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&#8217;s Operational Infrastructure Support Program. This work was partly supported by the Victorian Government&#8217;s Operational Infrastructure Support Program.

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.
...

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> World Health Organization, World Health Statistics. , (2008).</li><li>Jones, C. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M2+macrophage+polarization+is+associated+with+alveolar+formation+during+postnatal+lung+development.">M2 macrophage polarization is associated with alveolar formation during postnatal lung development.</a> Respir. Res. 14 (41), 14-41 (2013).</li><li>Campbell, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+factor-induced+airway+hyperreactivity+in+allergic+and+normal+mice.">Stem cell factor-induced airway hyperreactivity in allergic and normal mice.</a> Am. J. Pathol. 154 (4), 1259-1265 (1999).</li><li>Card, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclooxygenase-2+deficiency+exacerbates+bleomycin-induced+lung+dysfunction+but+not+fibrosis.">Cyclooxygenase-2 deficiency exacerbates bleomycin-induced lung dysfunction but not fibrosis.</a> Am. J. Respir. Cell. Mol. Biol. 37 (3), 300-308 (2007).</li><li>Berndt, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+unrestrained+plethysmography+and+forced+oscillation+for+identifying+genetic+variability+of+airway+responsiveness+in+inbred+mice.">Comparison of unrestrained plethysmography and forced oscillation for identifying genetic variability of airway responsiveness in inbred mice.</a> Physiol. Genomics. 43 (1), 1-11 (2011).</li><li>Flandre, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+somatic+growth,+strain,+and+sex+on+double-chamber+plethysmographic+respiratory+function+values+in+healthy+mice.">Effect of somatic growth, strain, and sex on double-chamber plethysmographic respiratory function values in healthy mice.</a> J. Appl. Physiol. 94 (3), 1129-1136 (2003).</li><li>Petak, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperoxia-induced+changes+in+mouse+lung+mechanics:+forced+oscillations+vs.+barometric+plethysmography.">Hyperoxia-induced changes in mouse lung mechanics: forced oscillations vs. barometric plethysmography.</a> J. Appl. Physiol. 90 (6), 2221-2230 (2001).</li><li>Drorbaugh, J. E., Fenn, W. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+barometric+method+for+measuring+ventilation+in+newborn+infants.">A barometric method for measuring ventilation in newborn infants.</a> Pediatrics. 16 (1), 81-87 (1955).</li><li>Milton, P. L., Dickinson, H., Jenkin, G., Lim, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+respiratory+physiology+of+C57BL/6+mice+following+bleomycin+administration+using+barometric+plethysmography.">Assessment of respiratory physiology of C57BL/6 mice following bleomycin administration using barometric plethysmography.</a> Respiration. 83 (3), 253-266 (2012).</li><li>Gargiulo, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+anesthesia,+analgesia,+and+care,+part+I:+anesthetic+considerations+in+preclinical+research.">Mice anesthesia, analgesia, and care, part I: anesthetic considerations in preclinical research.</a> ILAR J. 53 (1), 55-69 (2012).</li><li>Hildebrandt, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+and+other+considerations+for+in+vivo+imaging+of+small+animals.">Anesthesia and other considerations for in vivo imaging of small animals.</a> ILAR J. 49 (1), 17-26 (2008).</li><li>Meijer, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+restraint+and+injection+methods+on+heart+rate+and+body+temperature+in+mice.">Effect of restraint and injection methods on heart rate and body temperature in mice.</a> Lab Anim. 40, 382-391 (2006).</li><li>Hamelmann, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+measurement+of+airway+responsiveness+in+allergic+mice+using+barometric+plethysmography.">Noninvasive measurement of airway responsiveness in allergic mice using barometric plethysmography.</a> Am. J. Respir. Crit. Care Med. 156 (3), 766-775 (1997).</li><li>Lim, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+mesenchymal+stem+cells+reduce+lung+injury+in+immunocompromised+mice+but+not+in+immunocompetent+mice.">Human mesenchymal stem cells reduce lung injury in immunocompromised mice but not in immunocompetent mice.</a> Respiration. 85 (4), 332-341 (2013).</li><li>Murphy, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+amnion+epithelial+cells+prevent+Bleomycin-induced+lung+injury+and+preserve+lung+function.">Human amnion epithelial cells prevent Bleomycin-induced lung injury and preserve lung function.</a> Cell Transplant. 20, 909-923 (2011).</li><li>Murphy, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+amnion+epithelial+cells+do+not+abrogate+pulmonary+fibrosis+in+mice+with+impaired+macrophage+function.">Human amnion epithelial cells do not abrogate pulmonary fibrosis in mice with impaired macrophage function.</a> Cell Transplant. 21 (7), 1477-1492 (2012).</li><li>Wichers, L. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+exposing+rodents+to+resuspended+particles+using+whole-body+plethysmography.">A method for exposing rodents to resuspended particles using whole-body plethysmography.</a> Part. Fibre Toxicol. 13 (12), (2006).</li><li>Chong, B. T. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+bronchoconstriction+using+whole-body+plethysmograph:+comparison+of+freely+moving+versus+restrained+guinea+pigs.">Measurement of bronchoconstriction using whole-body plethysmograph: comparison of freely moving versus restrained guinea pigs.</a> J. Pharmacol. Toxicol. Methods. 39 (3), 163-168 (1998).</li><li>Lizuka, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+respiratory+function+using+whole-body+plethysmography+in+unanesthetized+and+unrestrained+nonhuman+primates.">Measurement of respiratory function using whole-body plethysmography in unanesthetized and unrestrained nonhuman primates.</a> J. Toxicol. Sci. 35 (6), 863-870 (2010).</li><li>McGregor, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+prenatal+exposure+to+carbon+monoxide+on+breathing+and+growth+of+the+newborn+guinea+pig.">The effect of prenatal exposure to carbon monoxide on breathing and growth of the newborn guinea pig.</a> Pediatr. Res. 43, 126-131 (1998).</li><li>Lundblad, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reevaluation+of+the+validity+of+unrestrained+plethysmography+in+mice.">A reevaluation of the validity of unrestrained plethysmography in mice.</a> J. Appl. Physiol. 93, 1198-1207 (2002).</li><li>Bartlett, D., Tenney, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+breathing+in+experimental+anemia.">Control of breathing in experimental anemia.</a> Respir. Physiol. 10 (3), 384-395 (1970).</li><li>Malan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventilation+measured+by+body+plethysmography+in+hibernating+mammals+and+in+poiiulotherms.">Ventilation measured by body plethysmography in hibernating mammals and in poiiulotherms.</a> Respir. Physiol. 17 (1), 32-44 (1973).</li><li>Seifert, E. L., Mortola, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+circadian+pattern+of+breathing+in+conscious+adult+rats.">The circadian pattern of breathing in conscious adult rats.</a> Respir. Physiol. 129 (3), 297-305 (2002).</li><li>DuBois, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+measuring+airway+resistance+in+man+using+a+body+plethysmograph:+Values+in+normal+subject+and+in+patients+with+respiratory+disease.">A new method for measuring airway resistance in man using a body plethysmograph: Values in normal subject and in patients with respiratory disease.</a> J. Clin. Invest. 35 (3), 327-335 (1956).</li><li>Enhorning, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-body+plethysmography,+does+it+measure+tidal+volume+of+small+animals.">Whole-body plethysmography, does it measure tidal volume of small animals.</a> Can. J. Physiol. Pharmacol. 76 (10-11), 945-951 (1998).</li><li>Zhang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+unrestrained+single-chamber+plethysmography+provide+a+valid+assessment+of+airway+responsiveness+in+allergic+BALB/c+mice.">Does unrestrained single-chamber plethysmography provide a valid assessment of airway responsiveness in allergic BALB/c mice.</a> Respir. Res. 10 (61), (2009).</li></ol>

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...

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 &#38; 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&#160;hr.

<table border="0" cellpadding="0" cellspacing="0" width="1031">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:263px;">LabChart 7 software (for Macintosh)</td>
			<td style="width:139px;">ADINSTRUMENTS</td>
			<td style="width:155px;">MLU60/7</td>
			<td style="width:475px;">used in protocol step 4</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">PowerLab 8/30 (model ML870)</td>
			<td>ADINSTRUMENTS</td>
			<td>PL3508</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Octal Bridge Amp (model ML228)</td>
			<td>ADINSTRUMENTS</td>
			<td>FE228</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Black BNC to BNC cable (1m)</td>
			<td>ADINSTRUMENTS</td>
			<td style="width:155px;">MLAC01&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Macintosh OS&#160;</td>
			<td>Apple Inc.&#160;</td>
			<td>-</td>
			<td style="width:475px;">Mac OS X 10.4 or later</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Surgipack Digital Rectal Thermometer&#160;</td>
			<td>Vega Technologies</td>
			<td>MT-918</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Grass volumeteric pressure transducer PT5A</td>
			<td>Grass Instruments Co.</td>
			<td style="width:155px;">Model number PT5A; serial No. L302P4.</td>
			<td style="width:475px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1ml Syringe</td>
			<td>Becton Dickinson (BD)</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:263px;">5ml serological syringe pipettes</td>
			<td>Greiner Bio One</td>
			<td>606160</td>
			<td style="width:475px;">Connected via plastic tubing</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Balance/Scales</td>
			<td>VWR International, Pty Ltd</td>
			<td>SHIMAUW220D</td>
			<td style="width:475px;">Any weighing balance with of 0.1 gram resolution</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">HM40 Humidity &#38; temperature meter&#160;</td>
			<td>Vaisala&#160;</td>
			<td>HM40A1AB</td>
			<td style="width:475px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Barometer</td>
			<td>Barometer World</td>
			<td>1586</td>
			<td></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">Laboratory tubing</td>
			<td>Dow Corning&#160;</td>
			<td>508-101</td>
			<td style="width:475px;">Used to connect water column to the syringe and pressure transducer</td>
		</tr>
		<tr height="136">
			<td height="136" style="height:136px;width:263px;">Cylindrical Perspex Chamber</td>
			<td>Dynalab Corp.</td>
			<td></td>
			<td style="width:475px;">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.&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">80% Ethanol (4L)</td>
			<td>VWR International, Pty Ltd</td>
			<td>BDH1162-4LP</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring respiratory function in mice using unrestrained whole-body plethysmography

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.

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...

Watch this Scientific Journal Video about Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography at JoVE.com

Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography

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 ...

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29.9K visualizações.  Monash Institute of Medical Research.  A avaliação da fisiologia respiratória, tradicionalmente invocado técnicas, que exigem contenção ou sedação do animal. Unrestrained pletismografia de corpo inteiro, no entanto, fornece uma análise precisa, não-invasivo, quantitativo de fisiologia respiratória em modelos animais. Além disso, a técnica permite a avaliação repetida respiratório de ratos permitindo estudos longitudinais. 