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 following guidelines established by the Institutional Animal Care and Use Committee at the University of California, Los Angeles (approval #2000-029-43) and approval for these studies was granted by the UCLA Animal Welfare Assurance (approval #A3196-01).
1. Preparation of Chamber
<ol>
	<li>Perform data collection in a quiet, temperature controlled room at a consistent time of day9. Perform the tests blinded to the genotype and treatment of the mouse being tested.</li>
	<li>Adapt any open field chamber system for this analysis. 
	NOTE: In this study, we use an inexpensive and easily transportable recording chamber constructed from a large trash bin. The open field arena is a tray set on a cage or similar elevated platform.</li>
	<li>Place the camera on a wire mesh above the chamber to record across the full arena.</li>
	<li>Clean the recording chamber with disinfectant before each trial.</li>
</ol>
2. Pre-exercise Protocol and Data Collection
<ol>
	<li>Optionally, immediately prior to activity recording, challenge each mouse with a muscle activation protocol. 
	NOTE: This is optional but recommended in experiments involving mdx mice.
	<ol>
		<li>Allow the mouse to grip the pull bar of a digital force gauge and gently pull back until the pull bar is released. Repeat this procedure five times per trial.</li>
		<li>Record peak tension (N) for each trial. 
		NOTE: In addition to serving as an exercise challenge for the ambulation assay, the force exerted by the animal during this grip strength assay can be used as an additional functional outcome measure2,10.</li>
		<li>Perform five total trials for each mouse with 1 min of rest between each trial.</li>
	</ol>
	</li>
	<li>Immediately following the grip strength assay or other exercise protocol, place the mouse in the activity chamber.</li>
	<li>Start the video recording of the movement of the mouse in the chamber arena. Allow the mouse to freely explore for 6 min.</li>
	<li>Stop the recording at 6 min and return the mouse to its home cage.</li>
</ol>
3. Video Analysis
<ol>
	<li>Optionally, prepare video for analysis by reducing the frame rate. In order to reduce the frame rate for tracking analysis, use the following protocol in a video editing software (e.g., iMovie or similar program) to decimate the video frame rate by a factor of 2. 
	NOTE: Depending on length and frame rate of the video, it may be useful to reduce the video frame rate prior to analysis. In this study, video was recorded at 30 frames/s (6 min recording, approximately 10,800 frames total).
	<ol>
		<li>Load the video into the software.</li>
		<li>Under the &#39;Speed&#39; menu, select &#39;Speed: Fast&#39; and set speed to 2x.</li>
		<li>Export the decimated video in .mp4 file format for tracking analysis.</li>
	</ol>
	</li>
	<li>Open the video for analysis in the software program. Set the calibration for the video recording by using the line tool. Draw a line along one side of the chamber. Right click on the line and select &#34;Calibrate measure&#8230;&#34; Enter the actual size of the chamber side in centimeters.</li>
	<li>To begin semi-automated tracking of the mouse position, click on the &#39;move&#39; cursor. 
	Starting at the initial frame in the video, right click on the point to be tracked; the tracking point is marked with a blue circle. 
	NOTE: In the present study, the position of each animal was tracked by tracing the base of the tail.</li>
	<li>Advance the frame by clicking the right arrow on the keyboard; the tracking point should automatically move based on the position of the tail base.
	<ol>
		<li>If the tracking position is not aligned with the point of interest in a given frame, manually align the blue circle to the base of the tail. 
		NOTE: Depending on the quality of the video and the speed of the mouse, the tracking may require variable levels of user input to maintain alignment with the point of interest. The path of the mouse should advance through the course of the video.</li>
	</ol>
	</li>
	<li>When tracking of the full video is complete, save the video and tracing overlay. Export the positional data of the tracing by selecting &#34;Export to Spreadsheet&#34;.</li>
</ol>
4. Data Analysis
<ol>
	<li>Open the positional data in a spreadsheet software program. The movement analysis software reports the X,Y coordinate position of the mouse in each frame.</li>
	<li>To calculate distance traveled by frame, use the following equation (where position in frame one is x1,y1 and frame two is x2,y2): 
	<img alt="Equation" src="/files/ftp_upload/56772/56772eq1.jpg" /> 
	NOTE: Cumulative distance over time can be calculated by adding the distance traveled between each frame.</li>
</ol>

<ol>
	<li>Nagaraju, K., Carlson, G., De Luca, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+and+locomotor+measurements+using+open+field+animal+activity+monitoring+system.">Behavioral and locomotor measurements using open field animal activity monitoring system.</a> TREAT-NMD SOP Number M2.1.002. 2. 2, (2010).</li><li>Tatem, K. 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=Behavioral+and+locomotor+measurements+using+an+open+field+activity+monitoring+system+for+skeletal+muscle+diseases.">Behavioral and locomotor measurements using an open field activity monitoring system for skeletal muscle diseases.</a> J Vis Exp. (91), e51785 (2014).</li><li>Kobayashi, Y. M., 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=Sarcolemma-localized+nNOS+is+required+to+maintain+activity+after+mild+exercise.">Sarcolemma-localized nNOS is required to maintain activity after mild exercise.</a> Nature. 456 (7221), 511-515 (2008).</li><li>Belanto, J. J., 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=Microtubule+binding+distinguishes+dystrophin+from+utrophin.">Microtubule binding distinguishes dystrophin from utrophin.</a> Proc Natl Acad Sci U S A. 111 (15), 5723-5728 (2014).</li><li>McDonald, C. M., 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+6-minute+walk+test+in+Duchenne/Becker+muscular+dystrophy:+longitudinal+observations.">The 6-minute walk test in Duchenne/Becker muscular dystrophy: longitudinal observations.</a> Muscle Nerve. 42 (6), 966-974 (2010).</li><li>Mazzone, 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=Star+Ambulatory+Assessment,+6-minute+walk+test+and+timed+items+in+ambulant+boys+with+Duchenne+muscular+dystrophy.">Star Ambulatory Assessment, 6-minute walk test and timed items in ambulant boys with Duchenne muscular dystrophy.</a> Neuromuscul Disord. 20 (11), 712-716 (2010).</li><li>Kobayashi, Y. M., Rader, E. P., Crawford, R. W., Campbell, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endpoint+measures+in+the+mdx+mouse+relevant+for+muscular+dystrophy+pre-clinical+studies.">Endpoint measures in the mdx mouse relevant for muscular dystrophy pre-clinical studies.</a> Neuromuscul Disord. 22 (1), 34-42 (2012).</li><li>Acosta, A. 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=Use+of+the+six-minute+walk+test+to+characterize+golden+retriever+muscular+dystrophy.">Use of the six-minute walk test to characterize golden retriever muscular dystrophy.</a> Neuromuscul Disord. 26 (12), 865-872 (2016).</li><li>Sousa, N., Almeida, O. F., Wotjak, C. 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+hitchhiker's+guide+to+behavioral+analysis+in+laboratory+rodents.">A hitchhiker's guide to behavioral analysis in laboratory rodents.</a> Genes Brain Behav. 5, 5-24 (2006).</li><li>Gibbs, E. M., 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=High+levels+of+sarcospan+are+well+tolerated+and+act+as+a+sarcolemmal+stabilizer+to+address+skeletal+muscle+and+pulmonary+dysfunction+in+DMD.">High levels of sarcospan are well tolerated and act as a sarcolemmal stabilizer to address skeletal muscle and pulmonary dysfunction in DMD.</a> Hum Mol Genet. 25 (24), 5395-5406 (2016).</li><li>Gillis, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multivariate+evaluation+of+the+functional+recovery+obtained+by+the+overexpression+of+utrophin+in+skeletal+muscles+of+the+mdx+mouse.">Multivariate evaluation of the functional recovery obtained by the overexpression of utrophin in skeletal muscles of the mdx mouse.</a> Neuromuscul Disord. 12, S90-S94 (2002).</li><li>Tinsley, J., 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=Expression+of+full-length+utrophin+prevents+muscular+dystrophy+in+mdx+mice.">Expression of full-length utrophin prevents muscular dystrophy in mdx mice.</a> Nat Med. 4 (12), 1441-1444 (1998).</li><li>Song, 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=Suite+of+clinically+relevant+functional+assays+to+address+therapeutic+efficacy+and+disease+mechanism+in+the+dystrophic+mdx+mouse.">Suite of clinically relevant functional assays to address therapeutic efficacy and disease mechanism in the dystrophic mdx mouse.</a> J Appl Physiol. 122 (3), 593-602 (2017).</li><li>Bolivar, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrasession+and+intersession+habituation+in+mice:+from+inbred+strain+variability+to+linkage+analysis.">Intrasession and intersession habituation in mice: from inbred strain variability to linkage analysis.</a> Neurobiol Learn Mem. 92 (2), 206-214 (2009).</li></ol>

The authors have nothing to disclose.

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 within vivaria where mice are housed, eliminating the need to transfer animals to core facilities. The versatility of this assay also enables the use of video tracking in a wide variety of research and educational settings, including undergraduate laboratory courses focused on animal physiology and behavior.
In this study, we found that differences in voluntary movement between wild-type and mdx mice can be readily detected following exacerbation by exercise. This robust difference has been reported by other groups looking at similar activity outcomes, including hour-long open field assays and overnight rearing assays. While we observed no difference in voluntary ambulation between unexercised wild-type and mdx mice, this is consistent with results from other groups13. Some researchers have reported a modest difference in voluntary ambulation distance between unexercised wild-type and mdx mice7, while others report no difference13. Our findings support the use of muscle activation prior to testing in order to reliably detect a statistically significant difference in mdx activity.
In this study, we presented mice with a novel open field environment. Using this novel environment protocol, we observed very little variability within groups. However, when mice are placed in an unfamiliar environment, they tend to have heightened activity14. If the same animals are to be tested across multiple sessions, animals should be habituated to the recording chamber and undergo the full test protocol multiple times before recording data.
One important consideration in evaluating data from open field assays is that it is highly sensitive to animal behavior and emotional conditions, particularly anxiety-like behaviors. It is possible that the mdx may have a different behavioral response to the novel environment than the wild-type control animals. However, we additionally tested a muscle-specific rescue, the mdx:utr-Tg mouse, which expresses utrophin under a muscle-specific promoter. These mice performed as well as wild-type animals, suggesting that any behavioral differences in mdx mice are not driving the changes in voluntary ambulation.
Open field assays are widely used in behavioral paradigms as well as in studies of motor function, and there are a variety of parameters that can be recorded from activity monitoring systems. Some of these measurements commonly used in commercial activity analysis software include the automated detection of specific behaviors, turn frequencies, vertical movement, and preferences for specific regions of interest. For the 6-minute ambulation distance assay used in this study, the output is largely limited to distance and speed of horizontal ambulation.
Additionally, due to the level of user input required for semi-automated tracking in this protocol, this assay would likely not be well suited for activity monitoring for longer periods of time or measuring multiple animals in parallel. In this study, we found that a trained user could process and extract data from videos in approximately 15-30 min per recording. While this represents only a moderate time burden for small-scale experiments, this level of analysis would rapidly become prohibitive for studies looking at large sample sets over a longer time period. While more fully automated commercial tracking systems typically cost more than 20,000 USD, these solutions may be more cost- and time-effective for laboratories where ambulation is frequently tested. However, for laboratory and educational settings that only test small numbers of animals, the simple set-up and reproducibility of this assay provides a valuable tool for assessing motor behavior.
The types of data analyses available are an important consideration in determining appropriate endpoint assays for a pre-clinical study. While we were able to consistently detect differences between three genotypes using 6 minute ambulation distance (wild-type, mdx, and mdx:utr-tg), a more complex analysis of longer-term activity may be desirable when looking at subtle changes in voluntary movement. However, the accessibility and simplicity of this low-cost set-up make it a useful and highly relevant functional test for pre-clinical studies in the mdx mouse model.

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&quot;W x 10&quot;D x 1.45&quot;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 (&lt;em&gt;mdx&lt;/em&gt;)</td><td>Jackson Laboratory</td><td>#001801</td><td>Male 6 month mice</td></tr><tr><td>&lt;em&gt;mdx:&lt;/em&gt; 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 with an exercise protocol immediately before testing show significantly less voluntary movement in a 6-minute open field task than wild-type mice (Figure 1C, Figure 2). Following the mild exertion of a grip strength challenge, mdx mice typically remain still for the first few minutes of the ambulation protocol, with a modest increase in movement by minute 4 to 5 (Figure 3A). When testing wild-type and mdx animals without an exercise challenge, we detected no difference in total distance traveled (Figure 2).
This is consistent with reports from other groups looking at similar movement assays, with unexercised mdx showing no difference or small decreases relative to wild-type mice. We additionally evaluated transgenic mice expressing high levels of utrophin in order to test the sensitivity of our assay (mdx:utr-tg, Fiona line)11,12. The mdx:utr-tg is a robust rescue model of the mdx phenotype with few dystrophic features. Following exercise, mdx mice expressing utrophin were indistinguishable from wild-type, with no significant difference in the total distance traveled (Figure 3B) or at any of the cumulative time points measured (Figure 3A).
Video-based tracking relies on frame-to-frame variability, measured by either manual observer scoring or by automated software detection. Free and open source movement analysis software allows for semi-automated detection of animal movement, in which undetected or overestimated movements can be manually corrected as the activity trace is generated. To minimize the data processing in this assay, we downsampled the original videos from the recorded 30 fps to 15 fps. To determine the effect of decreasing the sampling rate, we measured the total distance traveled from 1 min of full frame rate video and then compared distance measurements derived from downsampled versions of the data set (Figure 4A). We found that reducing the frame rate by half (decimation by 2) decreased the measured distance by 5.0%, and reducing the frame rate as low as 7.5 fps retained nearly 90% of the original distance calculated (89.2% of distance from 30 fps video). The accuracy of measured distance dropped off sharply after this point, although even highly-decimated datasets still closely approximated the path of the animal (Figure 4B). These observations highlight the importance of considering frame rate and signal processing in assessing animal activity, and demonstrate that high spatial resolution can be retained in downsampled video data.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56772/56772fig1.jpg" /> 
Figure 1: Representative open field traces of 6-minute ambulation distance. (A) Set-up of activity recording chamber used in this study. Grip meter was used for exercise challenge prior to assay, then free ambulation was recorded on the open field platform. (B) Composite image of 6 min video recording with tracing analysis in movement tracking software. (C) Overlay of two representative traces (WT, grey, n = 1; mdx, red, n = 1). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56772/56772fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56772/56772fig2.jpg" /> 
Figure 2: Effect of exercise on mdx ambulation. 6-minute ambulation distances were recorded at 6 months of age with or without prior exercise (non-exercised mdx n = 5, WT n = 4; exercised mdx n = 5, WT n = 5). No significant difference was observed between WT and mdx when unexercised, but a significant difference was seen in mice exercised prior to assay (no exercise: WT 1077.0&#177; 106.4 cm, mdx&#160;971.0 &#177; 36.16 cm; post-exercise: WT 770.2 &#177; 30.75 cm, mdx&#160;127.8 &#177; 36.16 cm). Data represent means &#177; SEM. Statistics calculated using two-way ANOVA followed by Tukey&#39;s multiple comparison&#39;s test (*p &#60; 0.05, **p &#60; 0.01, *** p &#60; 0.001 and ****p &#60; 0.0001). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56772/56772fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56772/56772fig3.jpg" /> 
Figure 3: Transgenic expression of utrophin improves ambulation in mdx mice. 6-minute ambulation distances were recorded at 6 months of age using an open field activity chamber (WT n = 5, mdx n = 5, mdx:utr-Tg n = 4). (A) Cumulative distance traveled by minute. No significant difference was detected between WT and mdx:utr-tg at any point. Statistics calculated using two-way ANOVA followed by Tukey&#39;s multiple comparison&#39;s test (*p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001 and ****p &#60; 0.0001). (B) Total open field distance traveled over 6 min (WT 770.2 &#177; 30.75 cm, mdx&#160;127.8 &#177; 36.16 cm, mdx:utr-Tg 701.3 &#177; 33.54 cm). Data represent means &#177; SEM. Statistics calculated using one-way ANOVA followed by Tukey&#39;s multiple comparison&#39;s test (*p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001 and ****p &#60; 0.0001). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56772/56772fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56772/56772fig4.jpg" /> 
Figure 4: Effect of sampling rate on ambulation tracking. In a wild-type mouse, 1 min of ambulation distance was recorded and tracked using movement tracking software at full frame rate (30.0 fps, 1,819 total frames). The two-dimensional position of the mouse in each frame was determined, and these data were then decimated to simulate tracking in lower frame rate video (30, 22.5, 20, 15, 10, 7.5, 6, 3, 1.5, and 0.75 fps). (A) Total distance calculated from coordinates in each downsampled data set. Dotted lines show values within 5% of the distance calculated from the full frame rate video. Data in this study were obtained from video decimated to 15 fps (red arrow). (B) Overlays of traces comparing full frame rate tracing with 1.5 fps (above) and 15 fps (below). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56772/56772fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

a-simple-low-cost-assay-for-measuring-ambulation-mouse-models

Nanyang Technological University

Research

JoVE Journal

Behavior

9.8K Views.  University of California. 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.

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

Assessment of Sensorimotor Function in Mouse Models of Parkinson's Disease

Sensitive and reliable behavioral outcome measures are essential to the evaluation of potential therapeutic treatments in preclinical trials for many neurodegenerative diseases. In Parkinson's disease, sensorimotor tests sensitive to varying degrees of nigrostriatal dysfunction are fundamental for testing the efficacy of potential therapeutics. Reliable and quite elegant sensorimotor measures exist for rats, however many of these tests measure sensorimotor asymmetry within the rat and are not entirely suitable for the newer genetic mouse models of PD. We have put together a battery of sensorimotor tests inspired by the sensitive tests in rats and adapted for mice. The test battery highlighted in this study is chosen for a) its sensitivity in a wide variety of mouse models of PD, b) its ease in implementing into a study, and c) its low expense. These tests have proven useful in characterizing novel genetic mouse models of PD as well as in testing potential disease-modifying therapies.

In Parkinson's disease and movement disorders in general, sensitive and reliable behavioral assays are essential for testing novel potential therapeutics. Here, we describe a manageable battery of sensorimotor tests for mice that are sensitive to varying degrees of injury to the nigrostriatal system and useful for preclinical studies.

Sensitive and  reliable behavioral outcome measures are essential to the evaluation of  potential therapeutic treatments in preclinical trials for many  ...

Behavioral and Locomotor Measurements Using an Open Field Activity Monitoring System for Skeletal Muscle Diseases

The open field activity monitoring system comprehensively assesses locomotor and behavioral activity levels of mice. It is a useful tool for assessing locomotive impairment in animal models of neuromuscular disease and efficacy of therapeutic drugs that may improve locomotion and/or muscle function. The open field activity measurement provides a different measure than muscle strength, which is commonly assessed by grip strength measurements. It can also show how drugs may affect other body systems as well when used with additional outcome measures. In addition, measures such as total distance traveled mirror the 6 min walk test, a clinical trial outcome measure. However, open field activity monitoring is also associated with significant challenges: Open field activity measurements vary according to animal strain, age, sex, and circadian rhythm. In addition, room temperature, humidity, lighting, noise, and even odor can affect assessment outcomes. Overall, this manuscript provides a well-tested and standardized open field activity SOP for preclinical trials in animal models of neuromuscular diseases. We provide a discussion of important considerations, typical results, data analysis, and detail the strengths and weaknesses of open field testing. In addition, we provide recommendations for optimal study design when using open field activity in a preclinical trial.

Open field activity levels are used to assess locomotive and behavioral activity levels. This protocol provides a well-designed, standardized protocol to use in preclinical trials for neuromuscular disorders.

The open field activity monitoring system comprehensively assesses locomotor and behavioral activity levels of mice. It is a useful tool for assessing ...

Low-cost Protocol of Footprint Analysis and Hanging Box Test for Mice Applied the Chronic Restraint Stress

Gait disturbance is frequently observed in patients with movement disorders. In mouse models used for movement disorders, gait analysis is important behavioral test to determine whether the mice mimic the symptoms of patients. Motor deficits are often induced by stress when no spontaneous motor phenotype is observed in the mouse models. Therefore, gait analysis followed by stress loading would be a sensitive method for evaluating the motor phenotype in mouse models. However, researchers face the requirement of an expensive apparatus to obtain quantitative results automatically from gait analysis. For stress, stress loading by simple methods without expensive apparatuses required for electric shock and forced running is desirable. Therefore, we introduce a simple and low-cost protocol consisting of footprint analysis with paper and ink, hanging box test to evaluate motor function, and stress loading defined by restraint with a conical tube. The motor deficits of mice were successfully detected by this protocol.

The low-cost protocol consisting of footprint analysis and hanging box test after restraint stress is useful for evaluating the movement disorders of mouse model.

Gait disturbance is frequently observed in patients with movement disorders. In mouse models used for movement disorders, gait analysis is important ...

Gait Analysis of Age-dependent Motor Impairments in Mice with Neurodegeneration

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. Specifically, gait analysis allows recapturing disease relevant phenotypes that are observed in human patients, especially in neurodegenerative diseases that affect motor abilities such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease (AD), amyotrophic lateral sclerosis (ALS), and others. In early studies along this line, the measurement of gait parameters was laborious and depended on factors that were hard to control (e.g., running speed, continuous running). The development of ventral plane imaging (VPI) systems made it feasible to perform gait analysis at a large scale, making this method a useful tool for the assessment of motor behavior in rodents. Here, we present an in-depth protocol of how to use kinematic gait analysis to examine the age-dependent progression of motor deficits in mouse models of neurodegeneration; mouse lines with decreased levels of endophilin, in which neurodegenerative damage progressively increases with age, are used as an example.

In this study, we demonstrate the use of kinematic gait analysis based on ventral plane imaging to monitor the subtle changes in motor coordination as well as the progression of neurodegeneration with advancing age in mouse models (e.g., endophilin mutant mouse lines).

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. ...

Developmental Biology

Low-Cost Gait Analysis for Behavioral Phenotyping of Mouse Models of Neuromuscular Disease

Measurement of animal locomotion is a common behavioral tool used to describe the phenotype of a given disease, injury, or drug model. The low-cost method of gait analysis demonstrated here is a simple but effective measure of gait abnormalities in murine models. Footprints are analyzed by painting a mouse&#8217;s feet with non-toxic washable paint and allowing the subject to walk through a tunnel on a sheet of paper. The design of the testing tunnel takes advantage of natural mouse behavior and their affinity for small dark places. The stride length, stride width, and toe spread of each mouse is easily measured using a ruler and a pencil. This is a well-established and reliable method, and it generates several metrics that are analogous to digital systems. This approach is sensitive enough to detect changes in stride early in phenotype presentation, and due to its non-invasive approach, it allows for testing of groups across life-span or phenotypic presentation.

Footprint analysis is a low-cost alternative to digitized gait analysis programs for researchers quantifying movement abnormalities in mice. Because of its speed, simplicity, and longitudinal potential, it is ideal for behavioral phenotyping of mouse models.

Measurement of animal locomotion is a common behavioral tool used to describe the phenotype of a given disease, injury, or drug model. The low-cost method ...

A Simple Technique to Assay Locomotor Activity in Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and diverse behavioral features. Identifying behavioral deficiency in animal models is a crucial measure of disease severity, for example, in neurodegenerative diseases where patients often experience impairments in motor function. However, with the availability of various systems to track and assess motor deficits in fly models, such as drug-treated or transgenic individuals, an economical and user-friendly system for precise evaluation from multiple angles is still lacking. A method based on the AnimalTracker application programming interface (API) is developed here, which is compatible with the Fiji image processing program, to systematically evaluate the movement activities of both adult and larval individuals from recorded video, thus allowing for the analysis of their tracking behavior. This method requires only a high-definition camera and a computer peripheral hardware integration to record and analyze behavior, making it an affordable and effective approach for screening fly models with transgenic or environmental behavioral deficiencies. Examples of behavioral tests using pharmacologically treated flies are given to show how the techniques can detect behavioral changes in both adult flies and larvae in a highly repeatable manner.

A Simple Technique to Assay Locomotor Activity in Drosophila

The present protocol assesses the locomotor activity of Drosophila by tracking and analyzing the movement of flies in a hand-made arena using open-source software Fiji, compatible with plugins to segment pixels of each frame based on high-definition video recording to calculate parameters of speed, distance, etc.

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and ...

Home-Based Monitor for Gait and Activity Analysis

Current outcomes in neuromuscular disorder clinical trials include motor function scales, timed tests, and strength measures performed by trained clinical evaluators. These measures are slightly subjective and are performed during a visit to a clinic or hospital and constitute therefore a point assessment. Point assessments can be influenced by daily patient condition or factors such as fatigue, motivation, and intercurrent illness. To enable home-based monitoring of gait and activity, a wearable magneto-inertial sensor (WMIS) has been developed. This device is a movement monitor composed of two very light watch-like sensors and a docking station. Each sensor contains a tri-axial accelerometer, gyroscope, magnetometer, and a barometer that record linear acceleration, angular velocity, the magnetic field of the movement in all directions, and barometric altitude, respectively. The sensors can be worn on the wrist, ankle, or wheelchair to record the subject&#8217;s movements during the day. The docking station enables data uploading and recharging of sensor batteries during the night. Data are analyzed using proprietary algorithms to compute parameters representative of the type and intensity of the performed movement. This WMIS can record a set of digital biomarkers, including cumulative variables, such as total number of meters walked, and descriptive gait variables, such as the percentage of the most rapid or longest stride that represents the top performance of patient over a predefined period of time.

This innovative device uses magneto-inertial sensors to permit gait and activity analysis in uncontrolled environments. Currently in the qualification process as an outcome measure in the European Medical Agency, one of the applications will be to serve as a clinical endpoint in clinical trials in neuromuscular diseases.

Current outcomes in neuromuscular disorder clinical trials include motor function scales, timed tests, and strength measures performed by trained clinical ...

Bioengineering

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

Neuroscience

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies

Critical to the evaluation of potential therapeutics for muscular disease are sensitive and reproducible physiological assessments of muscle function. Because many pre-clinical trials rely on mouse models for these diseases, isolated muscle function has become one of the standards for Go/NoGo decisions in moving drug candidates forward into patients. We will demonstrate the preparation of the extensor digitorum longus (EDL) and diaphragm muscles for functional testing, which are the predominant muscles utilized for these studies. The EDL muscle geometry is ideal for isolated muscle preparations, with two easily accessible tendons, and a small size that can be supported by superfusion in a bath. The diaphragm exhibits profound progressive pathology in dystrophic animals, and can serve as a platform for evaluating many potential therapies countering fibrosis, and promoting myofiber stability. Protocols for routine testing, including isometric and eccentric contractions, will be shown. Isometric force provides assessment of strength, and eccentric contractions help to evaluate sarcolemma stability, which is disrupted in many types of muscular dystrophies. Comparisons of the expected results between muscles from wildtype and dystrophic muscles will also be provided. These measures can complement morphological and biochemical measurements of tissue homeostasis, as well as whole animal assessments of muscle function.

Muscle function measurements contribute to the evaluation of potential therapeutics for muscle pathology, as well as to the determination of mechanisms underlying physiology of this tissue. We will demonstrate the preparation of the extensor digitorum longus and diaphragm muscles for functional testing. Protocols for isometric and eccentric contractions will be shown, as well as differences in results between dystrophic muscles, representing a pathological state, and wildtype muscles.

Critical to the evaluation of potential  therapeutics for muscular disease are sensitive and reproducible physiological  assessments of muscle function. ...

Biology

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

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

Summary

Explore More Videos

Chapters in this video

Assessment of Sensorimotor Function in Mouse Models of Parkinson's Disease

Behavioral and Locomotor Measurements Using an Open Field Activity Monitoring System for Skeletal Muscle Diseases

Low-cost Protocol of Footprint Analysis and Hanging Box Test for Mice Applied the Chronic Restraint Stress

Gait Analysis of Age-dependent Motor Impairments in Mice with Neurodegeneration

Low-Cost Gait Analysis for Behavioral Phenotyping of Mouse Models of Neuromuscular Disease

A Simple Technique to Assay Locomotor Activity in Drosophila

Home-Based Monitor for Gait and Activity Analysis

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice