrate its capability of quanitfying phenotypic changes in most of the most common models of lung disease in mice, including functional changes with lung development.
One assumption inherent in the approach that simplifies the DFCO measurement in mice is that the effect of unmixed inspired gas in the anatomic and equipment dead space is ignored. However, as previously described10, the use of the 0.8 ml inflation volume introduces a small but consistent error in all the measurements. The magnitude of this error is a function of the relative sizes of the inflation and dead space volumes. In the present approach, this error is minimized by eliminating the dead space in stopcocks or T-connectors, and as shown in the video, simply uses a fingertip to seal the syringe and facilitate gas transfer. This procedure results in a high repeatability between measurements. The effect of the smaller inflation volume required in the young mice is made clear in the 6 week old mice shown in Figure 2, where the initial 0.4 ml test was immediately followed with an 0.8 ml test in each mouse. With 0.8 ml the DFCO value in was in the range for C57BL/6 mice in the other groups, but the measurement with 0.4 ml is consistently smaller. This is a direct manifestation of the fact that with the smaller volume, the recovered 9 sec sample has a greater fraction of the dead space air, which just consists of the control gas concentrations. This fact manifests itself as a smaller fractional change in the CO concentration, which brings it closer the change in Ne concentration. With an increased ratio of CO/Ne, the calculated DFCO (1 &#8211; this ratio) is thus smaller.
In the many models of lung pathology shown in Figure 1, there were a significant decreases observed in DFCO. However, there are several different reasons why the DFCO decreases in these different models. In fibrosis caused by the single dose of bleomycin, there is inflammation and a thickening of the diffusion barrier that leads to a reduction in diffusing capacity16. In emphysema, there is a loss of surface area acts directly to decrease both the surface area for diffusion and the blood volume in capillaries that had been in the destroyed walls. No dose-response relations with the elastase are presented here, but unpublished data shows a good correlation of the DFCO with the level of emphysematous damage. With the influenza infection, there is reduced diffusing capacity likely as a result of both a thickening of the diffusion barrier and an increase in consolidated unventilated regions of lung. In the PR8 influenza model used here, this worsens over time (as reflected in the further significant decrease in DFCO at day 8), and the mice generally die at around day 10. In the CFTR null mice, there was a smaller DFCO at baseline, but these mice are made on a mixed genetic background17, so there may be structural differences with the C57BL6 controls. However, the Aspergillus infection caused a more substantial significant decrease in DFCO, and reasons for this decline with the fungal infection are likely similar to those with the influenza. The LPS results in acute inflammation that causes a significantly decreased DFCO, likely from edematous thickening of the diffusion barrier and unventilated regions of lung filled with fluid. With the dose of LPS used, the decrease in DFCO is greatest at day 4 or 5 and then recovers back to control by day 10 (data not shown).
For the analysis of changes in DFCO with lung growth in the young mice, the 0.4 ml inflation volume clearly allowed reproducible measurements, and was able to show a slow increase in diffusing capacity as the lung reached adult maturity (Figure 2). The effect of using a smaller 0.4 ml inflation volume in decreasing the calculated DFCO is also what was expected, but the larger 0.8 ml volume cannot be used in the smaller mice. But changes with development or pathology should still be reproducibly detectable even with the 0.4 ml volume.
In conclusion, this video and accompanying manuscript shows how to obtain a functional measurement in mice that is similar to what can be measured in humans. The metric reflects the ability of the lung to exchange gas with a variety of lung structural changes caused by the most commonly studied pathologies. This diffusion factor for carbon monoxide (DFCO) is highly reproducible, and sensitive enough to detect functional and structural changes with most experimentally induced pathologies in young or old mice. The fact that it is directly analogous to similar measurements made in humans to assess lung disease, makes it a simple way to obtain a very relevant metric to phenytype mouse lung pathologies.

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 reported3-7, 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&#8217;s ventilation as well as O2 and CO2 concentrations8,9. 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&#160;&#8211;&#160;(CO9&#160;/&#160;COc)&#160;/&#160;(Ne9&#160;/&#160;Nec), 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.

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

phenotyping mouse pulmonary function in vivo with the lung diffusing capacity

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.

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 inflation volume was also quite apparent at the 6 week time point. There was a tendency for the females to have a slightly higher DFCO, but this was only significant at the 5 week time point.
<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td style="width:40px;">Group</td>
			<td>Pathology or Condition</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>A</td>
			<td>C57BL/6 controls (8-10 weeks), n = 6</td>
			<td>Healthy mice</td>
		</tr>
		<tr>
			<td>B</td>
			<td>C57BL/6 mice given 25 TCID50 of influenza A virus (PR8) intranasally, n = 10</td>
			<td>Influenza model, studied 6 and 8 days post-infection</td>
		</tr>
		<tr>
			<td>C</td>
			<td>C57Bl/6 mice given 5.4 U pancreatic elastase intratracheally, n = 6</td>
			<td>Emphysema model10,13 studied 21 days after elastase challenge</td>
		</tr>
		<tr>
			<td>D</td>
			<td>C57BL/6 mice given 0.05 U bleomycin intratracheally, n = 6</td>
			<td>Fibrosis model14 studied 14 days after bleomycin challenge</td>
		</tr>
		<tr>
			<td>E</td>
			<td>CFTR null controls and those infected with aerosol inhalation of Aspergillus fumigatus (strain AF293), n = 6</td>
			<td>Fungal infection model11,17 studied 12 days after fungal infection</td>
		</tr>
		<tr>
			<td>F</td>
			<td>C57BL/6 mice given 3 &#181;g/g BW LPS (Escherichia coli) intratracheally, n = 6</td>
			<td>Acute Lung Injury (ALI) model15 studied on days 1 and 4 after the LPS insult</td>
		</tr>
		<tr>
			<td>G</td>
			<td>C57BL/6 male mice at 2 to 6 weeks of age, n = 5 at each age</td>
			<td>Lung development model</td>
		</tr>
	</tbody>
</table>
Table 1: Listing of the different mouse models where the DFCO was measured.
<img alt="Figure 1" src="/files/ftp_upload/52216/52216fig1highres.jpg" width="500" /> 
Figure 1: Measurement of DFCO in the control C57BL/6 mice (Group A) and in each of the 5&#160;different pathologic models. Shown are results 6 and 8 days after the PR8 influenza (Group B), 21 days after intratracheal elastase (Group C), 14 days after intratracheal bleomycin (Group D), 12 days after aspergillus infection in the CFTR null mice (Group E), and 1 and 4 days after LPS instillation (Group F). The * indicates P &#60;0.01 vs. control, the # indicates P &#60;0.01 between the 6 and 8 day influenza mice and the 1 and 4 day LPS mice, and the + indicates P &#60;0.05 vs. control.
<img alt="Figure 2" src="/files/ftp_upload/52216/52216fig2highres.jpg" width="500" /> 
Figure 2: Measurement of DFCO in male C57BL/6 mice from 2 through 6 weeks of age (Group G). The measurements were made in all mice with an inflation volume of 0.4 ml, and in the 6 week old mice a second measurement was made with 0.8 ml. With the 0.4 ml volume, there were significant increases in DFCO between the 2 week male and those at 4, 5, and 6 weeks (P &#60;0.05).

Watch this Scientific Journal Video about Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity at JoVE.com

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

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.

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

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 ...

phenotyping-mouse-pulmonary-function-vivo-with-lung-diffusing

Nanyang Technological University

Research

JoVE Journal

Biology

10.5K Views.  Johns Hopkins University Bloomberg School of Public Health. 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.

Video: Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation

Pulmonary morphology, physiology, and respiratory functions change in both physiological and pathological conditions. Internal lung surface area (ISA), representing the gas-exchange capacity of the lung, is a critical criterion to assess respiratory function. However, observer bias can significantly influence measured values for lung morphological parameters. The protocol that we describe here minimizes variations during measurements of two morphological parameters used for ISA calculation: internal lung volume (ILV) and mean linear intercept (MLI). Using ISA as a morphometric and functional parameter to determine the outcome of alveolar regeneration in both pneumonectomy (PNX) and prosthesis implantation mouse models, we found that the increased ISA following PNX treatment was significantly blocked by implantation of a prosthesis into the thoracic cavity1. The ability to accurately quantify ISA is not only expected to improve the reliability and reproducibility of lung function studies in injured-induced alveolar regeneration models, but also to promote mechanistic discoveries of multiple pulmonary diseases.

Internal lung surface area (ISA) is a critical criterion for assessing lung morphology and physiology in lung diseases and injury-induced alveolar regeneration. We describe here a standardized method that can minimize the measurement bias for ISA in both lung pneumonectomy and prosthesis implantation mouse models.

Pulmonary morphology, physiology, and respiratory functions change in both physiological and pathological conditions. Internal lung surface area (ISA), ...

Developmental Biology

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Medicine

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.

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 ...

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential.

The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice.

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

Immunology and Infection

Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high incidence in aging populations. The current conventional therapies for COPD focus mainly on symptom-modifying drugs; thus, the development of new therapies is urgently needed. Qualified animal models of COPD could help to characterize the underlying mechanisms and can be used for new drug screening. Current COPD models, such as lipopolysaccharide (LPS) or the porcine pancreatic elastase (PPE)-induced emphysema model, generate COPD-like lesions in the lungs and airways but do not otherwise resemble the pathogenesis of human COPD. A cigarette smoke (CS)-induced model remains one of the most popular because it not only simulates COPD-like lesions in the respiratory system, but it is also based on one of the main hazardous materials that causes COPD in humans. However, the time-consuming and labor-intensive aspects of the CS-induced model dramatically limit its application in new drug screening. In this study, we successfully generated a new COPD model by exposing mice to high levels of ozone. This model demonstrated the following: 1) decreased forced expiratory volume 25, 50, and 75/forced vital capacity (FEV25/FVC, FEV50/FVC, and FEV75/FVC), indicating the deterioration of lung function; 2) enlarged lung alveoli, with lung parenchymal destruction; 3) reduced fatigue time and distance; and 4) increased inflammation. Taken together, these data demonstrate that the ozone exposure (OE) model is a reliable animal model that is similar to humans because ozone overexposure is one of the etiological factors of COPD. Additionally, it only took 6 - 8 weeks, based on our previous work, to create an OE model, whereas it requires 3 - 12 months to induce the cigarette smoke model, indicating that the OE model might be a good choice for COPD research.

This study describes the successful generation of a new chronic obstructive pulmonary disease (COPD) animal model by repeatedly exposing mice to high concentrations of ozone.

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high ...

Evaluation of Respiratory System Mechanics in Mice using the Forced Oscillation Technique

The forced oscillation technique (FOT) is a powerful, integrative and translational tool permitting the experimental assessment of lung function in mice in a comprehensive, detailed, precise and reproducible manner. It provides measurements of respiratory system mechanics through the analysis of pressure and volume signals acquired in reaction to predefined, small amplitude, oscillatory airflow waveforms, which are typically applied at the subject's airway opening. The present protocol details the steps required to adequately execute forced oscillation measurements in mice using a computer-controlled piston ventilator (flexiVent; SCIREQ Inc, Montreal, Qc, Canada). The description is divided into four parts: preparatory steps, mechanical ventilation, lung function measurements, and data analysis. It also includes details of how to assess airway responsiveness to inhaled methacholine in anesthetized mice, a common application of this technique which also extends to other outcomes and various lung pathologies. Measurements obtained in na&iuml;ve mice as well as from an oxidative-stress driven model of airway damage are presented to illustrate how this tool can contribute to a better characterization and understanding of studied physiological changes or disease models as well as to applications in new research areas.

The present protocol provides a detailed step-by-step description of the procedures required to execute measurements of respiratory system mechanics as well as the assessment of airway responsiveness to inhaled methacholine in mice using the forced oscillation technique (flexiVent; SCIREQ Inc, Montreal, Qc, Canada).

The  forced oscillation technique (FOT) is a powerful, integrative and translational  tool permitting the experimental assessment of lung function in mice ...

Assessment of Respiratory Function in Conscious Mice by Double-chamber Plethysmography

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to non-invasively assess some features of the respiratory function in humans as well as in laboratory animals. The present article focuses on the application of the double-chamber plethysmography (DCP) in small animals. It provides background information on the methodology as well as a detailed step-by-step procedure to successfully assess respiratory function in conscious, spontaneously breathing animals in a non-invasive manner. The DCP can be used to monitor the respiratory function of multiple animals in parallel, as well as to identify changes induced by aerosolized substances over a chosen time period and in a repeated manner. Experiments on control and allergic mice are used herein to demonstrate the utility of the technique, explain the associated outcome parameters, as well as to discuss the related advantages and shortcomings. Overall, the DCP provides valid and theoretically sound readouts that can be trusted to evaluate the respiratory function of conscious small animals both at baseline and after challenges with aerosolized substances.

The objective of the present article is to provide a detailed description of the recommended procedures to evaluate respiratory function in conscious mice by double-chamber plethysmography.

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to ...

Hyperpolarized 129Xe Lung MRI and Spectroscopy in Mechanically Ventilated Mice

Hyperpolarized (HP) xenon-129 (129Xe) is an inhaled magnetic resonance imaging (MRI) contrast agent with unique spectral and physical properties that can be exploited to quantify pulmonary physiology, including ventilation, restricted diffusion (alveolar-airspace size), and gas exchange. In humans, it has been used to evaluate disease severity and progression in a variety of pulmonary disorders and is approved for clinical use in the United States and United Kingdom. Beyond its clinical applications, the ability of 129Xe MRI to noninvasively assess pulmonary pathophysiology and provide spatially resolved information is valuable for preclinical research. Among animal models, mice are the most widely used due to the accessibility of genetically modified disease models. Here, 129Xe MRI is promising as a minimally invasive, radiation-free, and sensitive technique to longitudinally monitor lung disease progression and therapy response (e.g., in drug discovery). This technique can extend to preclinical applications by incorporating an MRI-triggered, free-breathing apparatus or mechanical ventilator to deliver gas. Here, we describe the steps and provide checklists to ensure robust data collection and analysis, including creating a thermally polarized xenon gas phantom for quality control, optimizing polarization, animal handling (sedation, intubation, ventilation, and care for mice), and protocols for ventilation, restricted diffusion, and gas exchange data. While preclinical 129Xe MRI can be applied in various animal models (e.g., rats, pigs, sheep), this protocol focuses on mice due to the challenges posed by their small anatomy, which are balanced by their affordability and the availability of many disease models.

Hyperpolarized xenon MRI can quantify regional lung microstructure (air-space dimensions) and physiology (ventilation and gas exchange) in translational research and clinical care. Although challenging, it can provide comparable pulmonary insights in preclinical studies. This protocol describes the infrastructure and procedures needed to perform routine xenon lung MRI in mice.

Hyperpolarized (HP) xenon-129 (129Xe) is an inhaled magnetic resonance imaging (MRI) contrast agent with unique spectral and physical properties that can ...

Bioengineering

Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.

The assessment of respiratory physiology has traditionally relied upon techniques, which require restraint or sedation of the animal. Unrestrained whole-body plethysmography, however, provides precise, non-invasive, quantitative analysis of respiratory physiology in animal models. In addition, the technique allows repeated respiratory assessment of mice allowing for longitudinal studies.

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative ...

In vivo Evaluation of Mucociliary Clearance in Mice

Respiratory motile cilia, specialized organelles of the cell, line the apical surface of epithelial cells lining the respiratory tract. By beating in a metachronal, synchronal fashion, these multiple, motile, actin-based organelles generate a cephalad fluid flow clearing the respiratory tract of inhaled pollutants and pathogens. With increasing environmental pollution, novel viral pathogens and emerging multi-drug resistant bacteria, cilia generated mucociliary clearance (MCC) is essential for maintaining lung health. MCC is also depressed in multiple congenital disorders like primary ciliary dyskinesia, cystic fibrosis as well as acquired disorders like chronic obstructive pulmonary disease. All these disorders have established, in some case multiple, mouse models. In this publication, we detail a method using a small amount of radioactivity and dual-modality SPECT/CT imaging to accurately and reproducibly measure MCC in mice in vivo. The method allows for recovery of mice after imaging, making serial measurements possible, and testing potential therapeutics longitudinally over time. The data in wild-type mice demonstrates the reproducibility of the MCC measurement as long as adequate attention to detail is paid, and the protocol strictly adhered to.

In this publication, we describe protocols for assessing airway mucociliary clearance (MCC) in mice in vivo utilizing dual-modality radionuclide imaging. This protocol is designed for a single photon emission computed tomography (SPECT) and computed tomography (CT) acquisition protocol using mouse whole body (MWB) collimators in a dual SPECT/CT system.

Respiratory motile cilia, specialized organelles of the cell, line the apical surface of epithelial cells lining the respiratory tract. By beating in a ...

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

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