SOP - Whole Body Plethysmography - Buxco --PUBLIC

Whole Body Plethysmography

Purpose

To study breathing parameters (rate, tidal volume, irregularity)

Experimental Design

Minimum number of animals: Recommend at least 5 animals per group being compared

Age at test: recommended at least 6 wks of age – younger may be possible if they are mobile

Sex: either

Time of Day: typically during the light phase, but may vary depending on study requirements

Equipment

Buxco Plethysmogrpahy System with pull-flow bias flow and unrestrained plethysmography chambers FinePointe Software

Procedure

Turn on the Equipment

1. Turn on the Computer
2. Turn on the Amplifiers
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3. Turn on the Bias Flow (noe status lights for all bias flow sites should turn green – if not then the flow will not be at the correct setting)
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4. L ogin to computer (user: Buxco, pass: Buxco)
5. Start FinePointe Software (user: Buxco, pass: [leave blank])
6. Select or create the study folder
7. Launch the chambers (select “Vol” AHR vs Vol)

Calibration (Typically performed by core personnel)

1. Obtain small screw driver, and 1mL syringe with connector tube
2. Make sure plethysmography chambers are closed and connected to the bias flow pump
3. For all chambers that need calibration, set the amplifier toggle to the ‘DC’ setting
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4. In the calibration menu – click on calibrate flow
5. Load syringe with 1mL of air and connect to the 1st chamber for calibration (this will be repeated for each chamber)
6. Follow the onscreen instructions
   1. Balance the transducer so that baseline reads 0
   2. Inject 1mL of air into the chamber, confirm that the portion of the signal with the injection is correctly highlighted (note- a smooth injection over 3 seconds works well for this, not too fast, not too slow)
   3. Switch the toggle to the AC setting and allow the signal to settle
   4. Review the calibration notes (if error is >30% - redo calibration)
7. Repeat 4-6 for all remaining chambers
8. For Temperature Calibration – select the calibrate temperature command and follow the onscreen instructions
   1. Press the ‘Cal’ button on the amplifier and advance the calibration step in the software
   2. Confirm that the high and low signals are marked correctly
   3. Expose probe to room temperature (reopen chamber or remove probe from chamber)
   4. Enter the current ambient temperature value
   5. (error is typically ___%)
9. Repeat 8 for all remaining chambers
10. For Humidity Calibration – select the calibrate humidity command and follow the onscreen instructions
    1. Press the ‘Cal’ button on the amplifier and advance the calibration step in the software
    2. Confirm that the high and low signals are marked correctly
    3. Expose probe to room humidity (reopen chamber or remove probe from chamber)
    4. Enter the current ambient humidity value
    5. (error is typically ___%)
11. Repeat 10 for all remaining chambers

Protocol

(General Plethysmography)

(The suggested protocol is based on the BaSH Study – Volume protocol. This protocol has 30 minutes of acclimation which is discarded followed by 1 hour of recording that is included in the analysis. Modifications can be made if needed)

1. Locate the BaSH Study – folder, right click and ‘create new study based on selected study’___________
2. Rename study and place in appropriate folder (suggested naming format: YYYY-MM-DD-PI-user-brief_description)
3. Update Animals information and assign them to chambers _____________
4. Confirm/Finish updating study information to start the recording session
5. Session should start and status will reflect whether the mice are in the acclimation or recording phase
6. At the end of the recording the session will display “Task Sequence. Not Running”
7. Run another session with the next batch of animals – we suggest cleaning the chambers between groups of animals – (clean bases with running water and dry, wipe tops using damp paper towel)
8. End the Session
   1. File->End Session
   2. In the FinePointe Software – select your study and export/confirm data – copy to usb drive
   3. Exit software and turn off computer
9. Put away mice
10. Turn off bias flow
11. Turn off amplifier
12. Clean chambers (clean bottoms using detergent and water, rinse well and dry, wipe tops using damp paper towel)

(Video Recorded Plethysmography)

1. ...

(Gas Challenge Plethysmography)

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Data Outcome Measures Produced

Common parameters of interest

• Outcome Measure 1 : description of what the outcome measure is (how it is calculated, what common interpretations may be, etc.)
• Outcome Measure 2 : description of what the outcome measure is (how it is calculated, what common interpretations may be, etc.)

Troubleshooting

1. unable to detect hardware
   1. check USB hub, it may have become dislodged/disconnected