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....

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, d....

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)