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.
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.
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 a commonly used technique that requires the animal to be anesthetised3,4 and thus, resulting respiratory measurements are not necessarily reflective of a natural state. Further, the requirement for mechanical ventilation and chemical challenges preclude future measurements3,4. Another method of collecting respiratory data is by forced oscillation, which is more sensitive to finer changes in respiratory parameters compared to UWBP5. Forced oscillation is, however, an invasive technique and requires animal termination for data collection5-7.
UWBP involves placing an animal inside a specialized chamber. During inspiration, the tidal air is warmed and humidified within the lungs increasing water vapor pressure and causes thermal expansion of gas8. This effect causes a net change in air volume creating an increase in pressure within the plethysmograph chamber8. The opposite occurs during expiration creating a respiratory waveform from the animal. Waveform analysis is then used to measure from the respiratory trace: respiration rate (breaths/min), total breathing cycle time (sec), inspiration/expiration time (Ti/ Te, sec) and changes in pressure due to each tidal volume (PT). Figure 1 illustrates each measurements origin from a respiratory trace. These measurements are simple to calculate and multiple respiratory parameters may be derived from these measurements. These parameters include: Tidal volume (the volume of air moved between normal inhalation and exhalation), minute volume (volume of gas inhaled from the lungs per minute), inspiratory duty cycle (the percentage of inspiration time to the total breathing cycle duration) and inspiratory flow rate (the amount of air inspired in a given time).
UWBP provides precise, non-invasive, quantitative analysis of respiratory physiology in animal models and can be used for measuring the progression of respiratory disease and lung function6,9. Contrary to other plethysmography techniques, UWBP avoids the use of anesthesia, restraints and invasive manipulations that produce artefacts and experimental variability6,9. Anesthesia can suppress respiration, alter heart rate and can be challenging to regulate10. Restraints induce an increase in respiration due to additional stress via corticosterone and epinephrine release11,13. The key feature of UWBP is repeated physiological assessment making it amenable to longitudinal studies. UWBP is strongly recommended for the longitudinal assessment of lung physiology and offers a valuable skill for future respiratory drug assessment.
Bleomycin, ovalbumin, and hypoxia have been utilized to induce respiratory challenges in several studies and UWBP has successfully measured accurate lung physiological assessment7,9,13-16. The protocol described is designed for standard adult laboratory mice. However, UWBP has been adapted to other animals such as rats, guinea pigs, and non-human primates17-20. UWBP is not restricted only to assessing pulmonary dysfunction but has also been used for the assessment of lung maturation3. The versatility, simplicity and reproducibility of UWBP have established an excellent technique for assessing pulmonary function in animals. Various software (see materials and equipment table) will be required to follow this procedure. An experienced scientist would be able to perform this protocol with a mouse within 1 hr.
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
2. Bridge Amplifier Calibration
Note: To calibrate the bridge amplifier an injection of air into the water column is required to create a 1 cm H2O deflection. This will occur under a single set of conditions and is dependent upon the user's apparatus. For clarification these steps demonstrate how this laboratory would perform the calibration.
3. Recording Lung Function
4. Plethysmography Analysis
Note: To calculate respiratory parameters such as tidal volume (VT) and minute volume the following variables need to be measured: respiration rate (breaths/min), total breathing cycle time (sec), inspiration/expiration time (Ti/ Te, seconds) and change in pressure due to each tidal volume (PT). Figure 1 illustrates the variables that can be measured from a trace. The following steps use a software (see materials and equipment table) to measure these variables. When analyzing, avoid regions of the trace containing sniffing or movement. For reproducible results, at least 5 seconds of good breathing trace is required. For an example of different breathing traces refer to Figure 5 and 6.
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 assessment of lung function between control and pulmonary fibrosis cohort. Figure 7 demonstrates the lung function of a mouse with bleomycin-induced pulmonary fibrosis at day 14. In comparison to the control graph, Figure 7 illustrates a visual difference consistent with bleomycin administration7. As discussed previously, the procedure may be repeated allowing us to observe changes in respiratory parameters over time between these two groups.
Results obtained are to be expressed as means ± SEM. It is recommended to copy and paste the data collected into a simple Excel spreadsheet. This will become useful for carrying out calculations discussed in steps 4.13 & 4.14. Respiratory function can be compared visually between two groups as demonstrated in Figure 8.
Figure 1. Different components of the breathing cycle illustrated using the barometric plethysmography. This graph illustrates a) the change in pressure due to inspiration (∆Pi), b) the change in pressure due to each tidal volume (PT), c) the change in pressure due to expiration (∆Pe), d) total breathing cycle time (Ttot), e) inspiration time (Ti) and f) expiration time (Te). Click here to view larger image.
Figure 2. Visual representation of the barometer and water column setup. The figure is designed to aid the reader in setting up the barometer and water column for the calibration process. Notice the water is level within the two columns aided by the ruler. The two columns are connected via 15 cm of plastic tubing. The tubing on the right (65 cm) is connected to a 1 ml syringe and to the left (75 cm) the pressure transducer linked to the data acquisition machine. Note: the length of tubing determines the volume (300 μl) required to move 1 cm of water. Click here to view larger image.
Figure 3. Performing steps 2.4 and 2.5 of Bridge Amp Calibration. This figure illustrates the steps 2.7 and 2.8 for calibration of the equipment. It is crucial to correct the Bridge Amp to obtain precise results. Click here to view larger image.
Figure 4. An overall schematic of the UWBP setup. To the left is the humidity/temperature probe connected to one side of the plethysmography chamber containing the animal. To the right is the calibration syringe and pressure transducer leading from the plethysmography chamber to the data acquisition system producing a respiratory trace on the computer. Click here to view larger image.
Figure 5. An example of a respiratory breathing trace from a C57Bl/6 control mouse obtained when using UWBP. This breathing trace illustrates appropriate, consistent data from a control animal. Nine consecutive comments are added at the peaks and troughs of breathing oscillations to obtain the respiratory parameters listed by following steps 4.1-4.13. Time is represented along the x-axis (sec) and pressure changes along the y-axis (cm.H2O). Click here to view larger image.
Figure 6. Examples of different suboptimal traces obtained from a C57Bl/6 mouse when using UWBP. Suboptimal results can be confused as appropriate data and is the most common source for poor analysis. This figure illustrates the most common suboptimal traces that should never be used for analysis. These breathing traces demonstrate a) A breathing trace recorded while the animal is sniffing and moving altering the animal’s basal respiratory physiology. b) A trace recorded resulting oscillations gradually increasing over time is usually caused by condensation and humidity build up. However, the trace may be corrected by wiping the plethysmography chamber with ethanol or by repeating the calibration steps. c) A trace recorded during plethysmography chamber movement while the animal or researcher is engaging with the equipment. Time is represented along the x-axis (sec) and pressure changes along the y-axis (cm.H2O). Click here to view larger image.
Figure 7. An example of a breathing trace obtained from a C57Bl/6 mouse with induced pulmonary fibrosis when using UWBP. This breathing trace illustrates appropriate, consistent data from an animal with induced pulmonary obtained when using the UWBP procedure described in this article. Nine consecutive comments are added at the peaks and troughs of breathing oscillations to obtain the respiratory parameters listed by following steps 4.1-4.13. Time is represented along the x-axis (sec) and pressure changes along the y-axis (cm.H2O). Click here to view larger image.
Figure 8. Respiratory function compared between control and bleomycin challenged C57Bl/6 mice. Performing plethysmography analysis after using UWBP will allow the user to results similar to what is represented here. This figure demonstrates the physiological differences between bleomycin challenged animal (dotted grey line) and control animals (solid black line). These graphs show comparisons in a) Expiration time (sec), b) Inspiration time (sec), c) Inspiration duty cycle (%), d) Inspiratory flow rate (ml/sec) e) Respiration rate (breaths/min), f) Minute volume (ml/min/kg), g) Tidal volume (ml/kg) and h) Total cycle time (sec). Lung function data were collected longitudinally in the same cohort of animals on days 0, 7, and 14 following bleomycin challenge. Representative data has been adapted from Murphy et al. (2012)16. Click here to view larger image.
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 hence provide variable results (Figure 6a). This drawback will likely remain and is to be expected at times. However, replacing the mouse in its home-cage and allowing it time to resettle will easily correct this. It is crucial that the animal feels comfortable within the chamber environment to obtain five seconds of appropriate/useable data. Further consideration is required regarding the chamber environment. The environment, unrelated to respiratory mechanics, can significantly affect results. As the duration of assessment increases, the humidity and temperature of the chamber increases, as well as decreasing available oxygen, significantly impacting on ventilation. The timing of ventilation increases the humidity and temperature of the chamber as well as decreasing available oxygen21. A small leak in the chamber can aid in reducing thermal drifts created from heat production22,23. The protocol discussed is specific to the equipment and instrumentation listed. Calibration of the bridge amplifier in section two will depend upon the reader’s equipment. If factors such as the tubing length are different a 300 µl injection of air may not cause a 1 cm.H2O deflection.
There are also physiological differences depending on the time of analysis. Rodents are naturally nocturnal creatures and circadian cycles, which ultimately generate changes in respiration, should be taken into account when timing the experiments24. It is thus necessary to time and plan the experiments such that experimental data can be accurately compared between cohorts. It is also important to take note of the trace movement. If the oscillations are not running in a linear pattern, it is usually due to a build up of condensation or humidity within the chamber (Figure 6b), or ineffective seal on the chamber. Ultimately, these limitations can be accounted for and the UWBP process performed appropriately to provide precise respiratory measurement. It is also important to note that this method will require modification (smaller chamber size) for measuring respiratory changes in neonatal standard laboratory mice (e.g., <2 weeks C57Bl/6) in order to detect pressure changes in the respiration of animals of that size.
Although UWBP demonstrates considerable advantages it also carries controversy. Investigators should familiarize themselves with the debate and make an informed decision whether this technique is appropriate for the research question. Initially, Drorbaugh and Fenn (1955)8 believed that an increase in the chamber pressure is caused by inspired air being warmed and humidified to pulmonary values; the opposite occurred in expiration. This allowed the calculation of tidal volume. Subsequent research considered that the pressure changes were caused by changing alveolar pressure during the generation of airflow25. This work stated the use of plethysmography for airway resistance calculation. Enhorning et al. (1998)26 provided evidence that tidal volume, respiratory rate and airway resistance all influence pressure fluctuations within the plethysmography chamber. When the air in the chamber is heated and humidified to body conditions pressure fluctuations are reduced by two-thirds and are amplified through increased resistances21. As all these components reflect pressure fluctuations there is controversy whether measurements of a particular respiratory parameters are accurate. As a result it has been concluded that tidal volume obtained from plethysmography is a qualitative rather than quantitative assessment26. Both upper and lower airway resistances are components of the plethysmography system creating uncertainty in measuring bronchoconstriction27. It is of the author’s opinion that UWBP should be used reciprocally with invasive analyses. It is, in fact, the policy of certain journals that manuscripts solely based upon UWBP data will not be accepted. This will be another consideration for the reader.
In summary, UWBP is a useful method to measure changes to respiratory parameters in standard laboratory rodents, particularly amenable to longitudinal studies. Key advantages of this technique are the avoidance of invasive procedures, chemical challenges and requirement of anesthesia. This allows the researchers to collect physiological data that most closely represent naturally occurring events and reduce experimental variability.
The authors have nothing to disclose.
We would like to thank Prof David Walker for his technical advice and provision of equipment in the development of this technique. This work is supported by the Victorian Government’s Operational Infrastructure Support Program. This work was partly supported by the Victorian Government’s Operational Infrastructure Support Program.
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 (1m) | 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. | |
1ml Syringe | Becton Dickinson (BD) | 309628 | |
5ml 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: 50mm(w) x 1500mm(l). There are two lids for each side, with dimensions 80mm(l) x 80mm(w). Each lid has a 60mm wide circular hole cut on the face of the lid 50mm 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 5mm in diameter. For attachment of humidity probe, the openings are 25mm in diameter. | |
80% Ethanol (4L) | VWR International, Pty Ltd | BDH1162-4LP |