Measuring Breathing Patterns in Mice Using Whole-body Plethysmography

Materials and Reagents

1. SilicaGel Orange Indicator 2-4 mm (Bolaseca, catalog number: 8.1.002 )
2. Laboratory tissue paper, e.g., Wiping Paper Plus (Tork, catalog number: 130050 )
3. C57BL/6J background mice (The Jackson Laboratory, catalog number: 000 664 )
   Note: Other laboratory mice strains can be used. Please, see Procedure section.
4. Ethanol 70%, e.g., Ethanol absolute for analysis (PanReac AppliChem, catalog number: A1613 )
   

Equipment

1. Plethysmograph, Unrestrained Whole Body (90 mm diameter), for Mouse (EMMS, catalog number: PLY310 )
2. Pulmonary Flow Transducer (EMMS, catalog number: TPF100 )
3. Adaptive Amplifier, strain gauge type; single channel (EMMS, catalog number: AMP110 )
4. Large capacity drying column (0.5 kg indicating Silica desiccant, suitable for regeneration) complete with tubing and fitting, including 1ml Lock syringe connect to a plastic tube (EMMS, catalog number: ADR101 )
5. 2-channel Bias Flow Generator (EMMS, catalog number: AIR140 )
6. Acquisition Unit (laptop)
   

Software

1. USB Data Acquisition (eDacq) Single Subject Version 1.9.0 (Site) only version with Flow Derived Parameters Analyser (EMMS, catalog number: ESS101A)
   Note: EMMS company (Electro-Medical Measurement Systems), Bordon, UK, www.electromedsys.com (equipment and eDacq software).
2. Microsoft Office Excel 2016 or compatible
3. Statistical software (e.g., GraphPad Prism 6.0)
   

Procedure

Several parameters may critically influence WBP outcomes and reproducibility. Therefore, they might be considered in the experimental design. These include time of the day (considering mouse circadian cycle), mouse behavior during recording, and standardization of the habituation period (Quindry et al., 2016; Receno et al., 2019). Depending on the specific research question, strain-specific differences in pulmonary physiology might be evaluated (Cramer et al., 2015; Bates, 2017). Importantly, the parameters described in the following section may be adapted and validated considering the basal/control breathing activity of each mouse strain.
  Furthermore, environmental conditions must be kept constant during the entire procedure. Here, we maintained a constant temperature of 22 °C and a relative humidity of 55% (as recommended in the Guide for the Care and Use of Animals, National Research Council, 2011).

1. Setting up the equipment
   1. Install USB Data Acquisition (eDacq) to the computer.
   2. Connect through the USB connector the Adaptive Amplifier to the acquisition unit.
   3. Attach Pulmonary Flow Transducer into the transducer entrance from Adaptive Amplifier (Figure 1D).
   4. Join the Pulmonary Flow Transducer to the Plethysmograph apparatus (Figure 1A).
   5. Set up Flow 1 and Flow 2 levels at 1cc/min from the Bias Flow Generator (Figure 1C).
   6. Join Port 1 from Bias Flow Generator with Entrance 1 of Large capacity drying column and Port 2 to the top input from Plethysmograph chamber. Then, connect Entrance 2 of drying column to the lower input from Plethysmograph device (Figures 1A and 1C).
   Note: Silica beads must be maintained dehydrated, when hydrated (color indicator shifts from orange to green, Figure 1C) it is necessary to replace the beads or reactivate them by drying them at 150 °C for 24 h.
   
   
   Figure 1. Plethysmograph equipment. A. Plethysmograph chamber. 1: Top input (incoming airflow), 2: Pulmonary Flow Transducer, 3: Lower input (air exit), 4: Alternative lower input). B. Plethysmograph apparatus attached to the 1 ml Lock syringe by the alternative lower input (4). C. Bias Flow generator and drying column containing silica beads. 5: Port 1 (flow1), 6: Entrance 1, 7: Port 2 (flow2, incoming air), 8: Entrance 2 (air extraction). D. Adaptive amplifier connected to the chamber through transducer entrance (9). Fresh air is continuously administrated into the chamber trough the Port2 and released from the chamber trough the lower input (3). Then, the air passes through the drying column and it is extracted from the Bias Flow generator (air dehumidification is required to preserve bias flow generator).
   
   
2. Setting up the protocol (see Video 1)
   1. Start eDacq program.
   2. Select Protocol from left panel. A new window will pop up.
   3. Click Site 1 option and then Edit.
   4. Add and edit the different steps of the desired protocol:
      
      1. Record on all sites
      2. Wait for user advance
      3. Pause for 00:03:00 (time to leave the workspace/room)
      4. Record control period for 00:45:00 (habituation time)
      5. Record period for 00:15:00 (recording time)
      6. Record off all sites
   5. Select Site 1 from left panel. Add the following flow derived parameters (FDP) properties for recording:
      
      1. On the General tab: in the Input group select 20 ms of smoothing and in the Output group select 5 s of time based.
      2. On the Breath Analysis tab: in the Breath Detection group introduce 0.5 ml/s for Row Threshold and 20% TV for Start of Breath Extrapolation. In the Breath Rejection group, do not select Enable Breath Rejection.
      3. On the Filtering tab: do not select Enable-Pass Filter and Enable Mains Filter.
   6. Apply all changes.
   7. Save configuration.
   Note: Experimental parameters (from Steps B4 to B5) may be adjusted depending on the biological question, experimental design, and mouse phenotype. Please check manufacturer’s recommendations.
   
   
   
   Video 1. Setting up the protocol
   
   
3. Chamber calibration (see Video 2)
   
   1. Turn on the Bias Flow Generator and the Adaptive Amplifier (a white light will flash on standby) 15 min before starting calibration.
   2. Tightly close the plethysmography chamber.
   3. Start eDacq program.
   4. Introduce assigned password (select ok).
   5. Introduce study name (press close).
   6. Close the configuration file version window that appears.
   7. Accept calibration option (always recommended).
   8. Activate DC mode for calibration (AC mode is automatically set during recording).
   9. Set the high flow value to -10 ml/s.
   10. Press F8 (Zero button) to adjust basal parameters.
   11. Press F9 (Low) to adjust low calibration volume.
   12. Join 1ml lock syringe to the alternative lower stopcock input of plethysmography chamber (Figure1 A-B).
   13. Gently inject 1 ml air volume into the chamber using the connected syringe and 1.5 s after press F11 (Record volume) to adjust high calibration volume. Proper calibration range should be between -35 and +35 ml/s (repeat the process otherwise).
   14. Remove the syringe and place back the cap.
   15. Click Finish button and proceed to respiration recording.
   16. A white light should flash on ready from the adaptive amplifier machine.
   Notes:
   
   1. Calibration must be performed every time the equipment is switched on. There is no need to calibrate between each animal/trial in the same day. Calibration can also be initiated through the U option from toolbar at any time.
   2. High flow value must be ten times the injected calibration volume (step 13) as a correction factor for FDP analysis.
      
      
      
      Video 2. System calibration
   
   
4. Breathing recording (see Video 3)
   1. Weigh the mouse right before recording.
   2. Open the plethysmography device and place the animal inside.
      Note: It is critical not disturbing the transducer when manipulating the chamber device. Vibrations can affect the calibration. If this occurs recalibrate the system. Avoid installing the equipment in unstable surfaces or close to high-traffic areas.
   3. Close the chamber tightly and make sure the animal moves freely.
   4. Select Protocol option and then Site 1 choice (the pre-configured protocol will be open).
   5. Restart the protocol.
   6. Introduce mouse identification, weight and any other comments of interest. After assessing this step, recording will start, (green flashing window on screen may appear).
   7. Advance the protocol to wait period (3 min) step. After this period the protocol will automatically proceed to the following steps.
      Note: During this interval we recommend that the user leaves the workspace/room without disturbing the equipment to avoid interferences with the analysis. However, mouse must be monitored by visual resources or from outside the room through the whole session to record motor activity periods as well as any other behaviors.
   8. After recording, return the animal to its cage. Clean and wipe the equipment with 70% Ethanol. Recording will be automatically saved in the assigned project folder.
   9. Record next animal by restart button (start from Step D1) or proceed to data analysis.
   10. Turn off all the equipment.
       
       
       
       Video 3. Recording whole-body plethysmography

Data analysis

eDacq software displays the waveforms from the transducers in real-time. Data is finally recorded to a disk file for post experiment replay and analysis. Critically, to avoid background noise during the recording, especially from mouse motor activity, it is crucial to allow sufficient habituation time. Therefore, while the breathing patterns of the animal are recorded for 1 h, only data from the final 15 min are analyzed. Importantly, since no breath rejection parameters are applied, it is necessary to ensure data corresponds to resting periods (Figure 2).


Figure 2. Example of exported raw data from eDacq to Excel. Tidal volume and respiratory frequency values of a resting mouse from an experimental group (male, 33 days, 13.5 g) shown in blue. Corrected tidal volume by the animal weight and respiratory frequency from period values shown in darker blue.

1. Export data to an Excel file
   1. Start Excel with a blank workbook.
   2. On the Data tab, in the Get External Data group, click From Text.
   3. Browse your *.csv file (eDacq generates two files, choose the file without the period word) and click Import.
   4. Text import wizard will appear.
      
      1. Select Delimited option.
      2. Choose Comma as delimiter.
      3. In the Column data format section, select General and click finish.
   5. Data will appear in the workbook with column headings in the top row.
   6. Correct tidal volume values (ml) by the mouse weight (g).
   7. Average repose respiratory data (final 15 min values, named Period in exported Excel file) to calculate tidal volume (μl/g) and respiratory frequency (breaths/min) (Figure 2).
   8. Perform proper statistical test according to your experimental conditions.
   9. Values obtained by the eDacq software once exported in Excel file are reported in Table 1:
      
      Table 1. Breathing values obtained by eDacq software
      
      
      
      
2. (Optional) Represent breathing recording
   1. Initiate eDacq program.
   2. Select Experiment option from the toolbar.
   3. Press Data source.
   4. Select Recorded data file option from the available options.
   5. Choose the file of interest.
   6. Play recording using the toolbar (Play button).
   7. Pause at the frame time you want to represent (Pause button).
   8. Mouse right click on Site 1 window recording.
   9. Select Properties and freely change them.
   10. Save the file as .jpg selecting Save Chart from mouse right click menu (Figure 3).
   Note: Remember to set up Live data source when you are ready to start a new animal recording. Recorded data file source is only to analyze previous experiments.
   
   
   Figure 3. Representative breathing waveforms in a healthy resting animal (Top panel: Male, 62 days, 27 g) and in a resting experimental mouse presenting breathing alterations (Bottom panel: Male, 63 days, 12 g). These waveforms are not normalized by weight.

Acknowledgments

This work was supported by several pre-doctoral fellowships (BES-2015-073041; PPD, 2018FI_B 00452; AU, PRE2018-083179; LSB), and a Ramón y Cajal fellowship (RyC-2012-11873; AQ). AQ. received funds from the Seattle Children’s Research Institute and Northwest Mitochondrial Guild, and grants from the European Research Council (Starting grant NEUROMITO, ERC-2014-StG-638106), MINECO Proyectos I+D de Excelencia (SAF2014-57981P), MINECO Proyectos I+D “Retos Investigación” (SAF2017-88108-R), and AGAUR (2017SGR-323). This protocol was originally used in our previous study by Bolea et al., 2019.

Competing interests

Authors declare no conflict of interest.

Ethics

Original experimental data (Bolea et al., 2019) was collected following the recommendations in the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of the Seattle Children´s Research Institute and the Universitat Autònoma de Barcelona (Protocol P10127/CEEAH UAB 4155, valid until 10-2023).

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