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.

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The use of Simplified Whole Body Plethysmography can help shed light on the relationship between lung function and disease progression in research animals. The main advantage of this technique is the ability to collect respiratory data from non-restrained animals. Begin with the system setup by connecting the sampling chamber to a bridge amplifier using an eight-pin DIN connector and the bridge amplifier to the data acquisition device.

Connect the data acquisition device to a power supply and a computer with physiological data analysis software. When connected, initiate the software to interface with the data acquisition system. Download the optional spirometry module in the software and go to the spirometry tab to modify the default unit settings from liter per second to microliter per second in the settings window.

For the calibration of the system, create a four-channel window within the software. In channel one, set source data at 4, 000 data points per second sample rate and one millivolt range. Channel two will be the digital filter of the channel one using a high-pass, one-hertz, auto-adjust filter.

Create channel three in the smoothing tab of channel two data by averaging 100 samples. And create channel four in spirometry flow of channel three data. Next, set up data pad analysis of channel four with three columns.

For column one, go to channel four data and comments to access full comment text. Then set up column two and channel four data and cyclic measurements to select average cyclic frequency For column three and channel four data, select cyclic measurements and average cyclic height. Set the frame rate to 100 to 1 on the bottom right corner of the chart display and save the window configuration as a template for future studies.

When done, close the sample chamber lid and attach a 25-microliter gas-tight syringe to the lure bulkhead connector. Then fit the syringe with a Chaney adapter to use the depth stop of the Chaney adapter to draw 20 microliters of air into the syringe. Next, to zero the pleth in the software, access setup tab and select zero all input before starting a recording.

While recording, wait for a baseline to stabilize and then rapidly depress and withdraw the syringe plunger for 10 repetitions to replicate subject breathing with a measured 20-microliter breath. Once done, stop the recording by right clicking the beginning of numbered pleth recording and label the identity of the measured sample by clicking on add comment. Reset the syringe and zero the input, and repeat the recording measurements of 20-microliter pulses twice.

After completing all the measurements in three recording sessions, use the computer mouse to select a portion of the breathing pleth that accurately represents the artificial 20-microliter breaths. Next, review the column three data for average cyclic height and calculate the average measured breath volume from the three recordings. Open a master template outlined as described earlier.

Next, place a conscious 4-to-12 week, female, albino C57 black 6J mouse into the sampling chamber and latch the lid. Briefly loosen the lure bulkhead cap to equalize the atmospheric pressure in the chamber and re-tighten the cap again. Observe that the subject is not actively moving within the sampling chamber before zeroing all inputs and initiating a recording.

Label the subject's identity by right-clicking the beginning of the numbered pleth recording and clicking on add comment. Then return the mouse to the cage. Select a portion of the breathing pleth that accurately represents the subject breathing.

In the data pad module, data will appear in the preview header providing a temporary readout of the breath rate and breath volume. The data preview can be recorded into the data pad using the add to data pad icon. Continue measuring the parameters for a subject mouse one at a time, and recording representative sections of the breathing pleth to the data pad.

After data recording, export the data pad data to Excel and calculate the minute volume. In clinical use of whole-body plethysmography, the subject's own body temperature and environmental temperature, and humidity affect the complex respiration calculations. In the study, a Simplified Whole-Body plethysmography or SWBP approach controls for environmental temperature and humidity variation, and it was observed that the contribution to temperature and humidity of the host itself does not significantly impact the accuracy of breath measurements of a 20-microliter calibration volume.

The effect of inhaled anesthetics on the breathing of mice was investigated. The baseline breathing before anesthesia and the breathing while the mouse is recovering from anesthesia are shown here. The treatment with the preferred anesthetic caused the mice to exhibit a slow breath rate with a large tidal volume of air.

As the mice recover from sedation, the breath rate increased and breath volume decreased gradually. Similarly, the breath rate steadily increased until the baseline breathing is restored to 2 to 2.5 minutes post removal from anesthesia. The minute volume closely followed the effects of breath rate reaching baseline minute volume by 2.5 minutes post removal from anesthesia.

The breath changes during respiratory melioidosis infections were monitored in the host mice. The breath rate and total inspired air of the mice decreased rapidly during the first day of infection and remained low for the rest of the course of the disease. In contrast, the breath volume does not steeply drop during the first 24 hours and instead has a steady decrease over to the three-day course of the disease.

These data suggests that breath volume may provide a biometric readout of respiratory disease severity in mice. The most important thing to remember while attempting this procedure is to wait until the mouse is not moving before you start recording. Sometimes you must have patience while a curious mouse explores the sampling chamber.

This technique will pave the way to explore how bacterial mutants exhibit different effects on host lung function during their altered disease process, and how therapeutic intervention can accelerate the restoration of normal lung function in infected mice.

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Journal (Immunology and Infection)

Journal (Immunology and Infection)

Simplified Whole Body Plethysmography to Characterize Lung Function During Respiratory Melioidosis

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