assessing gait by footprint analysis: a method to test motor coordination in mice

Assessing Gait by Footprint Analysis: A Method to Test Motor Coordination in Mice

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-millimeter 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 hindlimbs and immerse the bottoms of the forelimbs in red ink and the bottom of the hindlimbs 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.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Footprint Analysis
<ol>
	<li>Set up&#160;the&#160;Runway&#160;(Figure 1A)
	<ol>
		<li>Cut a piece of white paper (29.7 cm x 42 cm x 0.09 mm) longitudinally into three lengths of equal width. Set a piece of white paper (9.9 cm x 42 cm) on the table.</li>
		<li>Put the dark goal box at the distal end of the paper. Put other boxes (approximately the same length as that of the paper) with the walls on both sides of the runway, preventing the escape of mice.</li>
		<li>Put black ink and red ink into separate Petri dishes (35 mm in diameter).</li>
	</ol>
	</li>
</ol>
2. Training Session
NOTE:&#160;Perform the training session only at 4 weeks of age.
<ol>
	<li>Put a mouse on the proximal end of the paper (face the head toward the goal box). Let the mouse walk from the proximal end to the goal box. Remove the mouse from the goal box. If the mouse stops on the paper, gently push the mouse to the goal box by finger.</li>
	<li>Hold the mouse by grasping the scruff between the thumb and forefinger to limit the movement of forelimbs. Then, grasp the back and the tail between the ball of the thumb and the other fingers to limit the movement of hindlimbs. 
	NOTE:&#160;Insufficient holding of mouse results in blots of ink on clothing.</li>
	<li>Immerse the bottoms of forelimbs in red ink and the bottoms of hindlimbs in black ink. Immediately put the mouse on the proximal end of the paper (Face the head toward the goal box). Let the mouse walk from the proximal end to the goal box. If the mouse stops on the paper, gently push the mouse to the goal box by finger.</li>
	<li>Remove the mouse from the goal box. Go to the test session.</li>
</ol>
3. Test Session
<ol>
	<li>Following the training session, set up the runway for footprints with a new cut piece of white paper.</li>
	<li>Hold the mouse by grasping the scruff between the thumb and forefinger to limit the movement of forelimbs. Then, grasp the back and the tail between the ball of the thumb and the other fingers to limit the movement of hindlimbs.</li>
	<li>Immerse the bottoms of forelimbs in red ink and the bottoms of hindlimbs in black ink. Immediately put the mouse on the proximal end of the paper. Let the mouse walk from the proximal end to the goal box. 
	NOTE:&#160;Because mice prefer the dark, walking becomes steadier as the mouse approaches the dark goal box. If the mouse stops on the paper, gently push the mouse to the goal box by finger. Then, if reliable footprints are not obtained for analysis because the mouse stopped, retry the test session.</li>
	<li>Return the mouse to the home cage from the goal box. Clean the goal box with 70% ethanol after each test session. Air-dry the foot-printed paper.</li>
</ol>
4. Analysis of Footprints
<ol>
	<li>Obtain three measurements of each parameter (stride lengths of forelimbs and hindlimbs, front and hind base widths, overlap between forelimb and hindlimb,&#160;Figure 1B) with a ruler from foot-printed paper. 
	NOTE:&#160;Because footprints of proximal and distal ends frequently show large variations because of stopping or running, choose the part with a steady gait pattern of footprints. The middle part of the foot-printed paper will usually be suitable for the analysis.
	<ol>
		<li>For the stride length, measure the distances between the same parts of the paw (e.g., paw pad or toe).</li>
		<li>For the front base width, draw a line between consecutive right (or left) front footprints. Then, measure the length of the vertical line from the pad of the left (or right) front footprint to the line drawn between the right (or left) footprints.</li>
		<li>For the hind base width, draw a line between consecutive right (or left) hind footprints. Then, measure the length of the vertical line from the pad of the left (or right) hind footprint to the line drawn between the right (or left) footprints.</li>
		<li>For overlap, measure the distance between pads of left (or right) front and hind footprints.</li>
	</ol>
	</li>
	<li>Average the three measurements for each individual. Use the individual average of each parameter for the statistical analysis.
	<ol>
		<li>For the stride length, use the average of the individual averages of the left and right strides.</li>
		<li>For asymmetry of stride length, use the absolute value of the difference between individual averages of left limb and right limb stride length.</li>
		<li>For the statistical analysis of the other parameters (front base width, hind base width, and overlap), use the individual average directly.</li>
	</ol>
	</li>
</ol>

<table><tbody><tr><td>Goal box</td><td>O&amp;rsquo;hara &amp;amp; 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&amp;rsquo;hara &amp;amp; Co.</td><td></td><td>Side wall of runway</td></tr><tr><td>Black ink&amp;nbsp;</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 dish (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)&amp;nbsp;</td></tr></tbody></table>

Source: Sugimoto, H. and Kawakami, K. <a href="https://www.jove.com/video/59027/low-cost-protocol-footprint-analysis-hanging-box-test-for-mice">Low-cost Protocol of Footprint Analysis and Hanging Box Test for Mice Applied the Chronic Restraint Stress.</a>&#160;J. Vis. Exp. (2019).
In this video, we describe the footprint testing procedure and related analysis to assess gait and motor coordination in mice.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/20029/20029_Figure1.jpg" /> 
Figure 1. Footprint analysis.&#160;(A) Runway for footprint analysis. A foot-painted mouse (forelimbs: red ink; hindlimbs: black ink) was allowed to walk from the proximal end to the goal box. (B) Representative image of footprint and measurement of parameters.&#160;Three measurements of each parameter (forelimb and hindlimb stride lengths, widths...

Source: Sugimoto, H. and Kawakami, K. Low-cost Protocol of Footprint Analysis and Hanging Box Test for Mice Applied the Chronic Restraint Stress.&#160;J. ...

The footprint test is performed on a flat surface covered with a white sheet of paper, like a runway, ending in a dark gold box and an escape hole. The runway is restricted on each side to prevent subjects from leaving the test area.
A mouse will avoid the bright setting by walking across the runway to enter the escape hole. After the mouse has learned to perform the task, paint its front paws in red and its back paws in black with non-toxic paints. As the mouse walks on the white paper, it will now leave color-labeled footprints.
To describe the animal&#39;s gait and motor coordination, measure the distance of the fore- and hindlimbs&#39; stride length, fore- and hindlimb base width, as well as the distance of their overlap.

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Assessing Gait by Footprint Analysis: A Method to Test Motor Coordination in Mice

Transcrição

Assessing Gait by Footprint Analysis: A Method to Test Motor Coordination in Mice