This work was supported by JSPS (Japan Society for the Promotion of Science) KAKENHI (Grant-in-Aid for Scientific Research C), Grant number 18K07373 (H.S.) and Subsidies for Private Universities.

All animal experiments were conducted in a humane manner. The Institutional Animal Experiment Committee of Jichi Medical University approved the study. The study was conducted in accordance with the Institutional Regulation for Animal Experiment and Fundamental Guideline for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the MEXT of Japan. Mice used in this protocol have been described previously21.
1...

<ol>
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The authors have nothing to disclose.

The footprint analysis and the hanging box test are simple and inexpensive behavioral tests for the motor function of mice. The neurobehavioral phenotypes in several mouse models have been successfully detected by these tests. For example, shortened stride length in amyotrophic lateral sclerosis24, increased length of asymmetrical stride in ataxia-telangiectasia25, increased length of overlap in Huntington&#39;s disease26 and dystonia<sup class="xref...

Movement disorders are defined as disturbances of the nervous system showing an excess or paucity of voluntary or automatic movements1. In particular, gait disturbance is frequently documented among patients with movement disorders2,3,4. Therefore, gait analysis is a suitable behavioral test for the validation of animal models of movement disorders. In mice, automated gait analyses have been performed for walking at natural speed5 and at adjustable speeds by treadmill6,7. These analyses provide quantitative results of gait automatically. An alternative method to detect gait disturbance is called footprint analysis. After labeling the bottoms of the feet with ink, mice walk on paper, and the footprints are analyzed. Initially, Vaseline and powdered charcoal were used to visualize the footprint8, and then were replaced by ink on polygraph paper9 and photographic developer on photographic paper10. A cheaper and less toxic method using ink and paper than the other methods remains to date11. Footprint analysis is less expensive compared with automated analysis5,6,7 and would be useful to evaluate the movement disorders in mouse models for the researchers without abundant research funds.
The hanging box test is a kind of four limb hanging tests using wire cage lid12 and wire mesh screen13. The box is an apparatus with rotatable mesh lid on the top of box along a center bar. In addition to gait analysis, the test can be inexpensively and easily performed. Therefore, we conducted the hanging box test to evaluate grip strength and balance, in addition to the footprint analysis in this protocol.
Stress induces the symptoms of movement disorders14,15. Motor deficits are often induced by several chronic stresses even when no spontaneous motor phenotype is observed in the mouse models of a movement disorder16,17,18. Restraint is one of the commonly used methods for stress loading in mice, because the animal is not physically harmed19 and cost is less compared with other methods such as electric shock with dedicated apparatus and forced running with use of a treadmill. Restraint by a tube, which is performed by confining a mouse in a holed 50 mL conical tube, is easier than other methods such as wire mesh strainer, taped limb, and wrapping of animal with gauze (reviewed20). In this paper, we summarize the protocols of footprint analysis and the hanging box test after restraint by a tube. This protocol would help us to use mouse models of movement disorders without spontaneous motor phenotype.

<table>
	<tbody>
		<tr>
			<td>Hanging box</td>
			<td>O&#8217;hara &#38; Co.</td>
			<td>http://ohara-time.co.jp/products/wire-hanging-test/</td>
			<td></td>
		</tr>
		<tr>
			<td>Marking pen</td>
			<td>ZEBRA</td>
			<td>MO-120-MC-BK</td>
			<td></td>
		</tr>
		<tr>
			<td>Goal box</td>
			<td>O&#8217;hara &#38; Co.</td>
			<td>http://ohara-time.co.jp/products/balanced-beam-test/</td>
			<td>Accessory for apparatus of balanced beam test</td>
		</tr>
		<tr>
			<td>Boxes</td>
			<td>O&#8217;hara &#38; Co.</td>
			<td>-</td>
			<td>Side wall of runway</td>
		</tr>
		<tr>
			<td>Black ink</td>
			<td>Shin-asahi</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Red ink</td>
			<td>Maruyamakogyo</td>
			<td>BC-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Petri Dish</td>
			<td>Corning</td>
			<td>351008</td>
			<td>Petri dishe (35 mm in diameter)</td>
		</tr>
		<tr>
			<td>Askul Multipaper Super White J Monochrome A3</td>
			<td>Askul</td>
			<td>701-712</td>
			<td>White paper (29.7 cm x 42 cm x 0.09mm)</td>
		</tr>
		<tr>
			<td>50 mL Conical tube</td>
			<td>Corning</td>
			<td>430829</td>
			<td></td>
		</tr>
		<tr>
			<td>Square drill</td>
			<td>KAKURI Corporation</td>
			<td>DIY FACTORY (K32-0313)</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 heterozygous male mice of Atp1a3 (Atp1a3+/&#8722;) that are the mouse model for rapid onset dystonia parkinsonism and wild-type littermates were used in this protocol. Atp1a3+/&#8722;&#160;showed significantly shorter stride lengths of forelimb and hindlimb than those of the wild type at 4 weeks of age (Figure 5A and Figure 5B, open circle and square). &#39;Stressed&#39; At...

Watch this Scientific Journal Video about Low-cost Protocol of Footprint Analysis and Hanging Box Test for Mice Applied the Chronic Restraint Stress at JoVE.com

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

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 ...

Our protocol using gait analysis is very useful in determining whether mouse motors of movement disorders mimic the symptom of patient with gait disturbance. So, gait analysis in the stress loading test can be performed easily, and without expensive equipment, to detect certain motor phenotypes in mice, with no spontaneous effects. Before initiating the behavioral test, record the weight of each mouse, and use a marking pen to label each tail.When all of the animals have been weighed and marked, place the mice in the experimental room for at least 30 minutes, and set a clear, 25 by 25 by 40 centimeter hanging box, with a rotatable mesh lid, onto the lab bench. When the mice have acclimated, place one mouse into the center of the mesh lid, and carefully turn the lid upside-down. Then, measure the fall latency of the mouse from the mesh lid, before returning the mouse to it's home cage.When all of the mice have been tested, place a 9.9 by 42 centimeter piece of white paper onto the lab bench, and place a dark goal box at the distal end of the paper. Place other boxes, approximately the same length as the paper, on both sides of the runway to prevent escape. And add black and red ink to separate 35 milliliter Petri dishes.To train the mice for the analysis, place a mouse at the proximal end of the paper, facing the goal box, and let the mouse walk from the proximal end of the paper to the goal box. When the mouse reaches the box, gently scruff the animal, tucking the back and tail between the ball of the thumb and the other fingers, to limit the movement of the hind limbs. And immerse the bottoms of the forelimbs in red ink, and the bottom of the hind limbs in black ink.Then, immediately place the mouse back onto the proximal end of the paper, facing the goal box, and let the mouse walk from the proximal end to the goal box. After air-drying the foot printed paper, use a ruler to obtain three measurements of the stride length, by measuring the distances between the same parts of the paw prints. Of the front base-width, by drawing a line between consecutive, same-side, front footprints, and measuring the length of the vertical line from the pad of the front footprint, to the line drawn between the footprints.And of the hind base-width, by drawing a line between consecutive right hind footprints, and measuring the length of the vertical line from the pad of the left hind footprint, to the line drawn between the other right footprints. For overlap, measure the distance between the pads of the front and hind footprints. To prepare a restraint tube, use a square drill to make 16 two millimeter holes in a 50 milliliter tube along the scale marks, and the backside of each scale mark.Cut off the tip of the tube to make a five millimeter hole, for breathing, and make a four millimeter hole in the tube cap, for the tail. For the stress-loading test, scruff a mouse, and place the animal into the restraining tube head first, with the forelimbs gently confined. Pass the tail through the hole in the cap before capping the tube.And place the mouse on the bench top for two hours at room temperature. At the end of the stress-loading period, remove the mouse from the tube, and return the animal to its home cage. As observed in these representative figures, heterozygous rapid-onset dystonia Parkinsonism model mice, demonstrate significantly shorter fore and hindlimb stride lengths, than do their wild-type litter mates at four weeks of age.Stress heterozygous rapid-onset dystonia Parkinsonism model mice, also exhibit significantly shorter stride lengths of both limbs than those of stressed wild-type litter mates at eight weeks of age. Asymmetries of the stride lengths of both limbs are not significantly different, in any group of mice, at any age. The front base and overlaps of both limbs are also similar, in all groups, at all ages.The hind base is significantly wider in these stressed heterozygous rapid-onset dystonia Parkinsonism model mice, than that observed in the stress wild-type animals at 10 weeks of age, indicating that stress causes motor deficits in heterozygous rapid-onset dystonia Parkinsonism model mice. No significant differences in hanging time, between wild-type and mutant, are observed in four to 10 week old animals. At 12 weeks of age, however, the hanging time of stress wild-type mice is significantly longer than that of the other animals, suggesting that the motor deficit in heterozygous rapid-onset dystonia Parkinsonism model mice, is distinguishable from wild-type mice by restraint test in older animals.Place the mouse onto the paper immediately after the mouse hangs the bottoms of limbs in the ink to avoid the ink drying before the walking test. Additional tests for emotional characteristics, such as open field, elevated plus-maze, and whole swim test, can be performed to answer whether the gait abnormality result from only motor deficit. Our protocol can be used to explore the susceptibility to stress, by changing the number and the duration of the stress loading, and by comparing motor phenotypes between various loading conditions.Our protocol can be used to explore the susceptibility to stress by changing the number and duration of the stress loadings, and by comparing motor phenotypes between various loading conditions.

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Research

JoVE Journal

Behavior

17.6K Views.  Jichi Medical University. Il nostro protocollo che utilizza l'analisi dell'andatura è molto utile per determinare se i motori del mouse dei disturbi del movimento imitano il sintomo del paziente con disturbo dell'andatura. Quindi, l'analisi dell'andatura nella prova di carico dello stress può essere eseguita facilmente e senza attrezzature costose, per rilevare alcuni fenotipi motori nei topi, senza effetti spontanei. Prima di iniziare il test comportamentale, registrare il peso di ogni mouse e utilizzare una penna ...