This work was funded by the Departments of Health and Human Physiology and Internal Medicine at the University of Iowa. This work was also supported by the Old Gold Fellowship (Bates) and Grant IRG-15-176-40 from the American Cancer Society, administered through The Holden Comprehensive Cancer Center at The University of Iowa (Bates)

This protocol has been previously approved by and follows the guidelines set by the University of Iowa Institutional Review Board. Data shown were collected as part of a project approved by the Institutional Review Board at the University of Iowa. Participants gave informed consent and the studies were performed in accordance with the Declaration of Helsinki.
1. Equipment
<ol>
	<li>Check the equipment table to verify that all required equipment is available. Double check the configuration using the graphic depiction of the equipment in Figure 2.</li>
</ol>
2. Plethysmography
NOTE: Barometric plethysmography is a well-described clinical tool and is performed using commercial equipment according to the consensus statements on standardizing lung volume measurements14,15. When necessary, lung flows and volumes are compared to predicted values from the NHANES data set and Goldman and Becklake16 that are included in the plethysmograph software.
<ol>
	<li>Perform calibration of the plethysmograph daily and prior to any experiments.
	<ol>
		<li>Measure the temperature, barometric pressure and relative humidity using a standard barometer prior to the calibration and enter these values into the plethysmograph software as correction factors.</li>
		<li>Calibrate the flow sensor using a calibrated 3 L syringe at variable flow rates. Calibrate the box pressure using a precise 50 mL pump. Box pressure transducers should be checked monthly and re-calibrated as needed, per manufacturer&#8217;s recommendation.</li>
	</ol>
	</li>
	<li>Immediately prior to the measurement, place the participant in the whole body plethysmograph and close the door. Make measurements after 30-60 s, which allows for thermal equilibration.
	<ol>
		<li>Instruct the participant to place their mouth on the mouthpiece, put on nose clips, and place their hands on their cheeks. Preventing &#8220;puffing&#8221; of the cheeks during the maneuver minimizes changes in volume that result from changing the mouth volume.</li>
		<li>Instruct the participant to breathe normally, allowing at least four tidal breaths to be acquired and functional residual capacity (FRC) to be established.</li>
		<li>At the end of a normal exhalation (FRC), close the shutter. Coach the participant to pant lightly at 0.5-1 breaths/s for 3-4 s. Evaluate the relationship between the mouth pressure and plethysmograph pressure to ensure that it is a series of overlapping, straight lines without thermal drift.</li>
		<li>Open the shutter and allow the participant to take a normal breath. Coach the participant to exhale to residual volume (RV), followed by a maximal inspiratory maneuver to total lung capacity. Repeat at least three times until FRC values that agree within 5% are obtained</li>
	</ol>
	</li>
</ol>
3. Volumetric Capnography
NOTE: Steps 3.1 &#8211; 3.4 are performed before the arrival of the research subject.
<ol>
	<li>Before proceeding, address the variables in Table 1 and modify if needed. It is important that these variables are adjusted during the study design phase and then held constant for the duration of the study.
	<ol>
		<li>Before beginning a new experimental protocol, take care to accurately measure the time delay between the gas analyzer, which measures CO2 concentration, and the pneumotach, which measures flow. This allows for the CO2 and flow signals to be aligned.</li>
		<li>Measure the time delay experimentally with a stream of 5% CO2. Attach the gas line to a stopcock, followed by the mouthpiece.</li>
		<li>Open the stopcock, introducing the gas at a rate of 10 L/min. Determine the mean time delay between the response of the pneumotach and gas analyzer over 10 trials and enter into the macro.</li>
		<li>Maintain the time delay constant by maintaining the analyzer sampling rate. The time delay is highly dependent on the sampling rate of the gas analyzer and it is critical that this remain constant through the experiment and between participants.</li>
	</ol>
	</li>
	<li>Define three &#8220;channels&#8221; for the collection of flow, exhaled CO2 (%), and volume. Flow and exhaled CO2 (%) are analog inputs and volume is the integral of flow.
	<ol>
		<li>Confirm that flow and CO2 (%) are measured directly from the pneumotach and gas analyzer and that volume is calculated as the integral of flow. Figure 3 shows that these are being collected in channels 1,2, and 6.</li>
	</ol>
	</li>
	<li>Calibrate the gas analyzer prior to each use. Include the O2 sensor if this is to be measured.
	<ol>
		<li>Zero the analyzer with an inert gas. 100% calibration grade (&#60;0.01% contaminant) N2 or He may be used, although helium is preferred because nitrogen may be contaminated with trace amounts of oxygen. Place the drying tube in a bag or connect to a mixing chamber. Flush the bag or chamber with inert gas at a rate of at least 10 L/min. Care should be taken not to pressurize the system as this can impact the calibration.</li>
		<li>Flood the bag or chamber with inert gas to displace O2 and the CO2. Once the displayed concentrations of CO2 and O2 stabilize, adjust the zero knobs until they both read zero.</li>
		<li>Repeat with 6% CO2 and room air (20.93% O2) as calibration gases. When the concentration of the desired gas stabilizes, adjust the span knob to match the concentration of the calibration gas.</li>
		<li>Recheck the inert gas and calibration gases and adjust the zero and span until both are accurate &#177;0.1%.</li>
	</ol>
	</li>
	<li>Calibrate the heated pneumotach according to the manufacturer&#8217;s instructions.
	<ol>
		<li>Briefly, allow the pneumotach to warm to 37 &#176;C for at least 20 min prior to the study.</li>
		<li>Select the drop-down menu of the flow channel (Channel 1), select the Spirometer menu option, and click Zero to zero the pneumotach. Finish by selecting Okay.</li>
		<li>Directly connect a 3L syringe to the pneumotach using a flow head adapter. Highlight the calibration breath. Again, select the drop-down menu of the flow channel. Select Spirometer flow | Calibrate, type in 3L, and select Okay&#34;\.</li>
		<li>Check the calibration by injecting 3L into the pneumotach at varying flow rates (0-4 L/s, 4-8 L/s, and 8-12 L/s). The difference from 3 L should be less than 5%.</li>
	</ol>
	</li>
	<li>Collect the maneuver, ensuring that two sequential breaths are collected and that they are made at the same flow rate.
	<ol>
		<li>Coach the subject to perform a single maneuver consisting of two pairs of breaths &#8211; a coaching breath and a breath for analysis. This is shown graphically in Figure 1 (bottom).</li>
		<li>During the maneuver, coach the participants to follow the flow guide on the computer monitor. The investigator may coach the subject by indicating &#8220;inhale now&#8221; or &#8220;exhale now&#8221;.</li>
		<li>Perform the maneuver so that there are two pairs of these breaths in a single maneuver. The first exhalation of the maneuver is 3 s and the second is 5 s. Consider adding a resistor in-line with the mouthpiece in order to make exhaled flow easier to control. A resistance with 5 cm H2O/L/s of resistance is generally well-tolerated. 
		NOTE: It is important that if a resistor is used, it is used throughout the study and for every participant because it increases mouth and airway pressure, which can change airway diameter. It is also important that participants not &#8220;puff out&#8221; their cheeks as this increases the dead space.</li>
	</ol>
	</li>
	<li>Measurement protocol
	<ol>
		<li>Instruct the participant to sit straight with both feet on the floor, put nose clips on their nose and place their mouth on the mouthpiece.</li>
		<li>Coach the participant to complete at least one minute of tidal breathing. This is for measures of metabolic function and allows the participant to familiarize themselves with the mouthpiece. After one minute, stop data collection.</li>
		<li>Next, coach the participants to vary their tidal volume, taking either normal, smaller- or larger than normal tidal breaths. This ensures that the capnograms are obtained at different lung volumes</li>
		<li>Coach the participant that they should transition to performing a capnogram maneuver as soon as they see the flow tracing appear on their screen.</li>
		<li>Resume data collection at a random point in the participant&#8217;s respiratory cycle. This allows for measurements to be made at different lung volumes.</li>
		<li>Finally, coach to perform a sigh at the end of each maneuver, completely relaxing the muscles of respiration. This allows for FRC to be determined.</li>
		<li>Stop data collection. Repeat Steps 3.6.3-3.6.5 until at least 6-8 maneuvers (12 -16 pairs of breaths for analysis) are completed.</li>
	</ol>
	</li>
</ol>
4. Data Analysis
<ol>
	<li>Exporting Data. To run through the macro, each pair of breaths must be exported as a single text file that is then imported into the macro. Screen shots of this process are given in Supplemental Figure 1.
	<ol>
		<li>Highlight each pair of breaths, taking to care to highlight a portion of the exhalation before the maneuver begins.</li>
		<li>Under the file menu, select Export, and name the subject&#39;s maneuver.</li>
		<li>Use the drop-down menu under Save As Type and save it as a data file. Then select Save.</li>
		<li>This will prompt an Export As Text box to appear. On the right deselect Block header Columns, Time, Date, Comments, and Event Markers.</li>
		<li>On the left, select Current Selection and Output NaN for Values. Select Downsample by and enter 10 into the box.</li>
		<li>Select the Flow Channel and the CO2 (%) Channel to be exported and click Okay. Consider making duplicates of these exported files as backups before beginning the analysis.</li>
	</ol>
	</li>
	<li>Perform the macro analysis. The Step-by-step annotated screen shots of for analyzing exported maneuvers with the macro and comparing to lung volume are given in Supplemental Figure 2 and may be used as a guide.
	<ol>
		<li>Open the macro, go to file, and select Open.</li>
		<li>Select the saved data file, saved with the .txt extension.</li>
		<li>A Text Import Wizard box will appear. In the upper left-hand corner, select Delimited and click Next. For step 2, select Tab under Delimiters and click Next. For step 3, select General under Column Data Format and click Finish.</li>
		<li>To run the macro, select View, Macro, View Macro, and Run in succession. Select Yes if there is a backup copy of the data.</li>
		<li>Allow the macro to run (approximately 90 s) and generate a workbook with four sheets. Of relevance to these measurements, Sheet 2 contains the numeric data and Chart 3 contains a plot of the capnogram.</li>
		<li>Return to the data and determine the volume for FRC. This is identified as the volume at the end of the sigh at which flow = 0 L/s.</li>
		<li>Determine the volume at which the second exhalation in each pair of breaths was begun. By subtracting this from the FRC volume, the starting volume above or below FRC can be determined for each breath.</li>
	</ol>
	</li>
</ol>

<ol>
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L., 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=Pulmonary+function+responses+to+ozone+in+smokers+with+a+limited+smoking+history.">Pulmonary function responses to ozone in smokers with a limited smoking history.</a> Toxicology and Applied Pharmacology. 278 (1), 85-90 (2014).</li><li>Bates, M. L., Brenza, T. M., Ben-Jebria, A., Bascom, R., Ultman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+distribution+of+ozone+absorption+in+the+lung:+comparison+of+cigarette+smokers+and+nonsmokers.">Longitudinal distribution of ozone absorption in the lung: comparison of cigarette smokers and nonsmokers.</a> Toxicology and Applied Pharmacology. 236 (3), 270-275 (2009).</li><li>Reeser, W. 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=Uptake+of+ozone+in+human+lungs+and+its+relationship+to+local+physiological+response.">Uptake of ozone in human lungs and its relationship to local physiological response.</a> Inhalation Toxicology. 17 (13), 699-707 (2005).</li><li>Taylor, A. B., Lee, G. M., Nellore, K., Ben-Jebria, A., Ultman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+carbon+dioxide+expirogram+in+response+to+ozone+exposure.">Changes in the carbon dioxide expirogram in response to ozone exposure.</a> Toxicology and Applied Pharmacology. 213 (1), 1-9 (2006).</li><li>Baker, L. G., Ultman, J. S., Rhoades, R. 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S., 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=Lumen+area+change+(Delta+Lumen)+between+inspiratory+and+expiratory+multidetector+computed+tomography+as+a+measure+of+severe+outcomes+in+asthmatic+patients.">Lumen area change (Delta Lumen) between inspiratory and expiratory multidetector computed tomography as a measure of severe outcomes in asthmatic patients.</a> J The Journal of Allergy and Clinical. , (2018).</li><li>Smith, B. M., 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=Human+airway+branch+variation+and+chronic+obstructive+pulmonary+disease.">Human airway branch variation and chronic obstructive pulmonary disease.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (5), E974-E981 (2018).</li><li>Farmery, A. 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The authors have nothing to disclose.

Here, a protocol for the measurement of VD and airways homogeneity (slope) is provided. These measurements can be made at FRC, or as a function of lung volume. Measuring FRC before the start of the experiment and after a perturbation allows VD and slope to be plotted as a function of lung volume and may provide useful information about the structure-function relationship of the lung that is not obtained from capnography at FRC alone.
Airways volume and high-resolution str...

Gas washout techniques have been used for decades to provide important information about the structure and uniformity of the airway tree. The lung is classically described as having two compartments &#8211; a conducting zone that is comprised of the anatomic dead space and the respiratory zone where gas exchange occurs in the alveoli. The conducting airways are termed as &#8220;dead space&#8221; because they do not participate in the exchange of oxygen and carbon dioxide. In the single breath gas washout method, the concentration profile of an exhaled gas can be used to determine the volume of the anatomic dead space and to derive information about the uniformity of ventilation. Some methods rely on the breathing of inert gases to make these measures (N2, argon, He, SF6, etc.). The use of inert gas is well-established, supported by scientific consensus statements1, and there are available commercial equipment with user friendly interfaces. However, the exhaled profile of carbon dioxide (CO2) can be used to derive similar information. Evaluating the profile of CO2 as a function of the exhaled volume, or volumetric capnography, does not require the participant to breathe special gas mixtures and allows the investigator to gather additional information flexibly about metabolism and gas exchange with minimal adjustment to the technique.
During a controlled exhalation, the concentration of CO2 can be plotted against the total exhaled volume. At the beginning of an exhalation, the dead space is filled with atmospheric gas. This is reflected in Phase I of the exhaled CO2 profile where there is an undetectable amount of CO2 (Figure 1, top). Phase II marks the transition to the alveolar gas, where gas exchange occurs and CO2 is abundant. The volume at the midpoint of Phase II is the volume of the anatomic dead space (VD). Phase III contains alveolar gas. Because airways with different diameters empty at different rates, the slope (S) of Phase III provides information about airways uniformity. A steeper slope of Phase III suggests a less uniform airway tree proximal to the terminal bronchioles, or convection-dependent inhomogeneity2. In the case where a perturbation may change the rate of CO2 production, and to make comparisons between individuals, the slope can be divided by the area under the curve to normalize for differences in metabolism (NS or normalized slope). Volumetric capnography has been used previously to evaluate the changes in airways volume and uniformity following air pollutant exposure3,4,5,6.
Gas transport in the lung is governed by both convection and diffusion. Single breath washout measures are highly dependent on air flow and the measured value of VD occurs at the convection-diffusion boundary. Changing the flow rate of the exhalation or preceding inhalation changes the location of that boundary7. Capnography is also highly dependent on the volume of the lung immediately preceding the maneuver. Larger lung volumes distend the airways, resulting in larger values of VD8. One solution is to consistently make the measurement at the same lung volume &#8211; usually functional residual capacity (FRC). An alternative, described here, is to couple volumetric capnography with barometric plethysmography, in order to obtain the relationship between VD and lung volume. The participant then performs the maneuver at constant flow rates, while varying the lung volume. This still allows for classic capnographic measures to be made at FRC, but also for the relationship between the lung volume and dead space volume and between the lung volume and homogeneity to be derived. Indeed, the added value of coupling capnography with plethysmography comes from the ability to test hypotheses about the distensibility of the airways tree and the structure-function relationship of the lung. This may be a valuable tool for investigators aiming to quantify the influence of airways mechanics versus lung compliance and elastance on pulmonary function in healthy and diseased populations9,10,11. Furthermore, accounting for the absolute lung volume at which the volumetric capnographic measurements are being performed allows investigators to characterize the effects of conditions that can alter the inflation state of the lung, such as obesity, lung transplant, or interventions like chest wall strapping. Volumetric capnography may ultimately have clinical utility in the intensive care setting12,13.

<table><tbody><tr><td>Computer with dual monitor</td><td>Dell Instruments</td><td></td><td></td></tr><tr><td>PowerLab 8/35*</td><td>AD Instruments</td><td>PL3508</td><td></td></tr><tr><td>LabChart Data Acquisition Software*</td><td>AD Instruments</td><td>Version 8</td><td></td></tr><tr><td>Gemini Respiratory Gas Analyzer* (upgraded option)</td><td>CWE, Inc</td><td>GEMINI 14-10000</td><td>*indicates that part is available in the Exercise Physiology package from AD Instruments</td></tr><tr><td>Heated Pneumotach with Heater Controller* (upgraded option)</td><td>Hans Rudolph, Inc</td><td>MLT3813H-V</td><td></td></tr><tr><td>3L Calibration Syringe</td><td>Vitalograph</td><td>36020</td><td></td></tr><tr><td>Nose Clip*</td><td>VacuMed</td><td>Snuffer 1008</td><td></td></tr><tr><td>Pulse Transducer*</td><td>AD Instruments</td><td>TN1012/ST</td><td></td></tr><tr><td>Barometer</td><td>Fischer Scientific</td><td>15-078-198</td><td></td></tr><tr><td>Flanged Mouthpiece*</td><td>AD Instruments</td><td>MLA1026</td><td></td></tr><tr><td>Nafion drying tube with three-way stopcock*</td><td>AD Instruments</td><td>MLA0343</td><td></td></tr><tr><td>Desiccant cartridge (optional for humid environments)*</td><td>AD Instruments</td><td>MLA6024</td><td></td></tr><tr><td>Resistor</td><td>Hans Rudolph, Inc</td><td>7100 R5</td><td></td></tr><tr><td>Flow head adapters*</td><td>AD Instruments</td><td>MLA1081</td><td></td></tr><tr><td>Modified Tubing Adapter (optional)</td><td>AD Instruments</td><td>SP0145</td><td></td></tr><tr><td>Two way non-rebreather valve (optional)*</td><td>AD Instruments</td><td>SP0146</td><td></td></tr><tr><td>Plethysmograph</td><td>Vyaire</td><td>V62J</td><td></td></tr><tr><td>High Purity Helium Gas</td><td>Praxair</td><td>He 4.8</td><td></td></tr><tr><td>6% CO&lt;sub&gt;2&lt;/sub&gt; and 16% O&lt;sub&gt;2&lt;/sub&gt; Calibration Gas</td><td>Praxair</td><td>Custom</td><td></td></tr><tr><td>Microsoft Excel</td><td>Microsoft</td><td>Office 365</td><td></td></tr></tbody></table>

combining volumetric capnography and barometric plethysmography to measure the lung structure-function relationship

Tools to measure lung and airways volume are critical for pulmonary researchers interested in evaluating the impact of disease or novel therapies on the lung. Barometric plethysmography is a classic technique to evaluate the lung volume with a long history of clinical use. Volumetric capnography utilizes the profile of exhaled carbon dioxide to determine the volume of the conducting airways, or dead space, and provides an index of airways homogeneity. These techniques may be used independently, or in combination to evaluate the dependence of airways volume and homogeneity on lung volume. This paper provides detailed technical instructions to replicate these techniques and our representative data demonstrates that the airways volume and homogeneity are highly correlated to lung volume. We also provide a macro for the analysis of capnographic data, which can be modified or adapted to fit different experimental designs. The advantage of these measures is that their advantages and limitations are supported by decades of experimental data, and they can be made repeatedly in the same subject without expensive imaging equipment or technically advanced analysis algorithms. These methods may be particularly useful for investigators interested in perturbations that change both the functional residual capacity of the lung and airways volume.

Representative plethysmography results are given in Figure 4. This participant required four attempts in order to collect three FRC values with &#60;5% variability from the mean.%Ref reflects the percent of the predicted value for each variable based on population regression equations that take into account sex, age, race, height and weight
Figure 1 (top) shows a repres...

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Combining Volumetric Capnography And Barometric Plethysmography To Measure The Lung Structure-function Relationship

Here, we describe two measures of pulmonary function &#8211; barometric plethysmography, which allows the measurement of lung volume, and volumetric capnography, a tool to measure the anatomic dead space and airways uniformity. These techniques may be used independently or combined to assess airways function at different lung volumes.

Tools to measure lung and airways volume are critical for pulmonary researchers interested in evaluating the impact of disease or novel therapies on the ...

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9.3K Views.  University of Iowa. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in de longfysiologie, met name over hoe de structuurfunctierelatie in de long bijdraagt aan de ziektepathologie. Het belangrijkste voordeel van deze techniek is dat het mogelijk maakt om herhaaldelijk maatregelen te nemen, binnen hetzelfde onderwerp, zonder dure beeldapparatuur of technisch geavanceerde analysealgoritmen. De implicaties van het meten van de longstructuur-functie relatie uit te breiden tot het begrijpen van de...

Video: Combining Volumetric Capnography And Barometric Plethysmography To Measure The Lung Structure-function Relationship

Expired CO2 Measurement in Intubated or Spontaneously Breathing Patients from the Emergency Department

Carbon dioxide (CO2) along with oxygen (O2) share the role of being the most important gases in the human body. The measuring of expired CO2 at the mouth has solicited growing clinical interest among physicians in the emergency department for various indications: (1) surveillance et monitoring of the intubated patient; (2) verification of the correct positioning of an endotracheal tube; (3) monitoring of a patient in cardiac arrest; (4) achieving normocapnia in intubated head trauma patients; (5) monitoring ventilation during procedural sedation. The video allows physicians to familiarize themselves with the use of capnography and the text offers a review of the theory and principals involved. In particular, the importance of CO2 for the organism, the relevance of measuring expired CO2, the differences between arterial and expired CO2, the material used in capnography with their artifacts and traps, will be reviewed. Since the main reluctance in the use of expired CO2 measurement is due to lack of correct knowledge concerning the physiopathology of CO2 by the physician, we hope that this explanation and the video sequences accompanying will help resolve this limitation.

Expired CO2 Measurement in Intubated or Spontaneously Breathing Patients from the Emergency Department

This video describes how to perform CO2 measurement in intubated as well as spontaneously breathing patients. The main clinical indications refer to emergency situations: (1) verifying adequate positioning of an endotracheal tube; (2) achieving normocapnia in trauma patients; (3) monitoring ventilation in the case of procedural sedation.

Carbon dioxide (CO2) along with oxygen (O2) share the role of being the most important gases in the human body. The measuring of expired CO2 at the mouth ...

Magnetic Resonance Imaging Quantification of Pulmonary Perfusion using Calibrated Arterial Spin Labeling

This demonstrates a MR imaging method to measure the spatial distribution of pulmonary blood flow in healthy subjects 
during normoxia (inspired O2, fraction (FIO2) = 0.21) hypoxia (FIO2 = 0.125), and hyperoxia 
(FIO2 = 1.00). In addition, the physiological responses of the subject are monitored in the MR scan environment. MR images 
were obtained on a 1.5 T GE MRI scanner during a breath hold from a sagittal slice in the right lung at functional residual capacity. An arterial 
spin labeling sequence (ASL-FAIRER) was used to measure the spatial distribution of pulmonary blood flow 1,2 and a multi-echo fast 
gradient echo (mGRE) sequence 3 was used to quantify the regional proton (i.e. H2O) density, allowing the quantification 
of density-normalized perfusion for each voxel (milliliters blood per minute per gram lung tissue). 
With a pneumatic switching valve and facemask equipped with a 2-way non-rebreathing valve, different oxygen concentrations 
were introduced to the subject in the MR scanner through the inspired gas tubing. A metabolic cart collected expiratory gas via expiratory tubing. Mixed expiratory O2 and CO2 concentrations, oxygen consumption, carbon dioxide production, respiratory exchange ratio, 
respiratory frequency and tidal volume were measured. Heart rate and oxygen saturation were monitored using pulse-oximetry. 
Data obtained from a normal subject showed that, as expected, heart rate was higher in hypoxia (60 bpm) than during normoxia (51) or hyperoxia (50) and the arterial oxygen saturation (SpO2) was reduced during hypoxia to 86%. Mean ventilation was 8.31 L/min BTPS during hypoxia, 7.04 L/min during normoxia, and 6.64 L/min during hyperoxia. Tidal volume was 0.76 L during hypoxia, 0.69 L during normoxia, and 0.67 L during hyperoxia. 
Representative quantified ASL data showed that the mean density normalized perfusion was 8.86 ml/min/g during hypoxia, 8.26 ml/min/g during normoxia and 8.46 ml/min/g during hyperoxia, respectively. In this subject, the relative dispersion4, an index of global heterogeneity, was increased in hypoxia (1.07 during hypoxia, 0.85 during normoxia, and 0.87 during hyperoxia) while the fractal dimension (Ds), another index of heterogeneity reflecting vascular branching structure, was unchanged (1.24 during hypoxia, 1.26 during normoxia, and 1.26 during hyperoxia). 
Overview. This protocol will demonstrate the acquisition of data to measure the distribution of pulmonary perfusion noninvasively under conditions of normoxia, hypoxia, and hyperoxia using a magnetic resonance imaging technique known as arterial spin labeling (ASL). 
Rationale: Measurement of pulmonary blood flow and lung proton density using MR technique offers high spatial resolution images which can be quantified and the ability to perform repeated measurements under several different physiological conditions. In human studies, PET, SPECT, and CT are commonly used as the alternative techniques. However, these techniques involve exposure to ionizing radiation, and thus are not suitable for repeated measurements in human subjects.

A MR imaging method to study the distribution of pulmonary blood flow under a variety of physiological conditions, in this case exposure to three different inspired oxygen concentrations: hypoxia, normoxia, and hyperoxia, is described. This technique utilizes human pulmonary physiology research techniques in an MR scanning environment.

This demonstrates a MR imaging method to measure the spatial distribution of pulmonary blood flow in healthy subjects 
during normoxia (inspired O2, ...

Method to Obtain Pattern of Breathing in Senescent Mice through Unrestrained Barometric Plethysmography

Unrestrained barometric plethysmography (UBP) is a method for quantifying the pattern of breathing in mice, where breathing frequency, tidal volume, and minute ventilation are routinely reported. Moreover, information can be collected regarding the neural output of breathing, including the existence of central apneas and augmented breaths. An important consideration for UBP is obtaining a breathing segment with a minimal impact of anxious or active behaviors, to elucidate the response to breathing challenges. Here, we present a protocol that allows for short, quiet baselines to be obtained in aged mice, comparable to waiting for longer bouts of quiet breathing. The use of shorter time segments is valuable, as some strains of mice may be increasingly excitable or anxious, and longer periods of quiet breathing may not be achieved within a reasonable timeframe. We placed 22 month-old mice in a UBP chamber and compared four 15 s quiet breathing segments between minutes 60&#8211;120 to a longer 10 min quiet breathing period that took&#160;2&#8211;3 h to acquire. We also obtained counts of central apneas and augmented breaths prior to the quiet breathing segments, following a 30 min familiarization period. We show that 10 min of quiet breathing is comparable to using a much shorter 15 s duration. Additionally, the time leading up to these 15 s quiet breathing segments can be used to gather data regarding apneas of central origin. This protocol allows investigators to collect pattern-of-breathing data in a set amount of time and makes quiet baseline measures feasible for mice that may exhibit increased amounts of excitable behavior. The UBP methodology itself provides a useful and noninvasive way to collect pattern-of-breathing data and allows for mice to be tested over several time points.

Unrestrained barometric plethysmography is used to quantify the pattern of breathing in awake mice. We show that 15 s segments under a standardized protocol display similar values to an extended duration of quiet breathing. This methodology also allows for the quantification of apnea and augmented breaths during the first hour in the chamber.

Unrestrained barometric plethysmography (UBP) is a method for quantifying the pattern of breathing in mice, where breathing frequency, tidal volume, and ...

Behavior

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

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 ...

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 ...

Biology

Evaluation of Capnography Sampling Line Compatibility and Accuracy when Used with a Portable Capnography Monitor

Capnography is commonly used to monitor patient&#8217;s ventilatory status. While sidestream capnography has been shown to provide a reliable assessment of end-tidal CO2 (ETCO2), its accuracy is commonly validated using commercial kits composed of a capnography monitor and its matching disposable nasal cannula sampling lines. The purpose of this study was to assess the compatibility and accuracy of cross-paired capnography sampling lines with a single portable bedside capnography monitor. A series of 4 bench tests were performed to evaluate the tensile strength, rise time, ETCO2 accuracy as a function of respiratory rate, and ETCO2 accuracy in the presence of supplemental O2. Each bench test was performed using specialized, validated equipment to allow for a full evaluation of sampling line performance. The 4 bench tests successfully differentiated between sampling lines from different commercial sources and suggested that due to increased rise time and decreased ETCO2 accuracy, not all nasal cannula sampling lines provide reliable clinical data when cross-paired with a commercial capnography monitor. Care should be taken to ensure that any cross-pairing of capnography monitors and disposable sampling lines is fully validated for use across respiratory rates and supplemental O2 flow rates commonly encountered in clinical settings.

The goal of this study was to evaluate the accuracy of capnography sampling lines used in conjunction with a portable bedside capnography monitor. Sampling lines from 7 manufacturers were evaluated for tensile strength, rise time, and ETCO2 accuracy as a function of respiratory rate or supplemental oxygen flow rate.

Capnography is commonly used to monitor patient&#8217;s ventilatory status. While sidestream capnography has been shown to provide a reliable assessment ...

Assessing Lung Function with Hyperpolarized Xenon-129 MRI

Quantitative Measure of Lung Structure and Function Obtained from Hyperpolarized Xenon Spectroscopy

Hyperpolarized Xenon-129 (HXe) magnetic resonance imaging (MRI) provides tools for obtaining 2- or 3-dimensional maps of lung ventilation patterns, gas diffusion, Xenon uptake by lung parenchyma, and other lung function metrics. However, by trading spatial for temporal resolution, it also enables tracing of pulmonary Xenon gas exchange on a ms timescale. This article describes one such technique, chemical shift saturation recovery (CSSR) MR spectroscopy. It illustrates how it can be used to assess capillary blood volume, septal wall thickness, and the surface-to-volume ratio in the alveoli. The flip angle of the applied radiofrequency pulses (RF) was carefully calibrated. Single-dose breath-hold and multi-dose free-breathing protocols were employed for administering the gas to the subject. Once the inhaled Xenon gas reached the alveoli, a series of 90&#176; RF pulses was applied to ensure maximum saturation of the accumulated Xenon magnetization in the lung parenchyma. Following a variable delay time, spectra were acquired to quantify the regrowth of the Xenon signal due to gas exchange between the alveolar gas volume and the tissue compartments of the lung. These spectra were then analyzed by fitting complex pseudo-Voigt functions to the three dominant peaks. Finally, the delay time-dependent peak amplitudes were fitted to a one-dimensional analytical gas-exchange model to extract physiological parameters.

Author Spotlight: Advancing Lung Disease Research with Free-Breathing Hyperpolarized Xenon-129 MRI

The manuscript presents a detailed protocol for using hyperpolarized Xenon-129 chemical shift saturation recovery (CSSR) to trace pulmonary gas exchange, assess the apparent alveolar septal wall thickness, and measure the surface-to-volume ratio. The method has the potential to diagnose and monitor lung diseases.

Hyperpolarized Xenon-129 (HXe) magnetic resonance imaging (MRI) provides tools for obtaining 2- or 3-dimensional maps of lung ventilation patterns, gas ...

Multi-modal Pulmonary Imaging: Using Complementary Information from CT and Hyperpolarized 129Xe MRI to Evaluate Lung Structure-Function

Hyperpolarized 129Xe gas MRI is an emerging technique to evaluate and measure regional lung function including pulmonary gas distribution and gas exchange. Chest computed tomography (CT) still remains the clinical gold standard for imaging of the lungs, though, in part due to the rapid CT protocols that acquire high-resolution images in seconds and the widespread availability of CT scanners. Quantitative approaches have enabled the extraction of structural lung parenchymal, airway and vascular measurements from chest CT that have been evaluated in many clinical research studies. Together, CT and 129Xe MRI provide complementary information that can be used to evaluate regional lung structure and function, resulting in new insights into lung health and disease. 129Xe MR-CT image registration can be performed to measure regional lung structure-function to better understand lung disease pathophysiology, and to perform image-guided pulmonary interventions. Here, a method for 129Xe MRI-CT registration is outlined to support implementation in research or clinical settings. Registration methods and applications that have been employed to date in the literature are also summarized, and suggestions are provided for future directions that may further overcome technical challenges related to 129Xe MR-CT image registration and facilitate broader implementation of regional lung structure-function evaluation.

Multi-modal Pulmonary Imaging: Using Complementary Information from CT and Hyperpolarized 129Xe MRI to Evaluate Lung Structure-Function

CT and 129Xe MRI provide complementary lung structure-function information that can be exploited for regional analysis using image registration. Here, we provide a protocol that builds from the existing literature for 129Xe MR to CT image registration using open-source platforms.

Hyperpolarized 129Xe gas MRI is an emerging technique to evaluate and measure regional lung function including pulmonary gas distribution and gas ...

Advanced Lung Imaging for Disease Detection and Monitoring

Phase-Resolved Functional Lung MRI for Pulmonary Ventilation and Perfusion (V/Q) Assessment

Fourier decomposition is a contrast agent-free 1H MRI method for lung perfusion (Q) and ventilation (V) assessment. After image registration, the time series of each voxel is analyzed with regard to the cardiac and breathing frequency components.
Using a standard 2D spoiled gradient-echo sequence with a temporal resolution of ~300 ms, an image-sorting algorithm was developed to produce phase-resolved functional lung imaging (PREFUL) with an increased temporal resolution. Thus, it is feasible to evaluate regional flow volume loops (FVL) during tidal volume breathing and depict the propagation of the pulse wave during the cardiac cycle. This method can be applied at 1.5T or 3T with standard MR hardware without the necessity for sequence programming, as the described protocol can be implemented with the default SPGRE sequence on most systems.
PREFUL ventilation MRI has been validated using 129Xe and 19F gas imaging with good regional agreement. Perfusion-weighted PREFUL MRI has been validated using SPECT as well as dynamic contrast enhanced (DCE) MRI. PREFUL has been tested in a dual center dual vendor setting and is currently applied in several ongoing multicenter trials. Furthermore, it is feasible across a range of field strengths (0.55T-3T) and different age groups, including newborns.
Quantitative V/Q PREFUL MRI has been used in patients with cystic fibrosis, chronic obstructive pulmonary disease, chronic thromboembolic pulmonary hypertension, and corona virus disease-2019 to quantify disease and monitor treatment change after therapy. Furthermore, PREFUL V/Q imaging has been shown to predict transplant loss due to chronic lung allograft dysfunction in patients after lung transplantation. In summary, PREFUL MRI is a validated technique for quantitative ventilation and pulmonary pulse wave/perfusion imaging for regional pulmonary disease detection, quantification, and treatment monitoring with potential added value to the current clinical routine.

Author Spotlight: Enhancing Diagnostic Strategies and Biomarker Development for Comprehensive Lung Function Analysis

Here, we describe the implementation of phase-resolved functional lung MRI as a contrast-agent-free proton MR technique for the assessment of pulmonary ventilation and perfusion dynamics. Validated and applicable across different field strengths and age groups, it could enhance clinical decision-making in the future by aiding in disease quantification and therapy monitoring.

Fourier decomposition is a contrast agent-free 1H MRI method for lung perfusion (Q) and ventilation (V) assessment. After image registration, the time ...

Combining Volumetric Capnography And Barometric Plethysmography To Measure The Lung Structure-function Relationship

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