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

Summary

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.

Abstract

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.

Introduction

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 disorders^2,3,4. 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 treadmill^6,7. 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 analysis^5,6,7 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 disorders^14,15. Motor deficits are often induced by several chronic stresses even when no spontaneous motor phenotype is observed in the mouse models of a movement disorder^16,17,18. 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.

Protocol

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. Hanging Box Test

1. Record the weight of each mouse. Mark the tail by marking pen for individual discrimination (e.g., a line, double lines, and triple lines).
   NOTE: Growth curves are used for an index of general health²².
2. Place the mice in the experimental room at least 30 min before the behavioral test. Set the hanging box, which consists of a clear box (25 x 25 x 40 cm³) with a rotatable mesh lid on the top (Figure 1). The mesh lid can be rotated along a central bar so that the top is flipped 180 degrees.
3. Put a mouse in the center of the mesh lid. Carefully turn the mesh lid up side down.
4. Measure the fall latency (hanging time) of the mouse from the mesh lid.
   NOTE: If the mouse does not fall within 5 min, record the latency as 5 min.
5. Return the mouse to the home cage. Clean the hanging box with 70% ethanol after every test.

2. Footprint Analysis

NOTE: Following the hanging box test, perform the footprint analysis.

1. Set up the Runway (Figure 2A).
   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 the 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 a 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 (see step 2.4. Analysis of footprints for details) 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 2B) 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.

3. Restraint Stress Loading

1. Preparation of Restraint Tubes.
   1. Make 16 holes (approximately 2 mm in diameter) in a 50 ml conical tube (30 mm in diameter x 115 mm in length) along the scale marks (5, 10, 15, 20, 25, 30, 35, 40 mL) and the backside of each scale mark by square drill (Figure 3). Make a hole on the tip of the 50 mL conical tube (approximately 5 mm in diameter) for breathing by cutting off the tip. Make a hole (approximately 4 mm in diameter) in the tube cap to pass the tail of mice.
2. Stress Loading
   1. Place the mice in the experimental room.
   2. Hold a mouse by grasping the scruff between the thumb and the forefinger. Enter the mouse into the restraint tube from the head. Pass the tail through the hole in the cap. Close the cap.
      NOTE: Limit the forelimb movement, because mice reject entering the tube by the forelimbs.
   3. Keep the mouse enclosed for 2 h on a desk at room temperature. Remove the mouse from the restraint tube and return to the home cage.
      NOTE: The restraint tubes can be reused after a wash and dry.

4. Experimental Schedule (Figure 4):

1. Perform the hanging box test and the footprint analysis on the same day at 4 weeks of age (see step 1. Hanging box test and step 2. Footprint analysis for details) as a baseline measurement on all mice prior to the grouping into ‘stress group’ and a ‘non-stress group’.
   NOTE: About 8-10 mice in 2-3 litters may be suitable to use in an experiment. Footprint analysis at 4 weeks of age consists of training and test sessions.
2. Randomly divide the mice into a ‘stress group’ and a ‘non-stress group’.
   NOTE: When the mice are used consisting of several litters, divide littermates evenly into both groups. Number in each group consists of about 4-5 mice.
3. Apply the restraint stress to the ‘stress group’ 6 times over the course of two weeks (see step 3. Restraint stress loading for details).
   NOTE: 6 times of restraint are applied every two weeks, followed by a hanging box test and footprint analysis from 6-12 weeks of age. Do not apply the restraint stress on the test day of the hanging box test and the footprint analysis.
4. Perform the hanging box test and the test session of footprint analysis on the same day at 6, 8, 10, and 12 weeks of age.

Results

The heterozygous male mice of Atp1a3 (Atp1a3^+/−) that are the mouse model for rapid onset dystonia parkinsonism and wild-type littermates were used in this protocol. Atp1a3^+/− 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). 'Stressed' At...

Discussion

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's disease²⁶ and dystonia

Materials

Name	Company	Catalog Number	Comments
Hanging box	O’hara & Co.	http://ohara-time.co.jp/products/wire-hanging-test/
Marking pen	ZEBRA	MO-120-MC-BK
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 dishe (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)
50 mL Conical tube	Corning	430829
Square drill	KAKURI Corporation	DIY FACTORY (K32-0313)