Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography

Summary

The assessment of respiratory physiology has traditionally relied upon techniques, which require restraint or sedation of the animal. Unrestrained whole-body plethysmography, however, provides precise, non-invasive, quantitative analysis of respiratory physiology in animal models. In addition, the technique allows repeated respiratory assessment of mice allowing for longitudinal studies.

Abstract

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.

Introduction

Lung dysfunction is one of the leading causes of morbidity and mortality in the world. The condition is characterized by inadequate oxygen exchange, synonymous with coughing, chest pains and dyspnea. Respiratory disease accounts for ~10% of mortality worldwide¹. According to the World Health Organization, mortality rates are set to rise due to persistent smoking, pollution & occupational irritants. UWBP is a useful addition for studying lung physiology, which strongly compliments traditional biochemical and histological analyses². Other procedures used for lung assessment do not provide the same advantages as UWBP. Invasive plethysmography is....

Protocol

NOTE: The following experimental procedure is approved by the Animal Ethics Committee at Monash University and conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (2006). Adult female C57BL/6 mice used to generate the representative results were obtained from the Monash Animal Services. The mice were housed in a specific pathogen free, temperature and humidity controlled room with a 12 hr light-dark cycle. These mice had free access to food and water.

1. Initial Setup

1. Connect the laptop/desktop to the data acquisition machine for recording via a USB cable.
2. Connect the Bridge ....

Results

When this procedure has been followed correctly, a consistent oscillating trace is created on the data analysis software. The procedure provides a respiratory trace within a few minutes after setup with simple computing calculations to determine respiratory parameters listed. Figure 5 represents a suitable breathing trace from a control (healthy) mouse. Appropriate oscillating data is produced when the animal is not actively moving.

UWBP is an extremely useful and reliable ass.......

Discussion

The technique described here is a non-invasive method for assessment of respiratory parameters of unrestrained and unanesthetized mice. The strengths of this protocol include its simplicity and precision to measure lung function longitudinally with minimal artefacts. There are, however, some limitations and critical steps to be noted about the procedure. Firstly and most importantly, the mouse must stay calm within the chamber for a minimum of five seconds. Added stress will disrupt the breathing pattern of the mouse and.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
LabChart 7 software (for Macintosh)	ADINSTRUMENTS	MLU60/7	used in protocol step 4
PowerLab 8/30 (model ML870)	ADINSTRUMENTS	PL3508
Octal Bridge Amp (model ML228)	ADINSTRUMENTS	FE228
Black BNC to BNC cable (1 m)	ADINSTRUMENTS	MLAC01
Macintosh OS	Apple Inc.		Mac OS X 10.4 or later
Surgipack Digital Rectal Thermometer	Vega Technologies	MT-918
Grass volumeteric pressure transducer PT5A	Grass Instruments Co.	Model number PT5A; serial No. L302P4.
1 ml Syringe	Becton Dickinson (BD)	309628
5 ml Serological syringe pipettes	Greiner Bio One	606160	Connected via plastic tubing
Balance/Scales	VWR International, Pty Ltd	SHIMAUW220D	Any weighing balance with of 0.1 gram resolution
HM40 Humidity & temperature meter	Vaisala	HM40A1AB
Barometer	Barometer World	1586
Laboratory tubing	Dow Corning	508-101	Used to connect water column to the syringe and pressure transducer
Cylindrical Perspex Chamber	Dynalab Corp.		Custom built cylindrical chamber with internal dimensions as follows: 50 mm(w) x 1,500 mm(l). There are two lids for each side, with dimensions 80 mm(l) x 80 mm(w). Each lid has a 60 mm wide circular hole cut on the face of the lid 50 mm deep. This allows the chamber to fit into the lid. A rubber ring is fitted around each hole of the lid where the chamber will fit. For attachment of syringe and pressure transducer, the openings are 5 mm in diameter. For attachment of humidity probe, the openings are 25 mm in diameter.
80% Ethanol (4 L)	VWR International, Pty Ltd	BDH1162-4LP