This work is supported by grants of OU General Fund G110 and Research Excellence Fund of Biomedical Research and OSU-HRS Fund 013000. The authors would like to thank Lauren Chen for her assistance in preparing this manuscript.

All procedures involving animal subjects were approved and completed in accordance and compliance with The Ohio State University Institutional Animal Care and Use Committee (IACUC) regulations and guidelines.
1.&#160;Mouse Anesthesia
<ol>
	<li>Set up a clean procedure table with a heated isothermal pad wrapped in a surgical towel. The heating pad should be maintained between 30 &#176;C and 34 &#176;C to stabilize the animal&#39;s core temperature while reducing potential stress to the animal...

<ol>
	<li>Snyder, C. 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=Complementarity+of+ultrasound+and+fluorescence+imaging+in+an+orthotopic+mouse+model+of+pancreatic+cancer.">Complementarity of ultrasound and fluorescence imaging in an orthotopic mouse model of pancreatic cancer.</a> BMC Cancer. 9, 106 (2009).</li><li>Franchi, 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=Non-invasive+assessment+of+cardiac+function+in+a+mouse+model+of+renovascular+hypertension.">Non-invasive assessment of cardiac function in a mouse model of renovascular hypertension.</a> Hypertension Research: Official Journal of the Japanese Society of Hypertension. , (2013).</li><li>Coatney, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+imaging:+principles+and+applications+in+rodent+research.">Ultrasound imaging: principles and applications in rodent research.</a> ILAR Journal / National Research Council, Institute of Laboratory Animal Resources. 42, 233-247 (2001).</li><li>Morenz, 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=Detection+of+air+trapping+in+chronic+obstructive+pulmonary+disease+by+low+frequency+ultrasound.">Detection of air trapping in chronic obstructive pulmonary disease by low frequency ultrasound.</a> BMC Pulmonary Medicine. 12, 8 (2012).</li><li>Gilliam, L. A., Moylan, J. S., Ann Callahan, L., Sumandea, M. P., Reid, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doxorubicin+causes+diaphragm+weakness+in+murine+models+of+cancer+chemotherapy.">Doxorubicin causes diaphragm weakness in murine models of cancer chemotherapy.</a> Muscle &amp; Nerve. 43, 94-102 (2011).</li><li>Ferreira, L. F., Campbell, K. S., Reid, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+sulfur-containing+antioxidants+in+delaying+skeletal+muscle+fatigue.">Effectiveness of sulfur-containing antioxidants in delaying skeletal muscle fatigue.</a> Medicine and Science in Sports and Exercise. 43, 1025-1031 (2011).</li><li>Zuo, L., Clanton, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+formation+in+the+transition+to+hypoxia+in+skeletal+muscle.">Reactive oxygen species formation in the transition to hypoxia in skeletal muscle.</a> American Journal of Physiology. Cell Physiology. 289, 207-216 (2005).</li><li>Helms, M. N., Torres-Gonzalez, E., Goodson, P., Rojas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+tracheal+instillation+of+solutes+into+mouse+lung.">Direct tracheal instillation of solutes into mouse lung.</a> J. Vis. Exp. , (2010).</li><li>Hedrick, W. R., Hykes, D. L., Starchman, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ultrasound Physics and Instrumentation. , 445 (2005).</li><li>von Sarnowski, B., Khaw, A. V., Kessler, C., Schminke, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+microconvex+array+transducer+for+the+ultrasonographic+examination+of+the+intrathoracic+segments+of+the+supraaortic+arteries.">Evaluation of a microconvex array transducer for the ultrasonographic examination of the intrathoracic segments of the supraaortic arteries.</a> Journal of Neuroimaging: Official Journal of the American Society of Neuroimaging. 20, 246-250 (2010).</li><li>Stocksley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Abdominal Ultrasound. , 7-8 (2001).</li><li>Kremkau, F. W., Taylor, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artifacts+in+ultrasound+imaging.">Artifacts in ultrasound imaging.</a> Journal of Ultrasound in Medicine: Official Journal of the American Institute of Ultrasound in Medicine. 5, 227-237 (1986).</li><li>Kremkau, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+Ultrasound:+Principles+and+Instruments.">Diagnostic Ultrasound: Principles and Instruments.</a> Saunders Elsevier. , 521 (2006).</li><li>Laing, F. C., Kurtz, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+ultrasonic+side-lobe+artifacts.">The importance of ultrasonic side-lobe artifacts.</a> Radiology. 145, 763-768 (1982).</li><li>Abu-Zidan, F. M., Hefny, A. F., Corr, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+ultrasound+physics.">Clinical ultrasound physics.</a> Journal of Emergencies, Trauma, and Shock. 4, 501-503 (2011).</li><li>Gargani, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+ultrasound:+a+new+tool+for+the+cardiologist.">Lung ultrasound: a new tool for the cardiologist.</a> Cardiovascular Ultrasound. 9, 6 (2011).</li><li>Sanders, R. C., Winter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Clinical Sonography A Practical Guide. , 632 (2007).</li></ol>

The current experimental protocol develops diagnostic ultrasound imaging techniques specific to the diaphragm activity in a mouse model via a non-invasive, in vivo approach. The anesthesia apparatus settings are approximated values, which may be slightly adjusted for each animal since individual mice may respond differently to anesthesia. To prevent improper anesthesia administration, it is important to regularly monitor the mouse&#39;s vital signs including the heart rate, respiration rate, and body temperature...

Recently, diagnostic ultrasound imaging techniques have been applied to mouse models of renovascular hypertension and pancreatic cancer1,2.&#160;However, these techniques have not been widely used in rodent respiratory muscle function assay. Therefore, we have developed a diagnostic ultrasound imaging method as a valuable tool for in vivo longitudinal assessments of diaphragm mobility in mice.
There are several advantages to diagnostic ultrasound imaging. For instance, it is noninvasive, safe, portable, and allows for real time measurements at a relatively low cost3. Particularly, certain low frequency ultrasound devices were able to detect air trapping, a clinical characteristic of chronic obstructive pulmonary disease (COPD) with mild to severe airflow limitation4. Thus, diagnostic ultrasound imaging may serve as an easily accessible and reproducible screening method for real-time monitoring of respiratory disorders.
Diagnostic ultrasound imaging techniques are frequently applied to larger animals or human subjects. However, there have been a limited number of ultrasound imaging studies on mouse models, which is likely due to the challenges of performing ultrasound on small-scale subjects. The current protocol outlines a novel procedure for measuring diaphragm function in the mouse. In addition, although there have been several rodent studies on diaphragm function, most of the results were generated by isolating muscle strips directly from the euthanized animal5-7. In contrast, using an in vivo diagnostic ultrasound imaging method for analyzing diaphragm activity would decrease the number of animals sacrificed for experimentation. Furthermore, long-term treatments focused on enhancing diaphragm contractility may be accurately assessed via ultrasound in rodent models without sacrificing animals.
In our lab, we have developed an effective method for visualizing as well as analyzing mouse diaphragm activity using an ultrasound machine, which helps the understanding of diaphragm function in vivo, avoids invasive methods to animals, and aids in the development of therapeutic treatments for respiratory dysfunction.

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	<tbody>
		<tr height="127">
			<td height="127" style="height:127px;width:147px;">Veterinary digital ultrasonic diagnostic imaging system</td>
			<td style="width:111px;">Edan</td>
			<td style="width:151px;">DUS 3 VET</td>
			<td style="width:196px;">Ultrasound parameters include: frequency of 6.5 MHz, Depth of 29 mm. Note: An equivalent ultrasound machine may be used for this protocol</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:147px;">Micro-convex array transducer</td>
			<td>Edan</td>
			<td>C611</td>
			<td>Or equivalent</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:147px;">GE Logiq i hand-carried unit (HCU)&#160;</td>
			<td>GE Healthcare</td>
			<td style="width:151px;">GE Logiq i hand-carried unit (HCU)&#160;</td>
			<td>Or equivalent</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:147px;">GE 12 MHz linear array probe</td>
			<td>GE Healthcare</td>
			<td style="width:151px;">12L-RS</td>
			<td>Or equivalent</td>
		</tr>
		<tr height="171">
			<td height="171" style="height:171px;width:147px;">Veterinary anesthetic vaporizer</td>
			<td style="width:111px;">Webster Veterinary</td>
			<td>Serial #: W422021</td>
			<td style="width:196px;">Isoflurane was exclusively used with this vaporizer (or equivalent). A custom made induction chamber for anesthesia was assembled for initial anesthesia. Maintenance anesthesia was performed using a nose cone</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:147px;">Isothesia (Isoflurane, USP)</td>
			<td>Butler Schein</td>
			<td>29405 250ML PVL</td>
			<td>Or equivalent</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:147px;">Enviro-pure anesthesia absorbing canister</td>
			<td style="width:111px;">Surgivet Smiths Medical PM, Inc.</td>
			<td>Part #: 32373B10</td>
			<td>Or equivalent</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:147px;">Ultrasound transmission gel</td>
			<td>HM Sonic</td>
			<td>N/A</td>
			<td>Or equivalent&#160;</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:147px;">Puralube vet ointment</td>
			<td>Puralube&#160;</td>
			<td>NDC 17033-211-38</td>
			<td>Or equivalent</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:147px;">Deltaphase isothermal pad</td>
			<td style="width:111px;">Braintree Scientific Inc.</td>
			<td>39DP</td>
			<td>Or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hair remover</td>
			<td>Nair</td>
			<td>N/A</td>
			<td>Or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Electric razor</td>
			<td>Remington</td>
			<td>HC-5015</td>
			<td>Or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical tape</td>
			<td>3M Micropore</td>
			<td>1530-1</td>
			<td>Or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gauze sponges</td>
			<td>Dynarex</td>
			<td>3262</td>
			<td>Or equivalent</td>
		</tr>
	</tbody>
</table>

diagnostic ultrasound imaging of mouse diaphragm function

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive surgical procedures. Although this is an effective method of assessing in vitro diaphragm activity, it involves non-survival surgery. The application of non-invasive ultrasound imaging as an in vivo procedure is beneficial since it not only reduces the number of animals sacrificed, but is also suitable for monitoring disease progression in live mice. Thus, our ultrasound imaging method may likely assist in the development of novel therapies that alleviate muscle injury induced by various respiratory diseases. Particularly, in clinical diagnoses of obstructive lung diseases, ultrasound imaging has the potential to be used in conjunction with other standard tests to detect the early onset of diaphragm muscle fatigue. In the current protocol, we describe how to accurately evaluate diaphragm contractility in a mouse model using a diagnostic ultrasound imaging technique.

A typical ultrasound image of a mouse diaphragm is shown in Figure&#160;1A. The mouse diaphragm maximal vertical displacement was recorded. This distance was calculated by precisely measuring the depth of diaphragm movement from relaxation to contraction using the electronic calipers that are part of the ultrasound software. Table 1 displays these distance measurements of diaphragmatic contractions from three different mice. After converting the cine loop file into a MPEG file, the respi...

Watch this Scientific Journal Video about Diagnostic Ultrasound Imaging of Mouse Diaphragm Function at JoVE.com

Diagnostic Ultrasound Imaging of Mouse Diaphragm Function

Diagnostic ultrasound imaging has proven to be effective in diagnosing various respiratory diseases in human and animal subjects. We demonstrate a comprehensive ultrasound protocol utilized by Dr. Zuo's lab to analyze diaphragm kinetics specifically in mouse models. This is also a non-invasive research technique which can provide quantitative information on mouse respiratory muscle function.

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive ...

diagnostic-ultrasound-imaging-of-mouse-diaphragm-function

Research

JoVE Journal

Medicine

15.8K vistas.  The Ohio State University College of Medicine.  De imágenes de ultrasonido de diagnóstico ha demostrado ser eficaz en el diagnóstico de diversas enfermedades respiratorias en sujetos humanos y animales. Demostramos un protocolo integral de ultrasonido utilizado por el laboratorio del Dr. Zuo para analizar la cinética de diafragma específicamente en modelos de ratón. Esta es también una técnica de investigación no invasiva que puede proporcionar información cuantitativa sobre la función de los músculos resp...