Method to Obtain Pattern of Breathing in Senescent Mice through Unrestrained Barometric Plethysmography

Summary

Unrestrained barometric plethysmography is used to quantify the pattern of breathing in awake mice. We show that 15 s segments under a standardized protocol display similar values to an extended duration of quiet breathing. This methodology also allows for the quantification of apnea and augmented breaths during the first hour in the chamber.

Abstract

Unrestrained barometric plethysmography (UBP) is a method for quantifying the pattern of breathing in mice, where breathing frequency, tidal volume, and minute ventilation are routinely reported. Moreover, information can be collected regarding the neural output of breathing, including the existence of central apneas and augmented breaths. An important consideration for UBP is obtaining a breathing segment with a minimal impact of anxious or active behaviors, to elucidate the response to breathing challenges. Here, we present a protocol that allows for short, quiet baselines to be obtained in aged mice, comparable to waiting for longer bouts of quiet breathing. The use of shorter time segments is valuable, as some strains of mice may be increasingly excitable or anxious, and longer periods of quiet breathing may not be achieved within a reasonable timeframe. We placed 22 month-old mice in a UBP chamber and compared four 15 s quiet breathing segments between minutes 60–120 to a longer 10 min quiet breathing period that took 2–3 h to acquire. We also obtained counts of central apneas and augmented breaths prior to the quiet breathing segments, following a 30 min familiarization period. We show that 10 min of quiet breathing is comparable to using a much shorter 15 s duration. Additionally, the time leading up to these 15 s quiet breathing segments can be used to gather data regarding apneas of central origin. This protocol allows investigators to collect pattern-of-breathing data in a set amount of time and makes quiet baseline measures feasible for mice that may exhibit increased amounts of excitable behavior. The UBP methodology itself provides a useful and noninvasive way to collect pattern-of-breathing data and allows for mice to be tested over several time points.

Introduction

UBP is a common technique for the assessment of breathing patterns^1,2,3,4. In this method, mice are placed in a closed chamber where pressure differences between the main chamber (where the animal is housed) and a reference chamber are filtered through a pneumotachograph to obtain values. The resulting UBP setup is noninvasive and unrestrained and allows for respiratory measures to be assessed without the requirement of anesthesia or surgery. Additionally, this technique is suitable for studies requiring multiple measurements in the same mouse over time. Variables such as breathing frequency, tidal volume, and minute ventilation can be quantified with this method, during a single trial or over several trials. Whole-body UBP also provides measures of peak flows and respiratory cycle duration. Together, these parameters quantify the pattern of breathing. The recorded breathing traces also make it possible to review the data and count the number of central apneas displayed within a given time period. This count can be used alongside an analysis of tidal volume and inspiratory times to gauge other alterations in the pattern of breathing.

While several noninvasive plethysmography techniques exist for the direct assessment of pulmonary physiological parameters, whole-body UBP allows for a way to screen for respiratory function with minimal undue stress to the mouse. Head-out plethysmography, which utilizes tidal midexpiratory flow measures and is also noninvasive, relies on restraint, like many other types of plethysmography (e.g., double-chamber plethysmography). While these methods have been used in rodent models to measure airway responsiveness⁵, the use of neck collars or small restraint tubes may take mice (vs. other species) longer to acclimate to and return their breathing to resting levels.

Obtaining an optimal air-breathing segment is an important consideration for baseline comparisons. The increased use of commercially available plethysmography systems makes collecting pattern-of-breathing data possible in many laboratories. Importantly, pattern of breathing is variable throughout the collection period, particularly for mice. With that said, it is necessary to standardize baseline analysis as a means of ensuring that the training level of experimenters does not confound results. There are numerous ways to collect an air-breathing segment, serving as one area of variation between experimental designs. One example includes averaging the final 10–30 min of data following a previously defined set of time within the chamber¹, while another method involves waiting until the mouse is visibly calm for 5–10 min⁶. The latter can take 2–3 h to achieve and in some cases, a trial may need to be abandoned if the mouse is not calm for long enough. This concern is an especially important consideration for strains of mice where observed behaviors are more anxious and excitable⁷. These mice may take longer to adjust to the chamber environment and only remain calm for short bursts of time. Limiting the time devoted to baseline collection standardizes the chamber time for each mouse.

It is crucial that experimenters obtain a suitable baseline that encompasses resting behavior values in the mouse but also occurs in a timely manner. Hence, the goal of this report is to provide a description of methods used to obtain short quiet baseline values for breathing parameters in mice. Moreover, we report that apneas and augmented breaths can be quantified during the first hour in the chamber.

Protocol

All procedures were approved by the Le Moyne College Institutional Animal Care and Use Committee. All use of animals was in agreement with the policies described in the Guide for the Care and Use of Laboratory Animals⁸.

NOTE: (Critical) Prior to experimentation, obtain all necessary approvals and training required for animal use. It is important the experimenters are familiarized with the mouse behaviors and activity levels, including signs of sleep, distress, and/or movement artifact vs. normal sniffing and breathing.

1. Whole-body Barometric Plethysmography Chamber

1. Read the appropriate user manuals for the barometric plethysmography chamber, including connectors, O-rings, etc., and create a standard protocol file to define analyzers (e.g., metabolic) and parameters specific to the software.
2. Make sure all hoses and tubes are connected to the chamber. Connect a gas flow tube (flow-in) and a vacuum tube (flow-out) directly to the barometric plethysmography chamber.
   NOTE: The inflow must be attached to the opening marked bias flow.
3. Attach CO₂, O₂, and N₂ gas tanks to the gas mixer. Make sure all gas tanks are in the open position prior to experimentation.

2. Calibration of the Barometric Plethysmograph Chamber

1. Calibrate a high and a low flow of gas by selecting the 7700-Amplifier Setup under the Hardware tab of the barometric plethysmography software.
2. Set a vacuum (flow out of the chamber) appropriate for the experimental design and gas analyzers (~0.1 L/min).
   NOTE: The outflow rate must remain the same throughout the calibrations and experiment for accurate metabolic recordings.
3. Set a low flow of air by removing the flow tube from the chamber and turning off the vacuum.
4. Record the zero flow by entering a 0 into the Low Unit cell for the corresponding chamber. Double-click the Low Cal cell, change the time to 3 s, and hit Measure.
5. Reattach the flow tube and allow gas (20.93% O₂, balanced N₂) to flow through the barometric plethysmography chamber from the gas mixer.
6. Convert the inflow from liters/minutes into milliliters/second. Click the High Unit cell for the corresponding chamber and enter the value in milliliters/second. Double-click High Cal, change the time to 3 s, and click Measure.
7. Leave the 7700-Amplifier Setup tab open to calibrate the metabolic analyzers to the barometric plethysmography software.

3. Metabolic Analyzer Calibration

1. In the gas mixer program, set the gas mixer to release a flow of gas containing 20.93% O₂ and 79.07% N₂.
2. On the metabolic analyzers, set the O₂ calibration level to 20.93% and the CO₂ to read 0%. Turn the dial back to Sample once the appropriate values are entered.
3. Set the high O₂ percentage. Click on the ABCD-4 tab of the barometric plethysmography software and then enter 20.93 under High Unit of the C2 line. Under High Cal, change the time to 3 s and hit Measure.
4. Set the low CO₂ percentage. Enter 0 under Low Cal of the C3 line, and then change the time to 3 s and click Measure under Low Cal.
5. In the gas mixer program, change the O₂ value to 10% and the CO₂ value to 5%. Wait several minutes for the gas flow to adjust to these values. On the metabolic analyzers, turn the adjustment knobs to calibrate CO₂ equal to 5%. Be sure to turn the dial back to Sample once the values are calibrated.
6. Set the high CO₂ percentage. Ensure the analyzer readings are stable before inserting appropriate values into the O₂ and CO₂ on the barometric plethysmography software. Click High Unit under C3 and enter 5. Change High Cal to 3 s and hit Measure.
7. Set the low O₂ percentage. Click Low Unit under the C2 option and enter 10. Click Low Cal, input 3 s, and click Measure.
8. Change the gas values on the gas mixer back to 20.93% O₂ and 79.07% N₂. Wait several minutes for the chamber to adjust to these values. Repeat the steps 3.1‒3.7 if the metabolic analyzers do not automatically read 20.93% O₂ and 0% CO₂, to ensure proper calibration. Routinely confirm proper calibration with certified gas tanks.
9. Recheck the flow meters connected to the barometric plethysmography chamber. Adjust the air flow into and out of the chamber to rates appropriate for the experiment (typically, 0.1–0.3 L/min).
10. Once all settings have been applied to the barometric plethysmography software, click OK to begin recording.

4. Unrestrained Barometric Plethysmography

1. Record the mouse’s weight and initial body temperature. Wait 10 min before placing the mouse in the chamber, to collect O₂ and CO₂ data from an empty chamber. Work in a quiet area familiar to the mice so noise and smells do not interfere with the data collection. Avoid any possible disruptions, including the opening and closing of doors or personnel moving in/out of the data collection room.
   NOTE: This specific protocol employed 22 month-old male C57BL/6J mouse.
2. During the first hour, document the behaviors of the mouse and take detailed notes, including specific values of the flow in/out of the chamber.
3. After 60 min of chamber habituation, watch for segments of quiet breathing for the following 60 min. List any segments lasting at least 15 s in length without sniffing and grooming. Take body temperature measures every 10 min when using an implantable device.
4. At the end of the experiment, remove the mouse from the chamber and place it back in its cage. All equipment should be cleaned and wiped down thoroughly. If droplets of water remain, use pressurized air to remove them.

5. Analysis of Pattern of Breathing and Metabolism

1. Open the barometric plethysmography review file and consult the recorded notes for the animal of interest.
2. Open the Metabolic panel in the software and take the average of the first 10 min of O₂ and CO₂, when the chamber was empty. Record these values as the FiO₂ and FiCO₂.
3. View the Flow panel of the barometric plethysmography software. Right-click Analyze Attributes and set appropriate parameters. Under the Meta 1 tab, enter the FiO₂ and FiCO₂ from step 5.2, as well as the flow into the chamber under Meta 2, to calculate VO₂ and VCO₂.
4. For pattern of breathing analysis, confirm the times for the 15 seconds of quiet breathing using notes about animal behavior as well as the flow panel tracing. Enter the times for the 15 s intervals of quiet breathing under Open Data Parser Dialogue from the Data Parser tab. Click Parser View Mode to only show the specific 15 s segments of interest.
5. Click Save Parsed Derived Data. Open the data file in a spreadsheet to obtain the binned data.

6. Analysis of Apneas and Augmented Breaths

1. In the open review file, exit Parser View Mode. Go into the Graph Setup option under Setup > P3 Setup and select Page View under Type. Select 5 for the number of panes. Enter -2 into the box labeled Low and 2 into the box labeled High for flow measures in milliliters/second. Apply the changes.
2. Scroll to the 30 min mark on the flow tracings panel.
3. Count apneas and augmented breaths for the 30–60 min after the mouse was placed in the chamber. Quantify periods of suspended breathing lasting longer than or equal to 0.5 s, indicative of an apnea. Augmented breaths are indicated by a sharp rise in the breathing trace above 1.25 mL/s followed by a sharp decrease below -0.75 mL/s.

Results

The results of UBP as an evaluation of pattern of breathing in 16 aged (22-month-old) mice performed under normal air gas (20.93% O₂ with balanced N₂) are reported. The analysis first included a comparison of a longer 10 min quiet breathing segment (which took over 2 h to obtain) versus the average of four short 15 s segments (quantified within minutes 60–120). A representative flow tracing of quiet breathing, where breathing is consistent with no active breathing behaviors, is provided in

Discussion

The protocol provides information regarding a quiet breathing baseline in mice, as well as collecting data about central apneas and augmented breaths. The representative results show that a 10 min quiet baseline has a similar pattern of breathing when compared to an average of four 15 s bouts for a cohort of old mice. Importantly, the 15 s bouts are not statistically different, nor do these groups have differences in variation from one another using Levene’s test. These data demonstrate that even one short bout is ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Carbon Dioxide Analyzer	AEI Technologies	CD-3A
Carbon Dioxide Sensor	AEI Technologies	P-61B
Computer			must be compliant with Ponemah requirements
Drierite beads	PermaPure LLC	DM-AR
Flow Control	AEI Technologies	R-1	vacuum
Flowmeter	TSI	4100	need one per chamber and one for vacuum
Gas Mixer	MCQ Instruments	GB-103
Gas Tanks	Haun		100% oxygen, 100% carbon dioxide, 100% nitrogen - food grade, or pre-mixed tanks for nomal room air and gas challenges
Oxygen Analyzer	AEI Technologies	S-3A
Oxygen Sensor	AEI Technologies	N-22M
Polyurethane Tubing	SMC	TUS 0604 Y-20
Ponemah Software	DSI
Small Rodent Chamber	Buxco/DSI
Thermometer (LifeChip System)	Destron-Fearing		any type of thermometer to take accurate body temperatures is appropriate, but the use of implantable chips allows for minimal disturbance to animal for taking several body temperature measurements while the animal is still in the UBP chamber
Transducers	Validyne	DP45	need one per chamber
Whole Body Plethysmography System	Data Science International (DSI)		Includes ACQ-7700, pressure/temperature probes, etc.