Name: a simple and low-cost assay for measuring ambulation in mouse models of muscular dystrophy
Uploaded: 2017-12-29T14:00:00
Description: Watch this Scientific Journal Video about A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy at JoVE.com

This work was supported by grants from the National Institutes of Health [R01 AR048179 and R01 HL126204 to R.C.W, T32 AR059033 and F32 AR069469to E.M.G] and the Muscular Dystrophy Association USA [274143 and 416364 to R.C.W.].

The methods described here were approved by the Institutional Animal Care and Use Committee at the University of California, Los Angeles. Wild-type (C57BL/6J) and mdx mice (C57Bl/10ScSn background) were purchased from a commercial source. mdx:utrophin-Tg mice were a gift from James Ervasti, with permission from Kay Davies, and were maintained on the mdx background. Ambulation assays were performed on male mice at 6 months of age. Mice were maintained in the Terasaki Life Sciences Vivarium follo...

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The open field assay provides an effective and non-invasive way to test motor function in mouse models of muscle disease, and variations of this assay can serve as a valuable endpoint measure for pre-clinical studies. One major limitation with this type of assay is the high-cost and limited accessibility of activity monitoring systems. In this study, we demonstrate a low-cost system for open field activity analysis that can generate similar results to existing commercial software and equipment. This system can be used wi...

Reliable and reproducible measurements of muscle function are critical for evaluating the efficacy of potential therapies for DMD. DMD is a genetic disorder caused by mutations in the dystrophin gene, leading to progressive muscle weakness, loss of ambulation, and eventual cardiorespiratory failure. The most widely used animal model of DMD is the dystrophin-null mdx mouse. A battery of functional tests have emerged as routine assays for evaluating disease progression in the mdx mouse as well as in similar animal models of other dystrophies and myopathies. Commonly used in vivo assays include measurements of forelimb grip strength, wire hanging time, rotarod maximum, time to exhaustion during treadmill running, and motor activity tracking. There has been a substantial effort in the field to standardize these tests, with the goal of reducing variability between preclinical studies and increasing the translational potential of therapeutics tested in mice1,2.
One important category of preclinical testing is the measurement of voluntary movement, a parameter that is frequently altered in murine models of muscular dystrophy. This is usually tested by assays based on open field activity monitoring, and may evaluate either horizontal (walking) or vertical (rearing) movements over a course of minutes or hours2,3,4. A number of studies have shown voluntary movement to be altered in mdx mice, particularly after exercise, and these measurements have been shown to be sensitive to drug treatment and disease progression. One major limitation in performing these assays is the need for specialized, high-cost equipment. Here, a low-cost method that tracks mouse ambulation using readily available resources is presented.
The 6-minute walk distance is a metric commonly used as a clinical assessment tool in individuals with Duchenne muscular dystrophy5,6. Modifications of this measure have been used to assess outcomes in animal models of Duchenne, including mdx mice7 and golden retriever muscular dystrophy (GRMD) dogs8. In this study, we record voluntary open field movement in the 6 minutes immediately following a mild exercise challenge. Ambulation distance was then calculated using free open-source software to measure horizontal movement over time.
The main advantage of this method is that animals can be tested in a variety of settings without needing specialized equipment or high-cost commercial software for analysis. One important aspect of this analysis is that it can be performed in a basic laboratory setting without needing to move or transfer mice out of the vivarium to a specialized core facility. The video-tracking protocol described here is well-suited to the assessment of ambulation over relatively short time-periods and can detect activity differences between wild-type and mdx mice, as well as reveal functional improvement in a rescue model of DMD.

<table>
	<tbody>
		<tr>
			<td>Video camera</td>
			<td>Apple Inc.</td>
			<td>ME553LL/A</td>
			<td>For recording ambulation video, iPhone 5S Plus (or equivalent)</td>
		</tr>
		<tr>
			<td>Kinovea software (version 0.8.15)</td>
			<td>Kinovea Association</td>
			<td></td>
			<td>Open source video analysis software. Free download, PC compatible (Version 0.8.15, www.kinovea.org)</td>
		</tr>
		<tr>
			<td>iMovie (version 10.0.6)</td>
			<td>Apple Inc.</td>
			<td></td>
			<td>Any similar software can be used to reduce video frame rate (optional)</td>
		</tr>
		<tr>
			<td>Roughneck 32 Gallon Black Round Trash Bin (Open field chamber)</td>
			<td>Rubbermaid</td>
			<td># 1778013</td>
			<td>Any open field chamber system can be adapted for recording. This study uses a recording chamber constructed out of a tray on a platform, at the bottom of a large trash bin.</td>
		</tr>
		<tr>
			<td>Avant White Plastic Tray 15&#34;W x 10&#34;D x 1.45&#34;H (Open field chamber)</td>
			<td>US Acrylic, LLC</td>
			<td></td>
			<td>Any open field chamber system can be adapted for recording. This study uses a recording chamber constructed out of a tray on a platform, at the bottom of a large trash bin.</td>
		</tr>
		<tr>
			<td>C57BL/6J</td>
			<td>Jackson Laboratory</td>
			<td>#000664</td>
			<td>Male 6 month mice</td>
		</tr>
		<tr>
			<td>C57BL/10ScSn-Dmd/J (mdx)</td>
			<td>Jackson Laboratory</td>
			<td>#001801</td>
			<td>Male 6 month mice</td>
		</tr>
		<tr>
			<td>mdx: utrophin-Tg (fiona)</td>
			<td>Gift from from James Ervasti, with permission from Kay Davies</td>
			<td></td>
			<td>Male 6 month mice</td>
		</tr>
	</tbody>
</table>

a simple and low-cost assay for measuring ambulation in mouse models of muscular dystrophy

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation in mouse models is a non-invasive and reproducible activity assay that is directly analogous to measures of patient ambulation such as the 6-minute walk test and related mobility scores. Many common methods for testing mouse ambulation speed and distance are based on the open field test, where an animal&#39;s free movement within an arena is measured over time. One major downside to this approach is that commercial software and equipment for high-resolution motion tracking is expensive and may require transferring mice to specialized facilities for testing. Here, we describe a low-cost, video-based system for measuring mouse ambulation that utilizes free and open-source software. Using this protocol, we demonstrate that voluntary ambulation in the dystrophin-null mdx mouse model for Duchenne muscular dystrophy (DMD) is decreased relative to wild-type mouse activity. In mdx mice expressing the utrophin transgene, these activity deficits are not observed and the total distance traveled is indistinguishable from wild-type mice. This method is effective for measuring changes in voluntary ambulation associated with dystrophic pathology, and provides a versatile platform that can be readily adapted to diverse research settings.

Variations of the open field assay have been shown to be an effective method to test the disease progression in models of muscular dystrophy. Here, we demonstrate a versatile video tracking method for the analysis of two-dimensional mouse movement using readily available recording equipment and open source software (Figure 1A-B). Consistent with results reported from similar assays, we show that mdx mice challenged w...

Watch this Scientific Journal Video about A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy at JoVE.com

A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy

This protocol describes a flexible, low-cost system for measuring mouse ambulation in an open field activity assay. We show that a 6-minute ambulation assay based on this system detects a decrease in voluntary movement in mdx mice, and accurately distinguishes improvement in a muscle-specific rescue of these animals.

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation ...

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