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 mou....

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

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.