Name: low-cost gait analysis for behavioral phenotyping of mouse models of neuromuscular disease
Uploaded: 2019-07-18T15:00:00
Description: Watch this Scientific Journal Video about Low-Cost Gait Analysis for Behavioral Phenotyping of Mouse Models of Neuromuscular Disease at JoVE.com

The authors wish to thank A.M. for animal identification assistance. This work was supported by grants from the US National Institutes of Health (R01 7 RF1 AG057264 to A.R.L.S. and C.J.C. and R01 NS100023 to A.R.L.S) and the Muscular Dystrophy Association (Basic Research Grant to A.R.L.S., Development Grant to C.J.C.).

All testing conducted with mice was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Duke University. Personnel responsible for testing and scoring must be blinded to animal genotype or experimental condition until gait analysis and scoring of papers has been completed for the entire cohort.
1. Testing material preparation
<ol>
	<li>Conduct testing with a tunnel built from 3 pre-cut clear acrylic panels that are 0.375 inches thick. Assemble tunnel by gluing...

<ol>
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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=RNA+interference+improves+motor+and+neuropathological+abnormalities+in+a+Huntington's+disease+mouse+model.">RNA interference improves motor and neuropathological abnormalities in a Huntington's disease mouse model.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (16), 5820-5825 (2005).</li><li>Lin, C. 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=Neurological+abnormalities+in+a+knock-in+mouse+model+of+Huntington's+disease.">Neurological abnormalities in a knock-in mouse model of Huntington's disease.</a> Human Molecular Genetics. 10 (2), 137-144 (2001).</li><li>Sopher, B. 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=Androgen+receptor+YAC+transgenic+mice+recapitulate+SBMA+motor+neuronopathy+and+implicate+VEGF164+in+the+motor+neuron+degeneration.">Androgen receptor YAC transgenic mice recapitulate SBMA motor neuronopathy and implicate VEGF164 in the motor neuron degeneration.</a> Neuron. 41 (5), 687-699 (2004).</li><li>Tillerson, J. L., Caudle, W. M., Reveron, M. E., Miller, G. W. <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+behavioral+impairments+correlated+to+neurochemical+deficits+in+mice+treated+with+moderate+doses+of+1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.">Detection of behavioral impairments correlated to neurochemical deficits in mice treated with moderate doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.</a> Experimental Neurology. 178 (1), 80-90 (2002).</li><li>Wheeler, V. C., 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=Early+phenotypes+that+presage+late-onset+neurodegenerative+disease+allow+testing+of+modifiers+in+Hdh+CAG+knock-in+mice.">Early phenotypes that presage late-onset neurodegenerative disease allow testing of modifiers in Hdh CAG knock-in mice.</a> Human Molecular Genetics. 11 (6), 633-640 (2002).</li><li>Clayton, J. A., Collins, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Policy:+NIH+to+balance+sex+in+cell+and+animal+studies.">Policy: NIH to balance sex in cell and animal studies.</a> Nature. 509 (7500), 282-283 (2014).</li><li>Maricelli, J. W., Lu, Q. L., Lin, D. C., Rodgers, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trendelenburg-Like+Gait,+Instability+and+Altered+Step+Patterns+in+a+Mouse+Model+for+Limb+Girdle+Muscular+Dystrophy+2i.">Trendelenburg-Like Gait, Instability and Altered Step Patterns in a Mouse Model for Limb Girdle Muscular Dystrophy 2i.</a> PLoS One. 11 (9), e0161984 (2016).</li><li>Castelhano-Carlos, M. 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Using the low-cost gait analysis method described above, we show successful identification of several parameters of gait dysfunction at post-symptomatic ages in the BAC fxAR121 mouse model of SBMA. Decreases in stride length are consistent with prior SBMA studies of mouse models and human patients9. We also show for the first time that there are significant differences in hind-limb toe spread in symptomatic SBMA mice compared to non-transgenic littermate controls. Interestingly, decreases in hind ...

Locomotion requires complex neurological and musculoskeletal coordination, and deficits in a single aspect of motor pathways can produce observable gait abnormalities1,2. Gait analysis is a critical tool for researchers testing mouse models because it provides quantifiable behavioral data on how a given disease, injury, or drug impacts an animal&#8217;s movement3. However, digitized gait analysis requires the purchase of a treadmill, a camera, and associated software, which can be prohibitively expensive for researchers. Gait analysis is often used intermittently to track longitudinal changes in motor function, hence it may be difficult to justify the expenditure if sporadically used4. Although digitized analyses may provide more detailed gait metrics than simple footprint analysis, these more complex measures are not always necessary or relevant for the characterization of a behavioral phenotype5.
Here we present a low-cost manual footprint analysis method as a quick and sensitive alternative to digitized gait analysis programs6,7. Manual footprint analysis has been demonstrated to detect significant gait differences in a multitude of murine disease models4,7,8,9,10,11,12,13,14,15,16,17, and in at least one case, this low-cost method identified changes in gait that were not detected by a common digitized gait analysis program12. The total cost of materials is nominal, and it can be easily adapted to other rodent research models.
While there are many different gait metrics from which data can be drawn, the method we describe focuses on three specific metrics: stride length, stride width (a.k.a. &#8220;track width&#8221;), and toe spread. It is important to note that the parameters to be assessed should be determined on a model-by-model basis. This method of gait analysis is not designed to measure cognitive function, and it is not recommended for studies that require complex biomechanical measurements of gait16.
We present behavioral data from a cohort of pre- and post-symptomatic mice modeling X-linked Spinal and Bulbar Muscular Atrophy (SBMA), a neuromuscular disease characterized by motor neuron degeneration and muscle atrophy. These mice develop progressive deficits in gait that coincide with the onset of other disease-specific phenotypes. This demonstrates the validity and specificity of this method, and confirms that it can reliably discriminate between affected and non-affected animals.
The experimental mice in this study were 2.5 (pre-symptomatic) and 9-month-old (post-symptomatic) BAC fxAR121 transgenic mice on a C57BL/6 background (nexpt=12). This model was generated in our lab and has been fully characterized as a powerful mouse model of SBMA9. Non-transgenic littermates were used as controls (nctrl=8). SBMA is a sex-limited disease which fully manifests in males only, so male mice were used exclusively for this study. During planning stages, researchers must take into account the National Institutes of Health&#8217;s considerations of sex as a biological variable to determine group sizes and composition18.

<table>
	<tbody>
		<tr>
			<td>Caliper</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>must have markings down to 0.1 mm</td>
		</tr>
		<tr>
			<td>Craft Glue</td>
			<td>E6000</td>
			<td>n/a</td>
		</tr>
		<tr>
			<td>Footprint Paint (Tempera Paint)</td>
			<td>Artmind</td>
			<td>n/a</td>
			<td>must be non-toxic</td>
		</tr>
		<tr>
			<td>Round Barrel Paintbrushes</td>
			<td>Symply Simmons</td>
			<td>n/a</td>
			<td>0.5 cm diameter</td>
		</tr>
		<tr>
			<td>Ruler</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>must have markings down to millimeters</td>
		</tr>
		<tr>
			<td>Scoring Paper (Watercolor Pads)</td>
			<td>Canson</td>
			<td>n/a</td>
			<td>cut to size</td>
		</tr>
		<tr>
			<td>Tunnel and Goal Chamber</td>
			<td>Interstate Plastics</td>
			<td>n/a</td>
			<td>cut to size</td>
		</tr>
	</tbody>
</table>

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.

With sufficient numbers of animals, this procedure is capable of detecting gait differences between mouse genotypes, within the same strain over time. Figure 1B shows representative traces of footprint images collected in our lab, using a mouse model of X-linked Spinal and Bulbar Muscular Atrophy (SBMA), a neurodegenerative disorder affecting lower motor neurons and skeletal muscle. We have previously reported that male BAC fxAR121 transgenic mice develop significant weight loss, impairments...

Watch this Scientific Journal Video about Low-Cost Gait Analysis for Behavioral Phenotyping of Mouse Models of Neuromuscular Disease at JoVE.com

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

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 ...

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