Comprehensive Understanding of Inactivity-Induced Gait Alteration in Rodents

Summary

The present protocol describes three-dimensional motion tracking/evaluation to depict gait motion alteration of rats after exposure to a simulated disuse environment.

Abstract

It is well known that disuse affects neural systems and that joint motions become altered; however, which outcomes properly exhibit these characteristics is still unclear. The present study describes a motion analysis approach that utilizes three-dimensional (3D) reconstruction from video captures. Using this technology, disuse-evoked alterations of walking performances were observed in rodents exposed to a simulated microgravity environment by unloading their hindlimb by their tail. After 2 weeks of unloading, the rats walked on a treadmill, and their gait motions were captured with four charge-coupled device (CCD) cameras. 3D motion profiles were reconstructed and compared to those of control subjects using the image processing software. The reconstructed outcome measures successfully portrayed distinct aspects of distorted gait motion: hyperextension of the knee and ankle joints and higher position of the hip joints during the stance phase. Motion analysis is useful for several reasons. First, it enables quantitative behavioral evaluations instead of subjective observations (e.g., pass/fail in certain tasks). Second, multiple parameters can be extracted to fit specific needs once the fundamental datasets are obtained. Despite hurdles for broader application, the disadvantages of this method, including labor intensity and cost, may be alleviated by determining comprehensive measurements and experimental procedures.

Introduction

Lack of physical activity or disuse leads to the deterioration of locomotor effectors, such as muscle atrophy and bone loss¹ and whole-body deconditioning². Moreover, it has recently been noticed that inactivity affects not only structural aspects of musculoskeletal components but also qualitative aspects of the movement. For example, the limb positions of rats exposed to a simulated microgravity environment were different from those of intact animals even 1 month after the intervention ended^3,4. Nevertheless, little has been reported on motion deficits caused by....

Protocol

The present study was approved by the Kyoto University Animal Experimental Committee (Med Kyo 14033) and performed in compliance with National Institute of Health guidelines (Guide for the Care and Use of Laboratory Animals, 8th Edition). 7-week-old male Wistar rats were used for the present study. A schematic representing the sequence of procedures is provided in Supplementary File 1.

1. Familiarizing rats with treadmill walking

NOTE: Please see the previously published report¹³ for details regarding the procedure.

1. Place the rat on a trea....

Results

12 animals were randomly assigned to one of two groups: the unloading group (UL, n = 6) or the control group (Ctrl, n = 6). For the UL group, the hindlimbs of the animals were unloaded by the tail for 2 weeks (UL period), whereas the Ctrl group animals were left free. 2 weeks after unloading, the UL group showed a distinct gait pattern compared with the Ctrl group. Figure 1 shows normalized joint trajectories of representative subjects. During the stance phase, the UL group exhibited further.......

Discussion

Alteration of environments leads to fluctuating functional aspects and musculoskeletal components of locomotor systems^26,27. Aberrations in contractile structures or environments may affect functional abilities, persisting even after resolving mechanical/environmental distortions¹⁹. Objective motion analysis helps to measure those functional abilities quantitatively. As shown above, video analysis is a powerful methodology for acquiring su.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Adhesive Tape	NICHIBAN CO.,LTD.	SEHA25F	Adhesive tape to secure thread on tails of rats for hindlimb unloading
Anesthetic Apparatus for Small Animals	SHINANO MFG CO.,LTD.	SN-487-0T
Auto clicker	N.A.	N.A.	free software available to download to PC (https://www.google.com/search?client=firefox-b-1-d&q=auto+clicker)
CCD Camera	Teledyne FLIR LLC	GRAS-03K2C-C	CCD (Charge-Coupled Device) cameras for video capture
Cotton Thread	N.A.	N.A.	Thread to hang tails of rats from the ceiling of cage
ISOFLURANE Inhalation Solution	Pfizer Japan Inc.	(01)14987114133400
Joint marker	TOKYO MARUI Co., Ltd	0.12g BB	6 mm airsoft pellets that were used as semispherical markers with modification
Kine Analyzer	KISSEI COMTEC CO.,LTD.	N.A.	Software for analysis
Konishi Aron Alpha	TOAGOSEI CO.,LTD.	#31204	Super glue to attach spherical markers on randmarks of rats
Motion Recorder	KISSEI COMTEC CO.,LTD.	N.A.	Software for video recording
Paint Marker	MITSUBISHI PENCIL CO., LTD	PX-21.13	Oil based paint marker to mark toes of animals
Three-dimensional motion capture apparatus (KinemaTracer for small animals)	KISSEI COMTEC CO.,LTD.	N.A.	3D motion analysis system that consists of four cameras (https://www.kicnet.co.jp/solutions/biosignal/animals/kinematracer-for-animal/ or https://micekc.com/en/)
Three-dimensional(3D) Calculator	KISSEI COMTEC CO.,LTD.	N.A.	Software fo marker tracking
Treadmill	MUROMACHI KIKAI CO.,LTD	MK-685	Treadmill equipped with transparent housing, electrical shocker, and speed control unit
Wistar Rats (male, 7-week old)	N.A.	N.A.	Commercially available at experimental animal sources