Assessment of Pulmonary Function:
Whole Body Plethysmograph (Buxco Electronics, Inc. Troy, NY)

Protocol Index

 

Prepared by: RVS
Date Prepared: 1/25/01
Code: MP-001
Reviewed by: KLS
Revision #: 2.0
Date Revised:  


0.0 Abstract: The Penh and Respiratory Rate are measured in a whole body plethysmograph. Base rate and from methacholine challenge are measured and recorded.

1.0 Instrument Layout:



Figure 1: Buxco 12 unit plethysmograph setup.



Figure 2: Buxco full body plethysmograph site cell.

2.0 Reagents and expendables:

2.1 Expendables:
2.1.1 - Dryerite: for the drying columns. Replace as needed when color indicator in the dryerite turns purple.
2.1.2 - 0.2 um filters: Replace as needed.
2.1.3 - Grease: for around the large o-ring on the plethysmograph.
White cloth disks: for the drying columns. Replace as needed.

2.2 Reagents:
2.2.1 - Methacholine (5, 10, and 20 mg/ml) stock solution. You will use approximately 4 ml. in the nebulizer.
Store pure powder in the freezer, with desiccant. Store the prepared solutions for no more than 5 weeks in the freezer at -20° C.
Prepare solutions only in a hood.
Wear protective equipment (mask and gloves).
Methacholine is potentially hazardous if inhaled so take proper precautions on when and where the pure powder is used. Have on site a bronchial inhaler and an epipen in case of accidental exposure.
Prepare 30 or more tubes with 200 mg ±40 mg (160 mg to 240 mg) of dry Methacholine at one time. Weigh out accurately to 1 mg and write the weight on the vial.
Weigh with the scale in the hood and with proper notification of all employees that you will be handling Methacholine at that time. Remove gloves immediately after weighing out the Methacholine by inverting the gloves and disposing them in the biohazard bag. Store the prepared tubes in a freezer at -20° C.
Solution A: Add 10 ml of 0.9 % Sodium Chloride solution to a 20 ml or larger vial containing 200 mg. of Methacholine (weighed to the nearest mg.). (20 mg/ml). To determine the exact volume of buffer, divide the weight of Methacholine in mg. by 20 to get the volume of buffer in ml. For example, 180 mg would require 180/20 = 9.0 ml of buffer.
Solution B: Add 4 ml of solution A to 4 ml of 0.9% Sodium Chloride solution (10 mg/ml).
Solution C: Add 4 ml of solution B to 4 ml of 0.9% Sodium chloride solution (5 mg/ml).
2.2.2 - 0.9 % NaCl buffer
Add 900 mg of dry Sodium Chloride (NaCl) to a 100 ml volumetric flask and dilute with 100 ml of DI water. Sterilize the stock solution.
2.2.3 - Cleaning solution. Use a mild soap and water for cleaning the cells.
2.2.4 - Gases:
a. 4.0% Carbon Dioxide, 21% Oxygen, Balance Nitrogen
b. 4.0% Carbon Dioxide, 10% Oxygen, Balance Nitrogen
c. 8.0% Carbon Dioxide, 21% Oxygen, Balance Nitrogen

3.0 Setup: Allow mice to acclimatize for 15 min. after putting them in the cells.

Calibration:
  1. Each site requires calibration with 1 ml of air.
  2. Activate calibration in software by going to the top menu and clicking on System, then Calibrate.
  3. Attach 1ml syringe to lower site port of the plethysmograph that you wish to calibrate.
  4. Turn top valve of each plethysmograph off (to horizontal position).
  5. Check high/low value (should be 0.0 and -1.0) for each lead number.
  6. Highlight lead number and make sure the box in front of it is checked.
  7. Set the Preamp to DC. Adjust 0 (knob if < 0.25 difference else use the potentiometer).
  8. Inject 1ml of air into site cell.
  9. If the injection is within the range (5.0 V to -5.0 V), press F7 or click In-Zero within 5 sec. If not then let the screen clear and re-inject.
  10. Check manual integration; check values. If values are within range (1.8 to 2.4) then OK, if not then check the site cell for leaks, etc. Then re-inject. If it continues to be out of range then adjust gain (Decrease gain to increase value).
  11. Set the Preamp to AC.
  12. Press F8 to re-zero. Try to get the values close to the same magnitude.
  13. Repeat for all sites.
4.0 Run:
  1. Put mice in Site cells.
  2. Open file (for the protocol that you wish to run)
    1.1 - HypercapMC
  3. Click the Record Tab and set the Record properties. Click the box in front of each site that will be recorded. Click the Run box. The Run Window will open. Enter the Subject ID for each Site and provide the Session Description and the Study Name, then click Connect (upper right corner). The Study Name will be the name of the folder in c:\program files\buxco\data\. Enter relevant information in the session diary (include any out of the ordinary occurrences.)
    3.1 - Click on the Protocol tab and then check each box in front of all sites to be recorded. On the tree, click on Experiment to view wave-forms. Carefully check to see if any of the sites are abnormal and correct for leaks, etc.
  4. Use the Go On to skip through a Pause.
  5. End the run with the Record tab with Stop. Record any out of the ordinary occurrences in the session diary (this is accessible from the tree by going to the bottom and clicking diary).
5.0 Clean up: After removing the mice, wipe out the cells with paper towels and soapy water. Then clean with wet paper towels and dry.

6.0 Data reduction: Copy the folder with the days Study Name from C:\program files\buxco\data\ to the removable ZIP drive and deliver this to the Data Processor.

7.0 Safety: See 2.2.1

8.0 Time and capacity:
Setup: 1 hr. for calibration, balancing, and getting mice.
Cleanup: 30 min. for cleaning up all 12 sites and transferring data files.
Run capacity and time per sample: 12 mice in 2.0 hr.

9.0 Protocols:

9.1 Protocol for starting the nebulizer: (used to check the nebulizer after initial setup).
  1. Double click the protocol on the Tree.
  2. Pause 001:00.0 or longer.
  3. Operate digital Port: Aerosol and drier control (with box 1 and 2 checked).
  4. Log period 010:00.0
  5. Operate Digital Port: Aerosol and drier control (with box 1 and 2 cleared).
  6. Note: Make sure Aerosol distribution box is set to "Computer Cntrl" on display. If not then use the menu button to set it.
9.2 Balance the flows: (used to balance the flows to the individual sites; to be checked daily).
  1. Open file to run
  2. Click box for all sites to be recorded. Click run. In the Run window click Connect: you do not need to fill out subject ID, Session Description, and Study Name. In the tree, click Experiment.
  3. Make sure all valves on Sites are open (in the vertical position). Make sure all pumps are on. Nebulizer control is set to Computer Ctrl. If not push Menu button next to power switch until led display shows Computer Ctrl. Air value set at 10.0 for nebulizer.
  4. Set nebulizer trickle to at least 20 times less (or procedure calls for 0.2 ml/min)
  5. Preamp set to DC
  6. Using the Experimental view pages, adjust flow at individual site cells to +1.0 V reading (plus or minus 0.1 V)
  7. Continue until all are in balance.
  8. Switch Preamp back to AC.
10.0 Notes: Protocol HypercapMC: (note: * for only site 1 , ** for only sites 1,2,and 3):

# Task Arguments  
1 Record
record off		 Record OFF Tables: Off Signals: Off  
2 Pause
begin room air		 Begin room air Period: 060:00.0  
3 Record
record on		 Record ON Tables: On Signals: On  
4 Reset Accumulators    
5 Reset Time    
6 Log On Time
Breathing Room Air		 Breathing Room Air Database Text Period ID: '' Comment: '' Control: No Descendent Control Period: 000:00.0 Log :10 lines at 000:30.0 intervals.  
7 Log Period
establish CONTROL FTB5.0		 Establish CONTROL FTB5.0 Database Text Period ID: 'Control' Comment: 'values are control and will be ' Control: Yes Descendent Control Period: 000:00.0 Timeout: 000:00.0 -- Start of Period -- Time before the end of the period: 005:00.0  
8 Record
record off		 Record OFF Tables: Off Signals: Off  
9 Beep Sound Filename: %%Beep%% **
10 Pause
Begin 4% CO2, 21% O2		 Begin 4%CO2, 21%O2 Period: 060:00.0  
11 Record
record on		 Record ON Tables: On Signals: On  
12 Reset Accumulators    
13 Log On Time
4% CO2, 21% O2, log on time		 4% CO2, 21% O2, log on time Database Text Period ID: '' Comment: '' Control: No Descendent Control Period: 000:00.0 Log :10 lines at 000:30.0 intervals.  
14 Log Period
4% CO2, 21% O2, FTB 5.0		 4% CO2, 21% O2, FTB 5.0 Database Text Period ID: '' Comment: '' Control: No Descendent Control Period: 000:00.0 Timeout: 000:00.0 -- Start of Period -- Time before the end of the period: 005:00.0  
15 Log On Time
Recovery 4% CO2, log on time		 Recovery 4% CO2, log on time Database Text Period ID: '' Comment: '' Control: No Descendent Control Period: 000:00.0 Log :10 lines at 000:30.0 intervals.  
16 Log Period
Recovery 4% CO2, FTB5.0		 Recovery 4% CO2, FTB5.0 Database Text Period ID: '' Comment: '' Control: No Descendent Control Period: 000:00.0 Timeout: 000:00.0 -- Start of Period -- Time before the end of the period: 005:00.0  
17 Record
record off		 Record OFF Tables: Off Signals: Off  
18 Beep Sound Filename: %%Beep%% **
19 Pause
Begin 8% CO2, 21% O2		 Begin 8% CO2, 21% O2 Period: 060:00.0  
20 Record
record on		 Record ON Tables: On Signals: On  
21 Reset Accumulators    
22 Log On Time
8% CO2, 21% O2, log on time		 8% CO2, 21% O2, log on time Database Text Period ID: '' Comment: '' Control: No Descendent Control Period: 000:00.0 Log :10 lines at 000:30.0 intervals.  
23 Log Period
8% CO2, 21% O2, FTB5.0		 8% CO2, 21% O2, FTB5.0 Database Text Period ID: '' Comment: '' Control: No Descendent Control Period: 000:00.0 Timeout: 000:00.0 -- Start of Period -- Time before the end of the period: 005:00.0  
24 Log On Time
Recovery 8% CO2, log on time		 Recovery 8% CO2, log on time Database Text Period ID: '' Comment: '' Control: No Descendent Control Period: 000:00.0 Log :10 lines at 000:30.0 intervals.  
25 Log Period
Recovery 8% CO2, FTB5.0		 Recovery 8% CO2, FTB5.0 Database Text Period ID: '' Comment: '' Control: No Descendent Control Period: 000:00.0 Timeout: 000:00.0 -- Start of Period -- Time before the end of the period: 005:00.0  
26 Record
record off		 Record OFF Tables: Off Signals: Off  
27 Beep Sound Filename: %%Beep%% **
28 Pause
Begin 4% CO2, 10%O2		 Begin 4%CO2, 10%O2 Period: 060:00.0  
29 Record
record on		 Record ON Tables: On Signals: On  
30 Reset Accumulators    
31 Log On Time
4% CO2, 10% O2, log on time		 4% CO2, 10% O2, log on time Database Text Period ID: '' Comment: '' Control: No Descendent Control Period: 000:00.0 Log :10 lines at 000:30.0 intervals.  
32 Log Period
4% CO2, 10% O2, FTB5.0		 4% CO2, 10% O2, FTB5.0 Database Text Period ID: '' Comment: '' Control: No Descendent Control Period: 000:00.0 Timeout: 000:00.0 -- Start of Period -- Time before the end of the period: 005:00.0  
33 Log On Time
Recovery 4% CO2, 10% O2 log on time		 Recovery 4% CO2, 10% O2 log on time Database Text Period ID: '' Comment: '' Control: No Descendent Control Period: 000:00.0 Log :10 lines at 000:30.0 intervals.  
34 Log Period
Recovery 4% CO2, 10% O2, FTB5.0		 Recovery 4% CO2, 10% O2, FTB5.0 Database Text Period ID: '' Comment: '' Control: No Descendent Control Period: 000:00.0 Timeout: 000:00.0 -- Start of Period -- Time before the end of the period: 005:00.0  
35 Record
record off		 Record OFF Tables: Off Signals: Off  
36 Beep Sound Filename: %%Beep%% **
37 Pause
Connect Nebulizer		 Connect Nebulizer Period: 060:00.0  
38 Beep Sound Filename: %%Beep%% **
39 Pause
Let clear for 5 min.		 Let clear for 5 min. Period: 005:00.0  
40 Record
record off		 Record OFF Tables: On Signals: On  
41 Reset Accumulators    
42 Reset Time    
43 Log On Time
Log On Time		 Log On Time Database Text Period ID: '' Comment: '' Control: No Descendent Control Period: 000:00.0 Log :6 lines at 000:30.0 intervals.  
44 Log Period
Establish Control FTB3.0		 Establish Control FTB3.0 Database Text Period ID: '' Comment: '' Control: Yes Descendent Control Period: 000:00.0 Timeout: 000:00.0 -- Start of Period -- Time before the end of the period: 003:00.0  
45 Record Tables: Off Signals: Off  
46 Beep Sound Filename: **
47 Pause
add Saline		 Add Saline Period: 060:00.0  
48 Record Tables: On Signals: On  
49 Reset Accumulators    
50 Operate Digital
Nebulizer on		 Nebulizer ON Operate Digital Output Word Name : aerosol dryer State: 3 *
51 Log Period
exposure Saline		 Exposure Saline Database Text Period ID: 'Saline exposure' Comment: 'Saline' Control: No Descendent Control Period: 000:00.0 Timeout: 001:00.0 -- Start of Period -- Time from the Start Of this Task: 000:00.0  
52 Operate Digital
aerosol off		 Aerosol OFF Operate Digital Output Word Name : aerosol dryer State: 0 *
53 Log On Time
response Saline log on time		 Response Saline log on time Database Text Period ID: 'response Saline' Comment: '' Control: No Descendent Control Period: 000:00.0 Log :6 lines at 000:30.0 intervals.  
54 Log Period
response Saline FTB3.0		 Response Saline FTB3.0 Database Text Period ID: 'response Saline' Comment: '' Control: No Descendent Control Period: 000:00.0 Timeout: 000:00.0 -- Start of Period -- Time before the end of the period: 003:00.0  
55 Record Tables: Off Signals: Off  
56 Beep Sound Filename: %%Beep%% **
57 Pause
add 5mg MC		 Add 5mg MC Period: 060:00.0  
58 Record Tables: On Signals: On  
59 Reset Accumulators    
60 Operate Digital
Nebulizer on		 Nebulizer ON Operate Digital Output Word Name : aerosol dryer State: 3 *
61 Log Period
exposure 5mgMC		 Exposure 5mgMC Database Text Period ID: '5mgMC exposure' Comment: '5mgMC' Control: No Descendent Control Period: 000:00.0 Timeout: 001:00.0 -- Start of Period -- Time from the Start Of this Task: 000:00.0  
62 Operate Digital
aerosol off		 Aerosol OFF Operate Digital Output Word Name : aerosol dryer State: 0 *
63 Log On Time
response 5mgMC log on time		 Response 5mgMC log on time Database Text Period ID: 'response 5mgMC' Comment: '' Control: No Descendent Control Period: 000:00.0 Log :16 lines at 000:30.0 intervals.  
64 Log Period
response 5mgMC FTB8.0		 Response 5mgMC FTB8.0 Database Text Period ID: 'response 5mgMC' Comment: '' Control: No Descendent Control Period: 000:00.0 Timeout: 000:00.0 -- Start of Period -- Time before the end of the period: 008:00.0  
65 Record Tables: Off Signals: Off  
66 Beep Sound Filename: %%Beep%% **
67 Pause
add 10mg MC		 Add 10mg MC Period: 060:00.0  
68 Record Tables: On Signals: On  
69 Reset Accumulators    
70 Operate Digital
Nebulizer on		 Nebulizer ON Operate Digital Output Word Name : aerosol dryer State: 3 *
71 Log Period
exposure 10mgMC		 Exposure 10mgMC Database Text Period ID: '10mgMC exposure' Comment: '10mg MC' Control: No Descendent Control Period: 000:00.0 Timeout: 001:00.0 -- Start of Period -- Time from the Start Of this Task: 000:00.0  
72 Operate Digital
aerosol on		 Aerosol OFF Operate Digital Output Word Name : aerosol dryer State: 0 *
73 Log On Time
response 10mgMC log on time		 Response 10mgMC log on time Database Text Period ID: 'response 10mgMC' Comment: '' Control: No Descendent Control Period: 000:00.0 Log :16 lines at 000:30.0 intervals.  
74 Log Period
response 10mgMC FTB 8.0		 Response 10mgMC FTB 8.0 Database Text Period ID: 'response 10mgMC' Comment: '' Control: No Descendent Control Period: 000:00.0 Timeout: 000:00.0 -- Start of Period -- Time before the end of the period: 008:00.0  
75 Record Tables: Off Signals: Off  
76 Beep Sound Filename: %%Beep%% **
77 Pause
add 20mg MC		 Add 20mg MC Period: 060:00.0  
78 Record Tables: On Signals: On  
79 Reset Accumulators    
80 Operate Digital
Nebulizer on		 Nebulizer ON Operate Digital Output Word Name : aerosol dryer State: 3 *
81 Log Period
exposure 20mgMC		 Exposure 20mgMC Database Text Period ID: '20mgMC exposure' Comment: '20mg MC' Control: No Descendent Control Period: 000:00.0 Timeout: 001:00.0 -- Start of Period -- Time from the Start Of this Task: 000:00.0  
82 Operate Digital
aerosol off		 Aerosol OFF Operate Digital Output Word Name : aerosol dryer State: 0 *
83 Log On Time
response 20mgMC log on time		 Response 20mgMC log on time Database Text Period ID: 'response 20mgMC' Comment: '' Control: No Descendent Control Period: 000:00.0 Log :16 lines at 000:30.0 intervals.  
84 Log Period
response 20mg FTB 8.0		 Response 20mgMC FTB 8.0 Database Text Period ID: 'response 20mgMC' Comment: '' Control: No Descendent Control Period: 000:00.0 Timeout: 000:00.0 -- Start of Period -- Time before the end of the period: 008:00.0  
85 Record Tables: Off Signals: Off  
86 Beep Sound Filename: %%Beep%% **

Protocol Index

 

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Supported by the National Heart, Lung, and Blood Institute (NHLBI) (Grant # HL66611)