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

Overview

In this video, we describe the footprint testing procedure and related analysis to assess gait and motor coordination in mice.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Footprint Analysis

1. Set up the Runway (Figure 1A)
   1. 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.
   2. 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.
   3. Put black ink and red ink into separate Petri dishes (35 mm in diameter).

2. Training Session

NOTE: Perform the training session only at 4 weeks of age.

1. 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.
2. 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: Insufficient holding of mouse results in blots of ink on clothing.
3. 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.
4. Remove the mouse from the goal box. Go to the test session.

3. Test Session

1. Following the training session, set up the runway for footprints with a new cut piece of white paper.
2. 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.
3. 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: 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.
4. 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.

4. Analysis of Footprints

1. Obtain three measurements of each parameter (stride lengths of forelimbs and hindlimbs, front and hind base widths, overlap between forelimb and hindlimb, Figure 1B) with a ruler from foot-printed paper.
   NOTE: 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.
   1. For the stride length, measure the distances between the same parts of the paw (e.g., paw pad or toe).
   2. 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.
   3. 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.
   4. For overlap, measure the distance between pads of left (or right) front and hind footprints.
2. Average the three measurements for each individual. Use the individual average of each parameter for the statistical analysis.
   1. For the stride length, use the average of the individual averages of the left and right strides.
   2. For asymmetry of stride length, use the absolute value of the difference between individual averages of left limb and right limb stride length.
   3. For the statistical analysis of the other parameters (front base width, hind base width, and overlap), use the individual average directly.

Representative Results

Figure 1. Footprint analysis. (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. Three measurements of each parameter (forelimb and hindlimb stride lengths, widths of front and hind base, overlap between forelimb and hindlimb) were obtained from foot-printed paper.

Materials

Name	Company	Catalog Number	Comments
Goal box	O’hara & Co.	http://ohara-time.co.jp/products/balanced-beam-test/	Accessory for apparatus of balanced beam test
Boxes	O’hara & Co.		Side wall of runway
Black ink	Shin-asahi
Red ink	Maruyamakogyo	BC-6
Disposable Petri Dish	Corning	351008	Petri dish (35 mm in diameter)
Askul Multipaper Super White J Monochrome A3	Askul	701-712	White paper (29.7 cm x 42 cm x 0.09mm)