These studies were supported by the National Institutes of Health COBRE grant P20GM125504-01 Sub-Project 8246.

The procedures described here were reviewed and approved by the University of Louisville Institutional Biosafety Committee (protocol # 14-038) and Institutional Animal Care and Use Committee (protocol # 19567).
1. Assembly of the sampling chamber
<ol>
	<li>Create two holes using a 3/4&#34; diamond drill bit on a drill press on the flat surfaces of a 600 mL square glass wide mouth Mason canning jar with 95 mm gasket and airtight lids, with bail and trigger clamp lids (Figure 1). 
	NOTE: A sampling chamber is not commercially available and must be constructed.</li>
	<li>Assemble a brass bulkhead (1/4&#34; NPT internal thread, 3/4-16 UNF external thread) through both the holes in the Mason jar using rubber washers (3/4&#34; inner diameter, 1&#34; outer diameter) on both faces of contact between the bulkhead and glass to ensure an airtight seal.</li>
	<li>Use one bulkhead assembly for a pressure sensor while attaching the other bulkhead by a Luer-connected syringe for calibration purposes.
	<ol>
		<li>For the pressure sensor, wrap the 1/4&#34; NPT threads of a high-performance gauge pressure transducer with Teflon tape and thread them into the bulkhead. Use a soldering iron to connect the pressure sensor wiring to an 8-pin male DIN connector, using the manufacturer wiring instructions for interface with a commercially available high-quality data acquisition device (see Table of Materials). 
		NOTE: This requires the use of a 150 K ohm 1/8 watt 1% metal film resistor within the DIN connector wiring.</li>
		<li>For the calibration port, use a 1/4&#34; male NPT to 1/8&#34; female NPT adaptor to connect a 1/8&#34; male NPT to a female Luer lock nickel-plated connector to the brass bulkhead wrapping threaded connections with Teflon tape. Use a polypropylene threaded male Luer cap to seal the Luer connector when not in use. 
		&#8203;NOTE: Do not overtighten the bulkhead connectors onto the glass jar, as this will develop cracks. If desired, silicone can be added to the rubber gaskets to ensure an airtight seal of the bulkheads to the glass jar.</li>
	</ol>
	</li>
</ol>
2. System setup
<ol>
	<li>Connect the sampling chamber to a bridge amplifier using an 8-pin DIN connector and the bridge amplifier to the data acquisition device, following the manufacturer&#39;s instructions.</li>
	<li>Connect the data acquisition device to a power supply and a computer running physiological data analysis software using the manufacturer&#39;s cables. 
	NOTE: Ensure that the data acquisition device is powered on and warmed up for at least 5 min before use to ensure that the sensor stabilizes its measurements.</li>
	<li>Initiate the software to interface with the data acquisition system.</li>
	<li>Download the optional Spirometry Module within the software, and modify the default unit settings from L/s to &#181;L/s in the Spirometry &#62; Settings window.</li>
</ol>
3. System calibration
<ol>
	<li>Within the software, create a 4-Channel window with the following data windows: Channel 1: Source data at 4 k/s sample rate and 1 mV range; Channel 2: Digital filter of Channel 1 using a high pass 1 Hz auto adjust filter; Channel 3: Smoothing of Channel 2 data by averaging of 100 samples; Channel 4: Spirometry Flow of Channel 3 data (Custom Flowhead, calibrated to Formula (&#181;L/s) = 120,000 x Voltage). 
	NOTE: 120,000 is a placeholder correlation coefficient that will be modified during calibration.</li>
	<li>Set up DataPad analysis of Channel 4 with the following Columns: Column 1: Channel 4 Data, Comments &#62; Full Comment Text; Column 2: Channel 4 data, Cyclic Measurements &#62; Average Cyclic Frequency; Column 3: Channel 4 data, Cyclic Measurements &#62; Average Cyclic Height.</li>
	<li>Set the frame rate to 100:1 on the bottom-right corner of the chart display. Save this window configuration as a template for all future studies.</li>
	<li>Close the Sample Chamber lid and attach a 25 &#181;L Gas Tight syringe to the Luer bulkhead connector. Fit the syringe with a Chaney adaptor set to repeatedly deliver a volume of 20 &#181;L. 
	NOTE: An optional short piece of 1/16&#34; tubing and Luer/barb connectors may be used to connect the syringe to Sample Chamber. However, long tubing should be avoided to avoid significant changes to the total air volume of the Sample Chamber.</li>
	<li>Draw 20 &#181;L of air into the syringe using the depth stop of the Chaney adaptor.</li>
	<li>Zero the pleth in the software (Setup &#62; Zero All Inputs (Alt-Z)) and start a recording.</li>
	<li>While recording, and with a stable baseline, rapidly depress/withdraw the Syringe plunger for approximately 10 repetitions to replicate subject breathing with a measured 20 &#181;L breath. Stop the recording. 
	NOTE: The frequency of the artificial breaths should exceed 2 Hz to maximize the reproducibility of the calibration.</li>
	<li>Label the identity of the measured sample by right-clicking the beginning of numbered pleth recording and click on Add Comment.</li>
	<li>Reset the Syringe, Zero the input, and repeat recording measurements of 20 &#181;L pulses two additional times (three total recording sessions).</li>
	<li>After completing all the measurements, use the computer mouse to select a portion of breathing pleth that accurately represents the artificial 20 &#181;L breaths. 
	NOTE: Within the DataPad module, data will appear in the preview header providing a temporary readout of the breath rate (Average Cyclic Frequency, Hz) and breath depth (Average Cyclic Height, &#181;L). The data preview can be recorded into DataPad using the Add to DataPad icon.</li>
	<li>Review the Column 3 data (Average Cyclic Height), and calculate the average measured breath volume from the three recordings. Perform the following calculation of the average measured breath volume: Calibration coefficient = delivered volume / measured volume x 120,000. 
	&#8203;NOTE: 120,000 was the placeholder Flow Head Calibration coefficient used in step 3.1 now modified from measured data. The system is now calibrated to typical mouse breathing using current environmental temperature and humidity. The system can now monitor the subject breathing, and calibration can be re-performed daily to account for any fluctuation in temperature/humidity.</li>
</ol>
4. Subject monitoring
<ol>
	<li>Open a master template as outlined in step 3.4, or complete steps 4.2 to 4.3.</li>
	<li>Within the software, create a 4-Channel window with the following data processing: Channel 1: Source data at 4 k/s sample rate and 1 mV range; Channel 2: Digital filter of Channel 1 using a High Pass 1 Hz Auto Adjust filter; Channel 3: Smoothing of Channel 2 data by Averaging of 100 Samples; Channel 4: Spirometry Flow of Channel 3 data (Custom Flowhead, calibrated to Formula (&#181;L/s) = 120,000 x Voltage). 
	NOTE: 120,000 is the correlation coefficient calculated for the current pressure sensor; however, the user should perform the system calibration described in step 3 and use this user-defined correlation coefficient instead.</li>
	<li>Set up DataPad analysis of Channel 4 with the following Columns: Column 1: Channel 4 Data, Comments &#62; Full Comment Text; Column 2: Channel 4 data, Cyclic Measurements &#62; Average Cyclic Frequency; Column 3: Channel 4 data, Cyclic Measurements &#62; Average Cyclic Height.</li>
	<li>Place subject in sampling chamber and close lid. For this experiment a conscious 4-12 week female albino C57BL/6J mouse (B6(Cg)-Tyrc-2J/J) was used.</li>
	<li>Equalize the atmospheric pressure in the chamber (from sealing the lid) by briefly loosening the Luer bulkhead cap and retighten.</li>
	<li>Observe that the subject is not actively moving within the sampling chamber before Zeroing All Inputs (Alt-Z shortcut) and beginning a recording. 
	NOTE: If the subject begins to move in the sampling chamber, the baseline may move off the scale, which may be addressed by re-zeroing all inputs in the middle of a recording, which will create a fresh recording on the scale. Suppose the subject engages in exploration or grooming during the recording; note which portion of the plethysmography recording most accurately reflects normal breathing.</li>
	<li>Label the subject&#39;s identity by right-clicking the beginning of numbered pleth recording and click on Add Comment.</li>
	<li>Return the subject to its cage. Limit the time spent in the sealed sampling chamber to 5 min to avoid asphyxiation and stress. 
	NOTE: The risk for asphyxiation is low given that the 600 mL air volume of the Sample Chamber will not be rapidly spent by a healthy mouse breathing at &#60;15 mL/min.</li>
	<li>Use the computer mouse to select a portion of breathing pleth that accurately represents the subject breathing. 
	NOTE: Within the DataPad module, data will appear in the preview header providing a temporary readout of the breath rate (Average Cyclic Frequency, Hz) and breath volume (Average Cyclic Height, &#181;L). The data preview can be recorded into DataPad using the Add to DataPad icon.</li>
	<li>Continue measuring subject mice one at a time and recording representative sections of breathing pleth to DataPad.</li>
	<li>After data recording, export DataPad data to Excel. Calculate the minute volume as follows: Minute Volume (mL/min) = Breath Rate (Hz) x Breath Volume (&#181;l) x 0.06.</li>
</ol>

<ol>
	<li>Warawa, J. M., Long, D., Rosenke, R., Gardner, D., Gherardini, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+for+the+Burkholderia+pseudomallei+capsular+polysaccharide+encoded+by+the+wcb+operon+in+acute+disseminated+melioidosis.">Role for the Burkholderia pseudomallei capsular polysaccharide encoded by the wcb operon in acute disseminated melioidosis.</a> Infection and Immunity. 77 (12), 5252-5261 (2009).</li><li>West, T. E., Myers, N. D., Liggitt, H. D., Skerrett, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+pulmonary+infection+and+inflammation+induced+by+inhalation+of+Burkholderia+pseudomallei.">Murine pulmonary infection and inflammation induced by inhalation of Burkholderia pseudomallei.</a> International Journal of Experimental Pathology. 93 (6), 421-428 (2012).</li><li>Lawrenz, M. B., 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=Development+and+evaluation+of+murine+lung-specific+disease+models+for+Pseudomonas+aeruginosa+applicable+to+therapeutic+testing.">Development and evaluation of murine lung-specific disease models for Pseudomonas aeruginosa applicable to therapeutic testing.</a> Pathogens and Disease. 73 (5), (2015).</li><li>Lim, R., 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=Measuring+respiratory+function+in+mice+using+unrestrained+whole-body+plethysmography.">Measuring respiratory function in mice using unrestrained whole-body plethysmography.</a> Journal of Visualized Experiments: JoVE. (90), e51755 (2014).</li><li>Drorbaugh, J. E., Fenn, W. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+barometric+method+for+measuring+ventilation+in+newborn+infants.">A barometric method for measuring ventilation in newborn infants.</a> Pediatrics. 16 (1), 81-87 (1955).</li><li>Simon, G., Pride, N. B., Jones, N. L., Raimondi, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relation+between+abnormalities+in+the+chest+radiograph+and+changes+in+pulmonary+function+in+chronic+bronchitis+and+emphysema.">Relation between abnormalities in the chest radiograph and changes in pulmonary function in chronic bronchitis and emphysema.</a> Thorax. 28 (1), 15-23 (1973).</li><li>Gassiep, I., Armstrong, M., Norton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+melioidosis.">Human melioidosis.</a> Clinical Microbiology Reviews. 33 (2), 06-19 (2020).</li><li>Gutierrez, M. G., Warawa, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuation+of+a+select+agent-excluded+Burkholderia+pseudomallei+capsule+mutant+in+hamsters.">Attenuation of a select agent-excluded Burkholderia pseudomallei capsule mutant in hamsters.</a> Acta Tropica. 157, 68-72 (2016).</li><li>Gutierrez, M. G., Pfeffer, T. L., Warawa, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+3+secretion+system+cluster+3+is+a+critical+virulence+determinant+for+lung-specific+melioidosis.">Type 3 secretion system cluster 3 is a critical virulence determinant for lung-specific melioidosis.</a> PLoS Neglected Tropical Diseases. 9 (1), 3441 (2015).</li><li>Rocco, P. R. M., Zin, W. A., Gullo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anaesthesia, Pain, Intensive Care and Emergency Medicine. , (2002).</li><li>Fodah, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+Klebsiella+pneumoniae+comparative+genetic+analyses+with+virulence+profiles+in+a+murine+respiratory+disease+model.">Correlation of Klebsiella pneumoniae comparative genetic analyses with virulence profiles in a murine respiratory disease model.</a> PLoS One. 9 (9), 107394 (2014).</li><li>Gotts, J. E., 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=Clinically+relevant+model+of+pneumococcal+pneumonia,+ARDS,+and+nonpulmonary+organ+dysfunction+in+mice.">Clinically relevant model of pneumococcal pneumonia, ARDS, and nonpulmonary organ dysfunction in mice.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 317 (5), 717-736 (2019).</li><li>Galan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+themes+in+the+design+and+function+of+bacterial+effectors.">Common themes in the design and function of bacterial effectors.</a> Cell Host &amp; Microbe. 5 (6), 571-579 (2009).</li></ol>

The authors have no conflicts of interest.

sWBP is an attractive approach for enhancing the understanding of respiratory infection in small animal models. Importantly, it is a non-invasive approach, and as such, it does not pose a significant risk of causing undue stress to research animals during an infection challenge. Indeed, the procedure of monitoring subject breathing is a rapid test requiring several minutes and minimal subject handling. The scientific benefit is the high-resolution understanding of how microbial pathogens influence lung function during disease. This approach will provide benefit to basic research, facilitating the understanding of how a pathogen causes disease, as well as providing a translational utility to understand how a novel therapeutic restores a research subject to a state of respiratory health.
In this manuscript, representative results are provided for the pathogen B. pseudomallei, which causes an early lethargic response. Not all bacterial lung infections present in the same manner in mouse infection models. Previous experience with other infection models has demonstrated that the bacterial pathogen Klebsiella pneumoniae presents as an asymptomatic infection up to the point at which mice succumb to infection, also at approximately Day 3 post-infection11. It is hypothesized that the host demand for inspired air (i.e., minute volume) may closely relate to the degree of lethargy with which a given disease presents. Future studies will be required to examine how different bacterial pathogens affect lung function during respiratory disease. It is understood that different pathogens have unique approaches to evade host defense, including differences in, (1) propensity to be intracellular or extracellular pathogens, (2) the ability to cause early/late hypothermic response, and (3) use of different repertoires of virulence determinants3,12,13. Therefore, it is likely that different disease strategies will result in unique effects on lung function and breathing during infection.
The recommended settings described in this protocol can be modified to accommodate unique challenges present during sWBP. One of the common issues experienced during an sWBP recording session is the subject's movement within the Sample Chamber. As mentioned, this movement modifies the baseline and can affect the accuracy of breathing measurements. A Digital filter was used to normalize the shifting baseline, allowing viable breath measurements despite small movements. Excessive movement can push a baseline measurement out of the range of a zeroed input. Recordings are recommended at 1 mV range (Channel 1 setting), which provides a compromise of still observing the peaks of the plethysmography while avoiding loss of data outside of range. For exceptionally active subjects, it may be necessary to extend the recording range &gt;1 mV to avoid persistent out of range signals.
The recommended procedure calls for daily calibration (or at each session) to accommodate environmental humidity/temperature fluctuations. Traditional WBP uses complex calculations that factor in the temperature/humidity of both environment and subject5,6. It has been demonstrated that in the present sWBP apparatus, the effects of the host temperature/humidity do not significantly alter the measured breath volume of a calibration source. Therefore, this approach in sWBP fundamentally differs from the &gt;50-year-old approach of Drorbaugh and Fenn. Here, sWBP directly relates pressure changes to a measured breath volume with no further correction from the host.
It is essential to contrast research animal WBP to that of clinical WBP. The types of biometric data that were attempted to collect by sWBP are breath volume and frequency. Such measurements are collected clinically using simple spirometry equipment in which a patient holds a breath monitor to their mouth and breaths normally into a device monitoring airflow. Similar spirometry in research animals requires restraint, thus contributing to stress and an inherent disruption in breathing. Therefore, simple spirometry is functional clinically but not for research animals. WBP serves an essential purpose in the clinic to collect advanced data, including such measurements as residual lung volume. Such data can only be contained in the context of a subject being able to follow instructions on how they breathe, including Forced Expiration (emptying of their lung by a deep exhale). Research animals cannot be relied upon to follow breathing instructions from a researcher. Many of the advanced measurements collected clinically during WBP cannot be reproduced in research animals. WBP in research animals is fundamentally different from clinical WBP. Animal WBP seeks to collect simple ventilation data (breath rate and volume) in a non-restrained fashion to avoid animal stress and breathing perturbation. Thus far, the use of WBP in research animals appears to replicate the techniques used in clinical WBP, including complex calculations based on environmental and subject temperature and humidity, but without the ability to collect the advanced data from a subject that can follow instructions on how to perform a forced expiration. With this in mind, it was sought to demonstrate whether a simplified version of WBP would suffice to collect the pertinent breathing frequency and volume relevant to respiratory disease studies. A calibration session was employed, which compensated for any variation in environmental temperature and humidity. Further, it was demonstrated with an artificial mouse that subject temperature, and humidity to a measured breath volume have no significant effect on accurately measuring breath volume. It was concluded that sWBP has excellent application to research animal studies, without the requirement of the user to employ cumbersome mathematical treatment of data.

Respiratory bacterial pathogens are often associated with an inflammatory response in the lung leading to lung pathology1,2. In the clinical setting, diagnosis of pneumonia typically includes culture techniques from sputum, blood-oxygen saturation analysis, and chest X-ray. These techniques can be translated for small animal infection models, but only oxygen saturation analysis represents a rapid, real-time analysis in mice for disease severity. The blood oxygen saturation (SpO2) was previously investigated as a method to track disease progression in respiratory disease studies; however, moribund mice have unexpectedly high SpO2 readings both in a Pseudomonas aeruginosa model3, which are not the predictive or moribund disease, likely because mice can modulate their physiological activity. To this end, diagnostic levels of SpO2 were not found for bacterial respiratory disease in mice thus far.
Therefore, this work investigated the use of other clinically relevant methods of detecting the effects of lung disease on lung function as a rapid physiologic measurement. Simplified Whole Body Plethysmography (sWBP) offers the opportunity to investigate breath rate and depth as a rapid, non-invasive biometric analysis. Previous studies have demonstrated how to assemble WBP apparatus in a laboratory4; however, several of the components shown in such studies are not currently commercially available. Further, traditional WBP requires complex data collection and data processing based on humidity and temperature5,6. Hence, it was decided to develop a simplified WBP apparatus that is calibrated daily to room temperature/humidity and assess whether or not the temperature/humidity contribution of the subject itself has any effect on the measured breath volume. Thus, a modified sWBP apparatus has been created which sources the currently available materials. Further, it has been investigated whether this lab-sourced apparatus can detect changes in breathing associated with disease progression during the model of lethal respiratory melioidosis in mice.
The sWBP apparatus constructed for this work used commercially available equipment and software to process analog pressure sensor data into a digital readout. The pressure sensor was fitted to an airtight glass jar with bulkhead connectors. The benefit of a glass jar is the structural rigidity of the material, which will resist changes to the internal pressure of the jar, affecting measurements of volume changes during the monitoring of breathing. The sampling chamber has been engineered to have two ports on the two flat surfaces of the square jar, one to access the chamber by a Luer connector for calibration and the other to house the pressure sensor. The pressure sensor selected has a highly sensitive gauge pressure transducer with a range for small changes in pressure (25 mbar range).
This protocol is demonstrated using a murine model of respiratory melioidosis. Burkholderia pseudomallei (Bp) is the bacterial agent of melioidosis - a disease associated with tropical regions of the world7. Bp is found in the environment, specifically in wet environments of standing water and moist soil, from which it typically causes subcutaneous infections of cuts/scratches of susceptible hosts. However, Bp is also infectious when inhaled and is a potential threat for use in bioterrorism by aerosol dispersal. While fully virulent Bp requires handling in a BSL-3 laboratory, an acapsular mutant strain was previously engineered, which can be safely handled at BSL-2 and excluded from the select agent criteria8. Further, an intubation-mediated intratracheal (IMIT) infection model of respiratory melioidosis has been developed to study respiratory disease progression of Bp5,9. We have used this infection model to characterize the change in breathing that occurs during disease progression through the moribund endpoint.

<table><tbody><tr><td>1/8&quot; NPT Luer adaptor</td><td>Amazon</td><td>B07DH9MY8W</td><td>Calibration port</td></tr><tr><td>1/8&quot; NPT to 1/4&quot; NPT adaptor</td><td>Amazon</td><td>B07T6CR6FS</td><td>Bulkhead to luer adaptor</td></tr><tr><td>150 kohm resistor</td><td>Amazon</td><td>B07GPRYL81</td><td>Pressure transducer excitation voltage selection</td></tr><tr><td>3/4&quot; diamond drill bit</td><td>Drilax</td><td>DRILAX100425</td><td>To drill bulkhead mounts in glass jar</td></tr><tr><td>Bridge Amp</td><td>AD Instruments</td><td>FE221</td><td>One channel option</td></tr><tr><td>Bulkhead fitting</td><td>Legines</td><td>3000L-B</td><td>1/4&quot; NPT, 3/4-16 UNF brass bulkhead coupling</td></tr><tr><td>Chaney adaptor</td><td>Hamilton</td><td>14725</td><td>Gas tight syringe adaptor for set volume</td></tr><tr><td>DIN connector</td><td>AD Instruments</td><td>SP0104</td><td>To connect pressure sensor to Bridge Amp</td></tr><tr><td>Gastight syringe, 25 uL</td><td>Hamilton</td><td>80201</td><td>Calibration syringe</td></tr><tr><td>LabChart</td><td>AD Instruments</td><td></td><td>Life Science Data Acquisition Software</td></tr><tr><td>Luer plug</td><td>Cole Parmer</td><td>45513-56</td><td>Calibration port closure</td></tr><tr><td>PowerLab 4/26</td><td>AD Instruments</td><td>PL2604</td><td>Digital interface to computer</td></tr><tr><td>Pressure transducer</td><td>Omega Engineering</td><td>PX409-10WGV</td><td>High accuracy oil filed gage pressure sensor</td></tr><tr><td>Rubber gasket</td><td>Amazon</td><td>B07LH4C8LS</td><td>To mount bulkheads (4 required per chamber)</td></tr><tr><td>Square glass jar</td><td>Amazon</td><td>B07VNSPR8P</td><td>600 ml with 95 mm silicone gasket</td></tr></tbody></table>

simplified whole body plethysmography to characterize lung function during respiratory melioidosis

Surrogate animal models of disease are subject to the 3Rs of Responsible Research. There is a frequent revisiting of refinements to animal models to ensure that both animal welfare and scientific insights advance with the availability of new technologies. This article demonstrates the use of Simplified Whole Body Plethysmography (sWBP) to non-invasively study respiratory failure in a model of lethal respiratory melioidosis. sWBP has the sensitivity to detect breathing in mice through the entirety of the course of the disease, allowing for the moribund-associated symptoms (bradypnea and hypopnea) to be measured and potentially used to develop humane endpoint criteria.
Some of the benefits of sWBP in the context of respiratory disease are that host breath monitoring comes the closest of any physiologic measurement at assessing dysfunction of the primary infected tissue, namely, the lung. In addition to biological significance, the use of sWBP is rapid and non-invasive, minimizing stress in research animals. This work demonstrates the use of in-house sWBP apparatus to monitor disease throughout the course of respiratory failure in the murine model of respiratory melioidosis.

System calibration 
The data analysis software allows for direct calibration of a custom flowhead, such as the one described herein. This is performed when setting the Spirometry Flow. As described in step 3.1, an option exists to input the known calibration air volume, which calculates the voltage to volume correlation coefficient within the system. This, however, generates a correlation coefficient based on a single read, and it has been observed that the inherent variation of calibration from an n = 1 standard has poor utility. The current approach can address this shortcoming and allows a user to perform a daily calibration using multiple readings averaged to calculate a calibration coefficient. Calibration with 20 &#181;L of injected air was demonstrated herein, representing a typical high-end breath volume in a typical mouse. The software assumes an origin intercept (0,0) and is thus calibrated from 0-20 &#181;L using this approach.
The methodology proposed here for sWBP calibrates daily, thus accounting for any fluctuations in environmental humidity/temperature. The original methods used for specific WBP, date back to the methodology of Drorbaugh and Fenn from 1955, who developed WBP for measuring ventilation in human infants5. The Drorbaugh and Fenn calculations account for variations in temperature and humidity of the environment and the subject. The current approach corrects for environmental fluctuations by calibrating each sWBP session. Still, it was decided to address whether the heating and humidification of breathing across the nasal cavity/lung of a mouse affect the measurement of a known volume of air. Thus, an artificial apparatus was created to mimic the subject&#39;s effect on heating and humidifying calibrated air measurements. Luer connectors were attached to a 15 mL conical tube and placed this sealed conical in-line between the sample chamber and the gas-tight calibration syringe. A 20 &#181;L calibration was performed using an empty conical tube held at room temperature (23 &#176;C). The conical tube was then partially filled with distilled water to just below the Luer connectors, allowing time to equilibrate the headspace of the conical; the calibration volume was then re-measured to investigate the effect of humidity. The conical tube was placed in a heating block and equilibrated at 37 &#176;C in a humid environment, and finally equilibrated to 37 &#176;C with no water to assess the effect of subject heating and with no additional contribution of humidity. Figure 2 demonstrates that all conditions tested did not significantly impact the calibrated 20 &#181;L measurement delivered by the gas-tight syringe. From this finding, it was concluded that sWBP offers an accessible approach to monitor breathing in research animals without the need for complex calculations predicated on the temperature and humidity of the animal subject, as these make no significant impact on measured breath volume.
Subject monitoring 
sWBP was used to monitor breathing during the disease of lethal respiratory infections with the bacterial pathogen B. pseudomallei. One challenge of monitoring breathing in conscious animals is the curiosity of normal healthy animals moving within the sample chamber. The movement of the mouse creates a constantly moving baseline that can be mitigated in part by pre-conditioning subjects to the chamber over a period of several days prior to measurement.&#160;This issue primarily affects the baseline measurement in healthy mice, as the subjects become lethargic during infection, making sWBP much more manageable with reduced subject activity. It may be tempting to attempt to use some form of restraint, whether physical or anesthesia. The use of physical restraint may affect natural breathing by causing stress. Further, the use of anesthetics is known to have pronounced effects on breath rate and depth10; thus, it was decided to investigate the impact of anesthesia with the in-house sWBP apparatus. Isoflurane is commonly used to perform in vivo diagnostic imaging during the infection models, and therefore, a C57BL/6 mouse was anesthetized and monitored progression until recovery out of anesthesia using sWBP. This trial was conducted with a juvenile 4-week old albino C57BL/6J mouse to prolong the window of recovery from anesthesia. Figure 3 demonstrates that the preferred anesthetic causes mice to exhibit a slow breath rate with a large tidal volume of air. As mice begin to recover from sedation, their breath rate increases and breath volume decreases, with the net effect that total inspired air slowly increases. In this trial, it was found that the breath volume is restored to pre-anesthesia levels within the first 30 s of recovery. The breath rate steadily increases until the baseline breathing is restored to 2-2.5 min post-removal from anesthesia. The minute volume closely followed the effects of breath rate, reaching baseline minute volume by 2.5 min post-removal from anesthesia. This finding supports that anesthesia should not be employed in the sWBP approach. It dramatically affects baseline breathing, not surprisingly, as anesthesia will slow the host metabolism, creating a reduced demand for inspired oxygen.&#160;Sanitation of the sample chamber should also be considered between subjects to address study-specific infection control as well as the impact of pheromones from urine or feces which could impact the stress between subjects.
WBP is an attractive strategy for monitoring lung function in respiratory disease models in a non-invasive manner. sWBP was used to study how breathing changes during lethal respiratory melioidosis infections (Figure 4), with time points mirroring bioluminescence monitoring in the lung. It was observed that this model is associated with an early onset of lethargy, which persists in a slowly progressive manner until the development of the moribund disease at approximately 3 days post-infection. It was also observed that the breath rate and total inspired air (minute volume) of the mice decrease rapidly during the first day of infection and remain low for the remainder of the course of infection (Figure 4A,C). This pattern is consistent with the early onset lethargy, which persists for the next 2 days of the infection. In contrast, the breath volume does not steeply drop during the first 24 h and instead has a slight and steady decrease, which approaches a linear decline over the 3-day course of the disease (Figure 4B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62722/62722fig01.jpg" /> 
Figure 1: sWBP apparatus. A custom Sample Chamber was constructed from a sealable square glass jar with bulkhead connectors on two flat faces. One bulkhead was used to mount a gauge pressure sensor connected to a bridge amplifier and data acquisition device digitizer via an 8-pin DIN connection. The second bulkhead was fitted with a Luer connector for calibration by a gas-tight syringe. The device was connected to a PC running the software. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62722/62722fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62722/62722fig02.jpg" /> 
Figure 2: Effect of subject temperature and humidity on breath volume. A 15 mL conical tube with Luer connectors was installed in-line between the 20 &#181;L calibration syringe and the Sample Chamber. The system was calibrated to 20 &#181;L with no additional temperature/humidity contribution from the conical tube. Other measurements were collected post-equilibration with saturated humidity from distilled water and/or warming of the conical tube from room temperature (23 &#176;C) to body temperature (37 &#176;C). No significant difference was detected from n = 5 measurements of each condition by One-way ANOVA with Tukey&#39;s Multiple Comparison post-test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62722/62722fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62722/62722fig03.jpg" /> 
Figure 3: Effect of gas anesthesia on breathing in mice. Representative data from a 4-week old female albino C57BL/6J mouse (8.6 g) was sedated for 5 min with 3% isoflurane in oxygen and transferred to an sWBP Sample Chamber. Pleth data was collected for 150 s following removal from anesthesia. The subject began initial ambulation by 100 s following removal from anesthesia. (A) Baseline breathing before anesthesia, measuring a 4.97 Hz breath rate, 9.74 &#181;L breath volume, and 2.91 mL minute volume. (B) The first 60 s of changes to breathing during recovery from anesthesia. (A-B) Vertical axis measuring &#181;L per breath and horizontal axis in seconds. (C-E) Ventilation data was collected during the 150 s of recovery from anesthesia, averaged from &#8805;3 breath cycles per time point for (C) Breath Rate, (D) Breath Volume, and the (E) calculated Minute Volume. The pre-anesthesia baseline values are indicated with a horizontal dotted line in each respective graph. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62722/62722fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62722/62722fig04.jpg" /> 
Figure 4: Effect of respiratory melioidosis on host breathing. Five 8-week female C57BL/6 mice were infected with 4.9 log CFU of bioluminescent B. pseudomallei strain JW270. sWBP was conducted throughout the 3-day course of infection, measuring Breath Rate (A) and Breath Volume (B). The total inspired air was calculated as the Minute Volume (C). Data for each of the five subjects are independently plotted with third-order polynomial regression. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62722/62722fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Simplified Whole Body Plethysmography to Characterize Lung Function During Respiratory Melioidosis at JoVE.com

Simplified Whole Body Plethysmography to Characterize Lung Function During Respiratory Melioidosis

This protocol presents the construction and use of a Simplified Whole Body Plethysmography apparatus to monitor bacterial respiratory disease progression non-invasively.

Surrogate animal models of disease are subject to the 3Rs of Responsible Research. There is a frequent revisiting of refinements to animal models to ...

simplified-whole-body-plethysmography-to-characterize-lung-function

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

3.1K Views.  University of Louisville. This protocol presents the construction and use of a Simplified Whole Body Plethysmography apparatus to monitor bacterial respiratory disease progression non-invasively.

Video: Simplified Whole Body Plethysmography to Characterize Lung Function During Respiratory Melioidosis

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, ...

Medicine

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 ...

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 ...

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.

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 ...

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

Employing the Forced Oscillation Technique for the Assessment of Respiratory Mechanics in Adults

There is increasing interest in the use of the forced oscillation technique (FOT) or oscillometry to characterize respiratory mechanics in healthy and diseased individuals. FOT, a complementary method to traditional pulmonary function testing, utilizes a range of oscillatory frequencies superimposed on tidal breathing to measure the functional relationship between airway pressure and flow. This passive assessment provides an estimate of respiratory system resistance (Rrs) and reactance (Xrs) that reflect airway caliber and energy storage and dissipation, respectively. Despite the recent increase in popularity and updated Technical Standards, clinical adoption has been slow which relates, in part, to the lack of standardization regarding the acquisition and reporting of FOT data. The goal of this article is to address the lack of standardization across laboratories by providing a comprehensive written protocol for FOT and an accompanying video. To illustrate that this protocol can be utilized irrespective of a particular device, three separate FOT devices have been employed in the case examples and video demonstration. This effort is intended to standardize the use and interpretation of FOT, provide practical suggestions, as well as highlight future questions that need to be addressed.

As the use of forced oscillation technique (FOT) is increasingly utilized to characterize respiratory mechanics, there is a need to standardize methods with respect to nascent technical guidelines and various manufacturer's recommendations. A detailed protocol is provided including FOT assessment and interpretation for two cases to facilitate the standardization of methods.

There is increasing interest in the use of the forced oscillation technique (FOT) or oscillometry to characterize respiratory mechanics in healthy and ...

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 ...

Assessing Pulmonary Alveolar-Capillary Reserve During Exercise

Dual Test Gas Pulmonary Diffusing Capacity Measurement During Exercise in Humans Using the Single-Breath Method

The combined single-breath measurement of the diffusing capacity of carbon monoxide (DL,CO) and nitric oxide (DL,NO) is a useful technique to measure pulmonary alveolar-capillary reserve in both healthy and patient populations. The measurement provides an estimate of the participant&#39;s ability to recruit and distend pulmonary capillaries. The method has recently been reported to exhibit a high test-retest reliability in healthy volunteers during exercise of light to moderate intensity. Of note, this technique permits up to 12 repeated maneuvers and only requires a single breath with a relatively short breath-hold time of 5 s. Representative data are provided showing the gradual changes in DL,NO and DL,CO from rest to exercise at increasing intensities&#160;of up to 60% of maximal workload. The measurement of diffusing capacity and evaluation of alveolar-capillary reserve is a useful tool to evaluate the lung&#39;s ability to respond to exercise both in the healthy population as well as in patient populations such as those with chronic lung disease.

Author Spotlight: Integrating Alveolar-Capillary Reserve Measurements in Exercise Adaptation and Therapeutic Strategies

This protocol presents a method to assess pulmonary alveolar-capillary reserve measured by combined single-breath measurement of the diffusing capacity to carbon monoxide (DL,CO) and nitric oxide (DL,NO) during exercise. Assumptions and recommendations for using the technique during exercise form the foundation of this article.

The combined single-breath measurement of the diffusing capacity of carbon monoxide (DL,CO) and nitric oxide (DL,NO) is a useful technique to measure ...

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 ...

Dynamic Lung Ventilation Assessment with 3D Phase Resolved Functional Lung MRI

Three-Dimensional Phase Resolved Functional Lung Magnetic Resonance Imaging

Pulmonary magnetic resonance imaging (MRI) offers a variety of radiation-free techniques tailored to assess regional lung ventilation or its surrogates. These techniques encompass direct measurements, exemplified by hyperpolarized gas MRI and fluorinated gas MRI, as well as indirect measurements facilitated by oxygen-enhanced MRI and proton-based Fourier decomposition (FD) MRI. In recent times, there has been substantial progress in the field of FD MRI, which involved improving spatial/temporal resolution, refining sequence design and postprocessing, and developing a comprehensive whole-lung approach.
The two-dimensional (2D) phase-resolved functional lung (PREFUL) MRI stands out as an FD-based approach developed for the comprehensive assessment of regional ventilation and perfusion dynamics, all within a single MR acquisition. Recently, a new advancement has been made with the development of 3D PREFUL to assess dynamic ventilation of the entire lung using 8 min exam with a self-gated sequence.
The 3D PREFUL acquisition involves employing a stack-of-stars spoiled-gradient-echo sequence with a golden angle increment. Following the compressed sensing image reconstruction of approximately 40 breathing phases, all the reconstructed respiratory-resolved images undergo registration onto a fixed breathing phase. Subsequently, the ventilation parameters are extracted from the registered images.
In a study cohort comprising healthy volunteers and patients with chronic obstructive pulmonary disease, the 3D PREFUL ventilation parameters demonstrated strong correlations with measurements obtained from pulmonary function tests. Additionally, the interscan repeatability of the 3D PREFUL technique was deemed to be acceptable, indicating its reliability for repeated assessments of the same individuals.
In summary, 3D PREFUL ventilation MRI provides a whole lung coverage and captures ventilation dynamics with enhanced spatial resolution compared to 2D PREFUL. 3D PREFUL technique offers a cost-effective alternative to hyperpolarized 129Xe MRI, making it an attractive option for patient-friendly evaluation of pulmonary ventilation.

Three-dimensional (3D) phase-resolved functional lung (PREFUL) is a functional magnetic resonance imaging (MRI) technique that allows for quantification of regional ventilation of the whole human lung volume, using tidal breathing and contrast-agent free acquisition for 8 min. Here, we present an MR protocol to collect and analyze 3D PREFUL imaging data.

Pulmonary magnetic resonance imaging (MRI) offers a variety of radiation-free techniques tailored to assess regional lung ventilation or its surrogates. ...