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 ended3,4. Nevertheless, little has been reported on motion deficits caused by inactivity. Also, comprehensive motion characteristics of the deteriorations have not been fully determined.
The current protocol demonstrates and discusses the application of kinematic evaluation to visualize motion alterations by referring to gait motion deficits evoked through disuse in rats subjected to hindlimb unloading.
It has been shown that hyperextensions of limbs in walking after a simulated microgravity environment are observed both in humans5 and animals4,6,7,8. Therefore, for universality, we focused on general parameters in this study: angles of the knee and ankle joints and vertical distance between the metatarsophalangeal joint and hip (roughly equivalent to the height of the hip) at the middle point of the stance phase (midstance). Further, potential applications of video kinematic evaluation are suggested in the discussion.
A series of kinematic analyses may be an effective measure to assess functional aspects of neural control. However, although motion analyses have been developed from footprint observation or simple measuring on captured video9,10 to multiple camera systems11,12, universal methods and parameters are yet to be established. The method in this study is intended to provide this joint motion analysis with comprehensive parameters.
In the previous work13, we tried illustrating gait alterations in nerve lesion model rats using comprehensive video analysis. However, in general, the potential outcomes of motion analyses are often restricted to predetermined variables provided in the analysis frameworks. For this reason, the present study further detailed how to incorporate user-defined parameters that are broadly applicable. Kinematic evaluations using video analyses may be of further use if proper parameters are implemented.
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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.
- Place the rat on a treadmill designed for rodents (see Table of Materials). In the first session, allow the animal to explore the treadmill to become accustomed to the environment.
NOTE: This process takes approximately 5 min. - Gradually increase the belt's velocity to the desired level (20 cm/s) and walk the rat. Use an electric shock at the end of the treadmill if needed14.
NOTE: One walking session lasts approximately 10-20 min. - Repeat this process every other day for 1 week or more frequently if needed15,16,17.
NOTE: Start the familiarization period 1 week prior to step 2. - Keep the rats in groups in cages (2-3 rats in each cage) with a 12 h light-dark cycle. Provide food and water ad libitum.
2. Application of hindlimb unloading to the rats and setting up of joint markers
NOTE: Elevate the hindlimbs of the rat using thread and adhesive tape attached to the tail as described in previous reports18,19,20. Make sure the thread and tape are attached at the base of the tail to prevent slippage of the tail skin. Monitor the animals thoroughly and adjust the unloading height or tightness of the tape if needed.
- Under 2-5% isoflurane inhalation with an anesthetic mask, wrap the first half of a 30 cm long strip of adhesive tape around the proximal portion of the rat's tail.
- Fold a 1 m long cotton thread (cotton kitchen twine, approximately 1 mm diameter) into half. Make a loop by tying a knot at the folded 50 cm midpoint. The knot must be approximately 5 cm from the tip to leave a 10 cm circumference loop.
- Allow the remaining 15 cm of the adhesive tape to pass once through the thread loop to secure the tape. Wrap the remaining tape around the distal portion of the tail.
- Secure the other tip of the thread on the overhead platform of the cage. Keep the animals in a cage that is high enough to elevate their hindlimbs by their tails. Other than the unloading, provide the same environment as those for the Ctrl group, such as food, water, and floor bedding.
- Set up the joint markers and software (see Table of Materials) following the steps below.
NOTE: For details regarding this step, please see Wang et al.13.- Under 2-5% isoflurane inhalation, attach colored semispherical markers (3 mm diameter) to the shaved skin corresponding to bony landmarks. Keep the isoflurane level as low as possible to prevent very deep anesthesia.
- Ensure that the landmarks are the anterior superior iliac spine (ASIS), trochanter major (hip joint), knee joint (knee), lateral malleolus (ankle), and fifth metatarsophalangeal joint (MTP)21.
NOTE: Paint the tip of the toe if the angle of the toe is needed. Use an oil-based paint marker (see Table of Materials). Liquid glue is preferable for adhesive since the liquid form dries faster.
3. Marker tracking using captured videos
- Open the MotionRecorder app (see Table of Materials) and turn on the treadmill. Place the rat on the treadmill belt.
NOTE: The four cameras for video capture (see Table of Materials) are laid out along the long edges of the treadmill: two cameras on each edge, approximately 50 cm x 50 cm apart, facing the center of the treadmill belt area. - Increase the belt speed up to 20 cm/s. As the rat begins walking normally at the desired speed, click on the Record icon to start the video capture. Once enough steps (5 consecutive steps, preferably 10 steps) are obtained, stop the capture by clicking on the Record icon again.
NOTE: Capture data on multiple animals in one experiment. Try up to five times for each rat. If a rat does not walk, capture a different one and try the first one later. The capturing rate of the camera was 120 frames/s. - Open the 3DCalculator app (see Table of Materials) and the video file to be analyzed.
- Crop the video by adjusting the horizontal slider bar on the top to contain enough numbers of consecutive steps. Captured image changes by dragging the yellow slide bar's end tip icon(s).
- To capture the markers, select the marker legends by clicking on the marker legends on the stick picture model, dragging them to the corresponding marker on the captured video, and releasing the button. This process allocates the color of the marker to the marker legend in the stick picture. Repeat this process for every marker to be tracked.
- Click on the Automatic trace icon. If the system does not accurately track markers or the tracking process halts due to marker loss, switch to manual mode.
NOTE: This automatic process does not halt unless the markers are missed. If halts happen more often than every few frames, consider repositioning the lost markers. - If manual mode is needed, click the Manual icon to switch. Click the missing marker legend on the stick picture and the corresponding marker on the video. The video proceeds with one frame for each click in the manual mode.
NOTE: Utilize freely available apps that enable auto click to prevent fatigue of those who track (digitize) the markers (see Table of Materials).
4. Computation of desired parameters
- Open the KineAnalyzer app (see Table of Materials) and load the file.
- Go to the View > Edit Marker Master menu.It opens the "Marker master edit" window.
NOTE: Captured markers have simple numbers until they are labeled. - Click on the desired label (landmark) on the marker tab, then click on the desired color. This process designates each marker to a specific landmark.
- Go to the link tab. Create lines by clicking on two markers consecutively. This process creates lines that correspond to each limb using labeled markers.
- Assign colors to the created lines by selecting the desired color from the Color column.
- Define angles by assigning reference/moving lines and directions of the angles. Go to the angle tab. After naming the angle, assign Vector A (reference line) and Vector B (moving line) by clicking on the markers corresponding to each landmark. Then, define the direction of the angle with a value in the operate section in the same tab.
NOTE: For the present study, the parameters mainly focused on were at the middle of the stance phase (midstance): KSt (knee angle), ASt (ankle angle), MHD (metatarso hip distance: equivalent to the height of the hip, see the next section). Knee angle and ankle angle were defined as the angle between the femur and tibia and the tibia and fifth metatarsal bone, respectively. A 0° angle means that the joint was fully flexed. - On the distance tab, define the distance parameter (MHD). Select two corresponding markers in the Distance Setting section. Joint trajectories as a function of normalized step cycle will also be available.
NOTE: Defining angles/parameters needs to be performed only one time. The settings of the parameters will be available for later evaluations once this defining process is completed.
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Representative 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 extensions in the knee and ankle (i.e., plantarflexion for the ankle) than the Ctrl group, called "toe walking"3,16. The goal of this study was to determine the comprehensive characteristics of these motion deteriorations. To elucidate quantitative measures out of these overall outcomes, three parameters were implemented as stated above: KSt, knee angle at the midstance; ASt, ankle angle; MHD, metatarso hip distance (vertical distance between the fifth metatarsophalangeal joint and hip joint), which is virtually equivalent to the height of the hip joint at midstance.
At 2 weeks (2 weeks after unloading), both the KSt and ASt of the UL group were significantly greater than those of the Ctrl group (Figure 2A,B, unpaired t-test: p < 0.01). In addition, MHD was considerably higher in the UL group (Figure 3, unpaired t-test: p < 0.01). The paw position during midstance is shown in Supplementary Figure 1.
Less activity through unloading might cause neural alterations22,23,24,25. Those alterations could lead to deterioration in functional features of locomotor systems3,4 and musculoskeletal features. Significant changes in the parameters described above may be attributed to those neural alterations.
Figure 1: Normalized joint trajectories of the representative subjects. The ordinate is adjusted so that the trajectories in the diagram appear approximately in the center. (A) Knee and (B) ankle joints in the unloading group exhibited further extension (plantar flexion for the ankle) than the control group during the stance phase. Please click here to view a larger version of this figure.
Figure 2: Joint angles of the knee and ankle at midstance. The unloading group showed significantly greater angles both in (A) KSt (knee) and (B) Ast (ankle) than the control group (unpaired t-test: p < 0.01). The error bar represents the 95% confidence interval. Please click here to view a larger version of this figure.
Figure 3: Height of the hip joint at midstance. The metatarso hip distance of the unloading group was significantly higher than that of the control group (unpaired t-test: p < 0.01). The error bar represents the 95% confidence interval. Please click here to view a larger version of this figure.
Supplementary File 1: A schematic representing the sequence of procedures. Please click here to download this File.
Supplementary Figure 1: The paw position of the rat during midstance. Please click here to download this File.
Supplementary Video 1: Footstep tracking from the bottom. Please click here to download this Video.
Supplementary Video 2: Evaluation of reaching motions. Please click here to download this Video.
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Discussion
Alteration of environments leads to fluctuating functional aspects and musculoskeletal components of locomotor systems26,27. 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 such parameters.
In order to track joint landmarks for video analysis, using infrared markers and cameras is prevalent, while manual tracking is also common10,28. Utilizing colored semispherical markers combined with the automated capturing process would make this tracking process simpler and more cost-effective. This tracking method was incorporated in the present study despite the potential fluctuation of the outcomes due to skin slippage. To address this skin slippage, Bojados et al. also tried a radiographic approach with markers implanted directly on the bone underneath the skin17.
Another advantage of motion analysis is that it extracts multiple functional aspects once the fundamental dataset is obtained. Since characteristic motions differ in terms of affected functions, data transformation to distinct parameters even after data collection would be a substantial benefit. Even footstep tracking is achievable with a mirror placed at 45º slanted underneath the walking platform. Furthermore, the application of video analysis is not limited to walking motion (Supplementary Videos 1, Supplementary Video 2).
Despite these advantages, motion analysis, especially the 3D analysis approach, has limitations. First, since the methodology works as a constellation of devices (i.e., a treadmill for animals, multiple cameras, apps), the whole setup of apparatuses can be expensive. Second, the experimental procedure is labor-intensive, and operators need to become fully accustomed to the procedures.
However, considering its applicability to both gait analysis and joint angle, its benefits outweigh its disadvantages if it becomes widely available. Future work may utilize video analysis in a broader range of functional assessments to afford this analysis series.
3D motion tracking/evaluation is a strong tool for quantitatively assessing functional alterations of movements. Obstacles to implementing this methodology may be resolved through further studies.
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Disclosures
The authors declare that there is no conflict of interest.
Acknowledgments
This study was supported in part by the Japan Society for the Promotion of Science (JSPS) KAKENHI (no. 18H03129, 21K19709, 21H03302, 15K10441) and the Japan Agency for Medical Research and Development (AMED) (no. 15bk0104037h0002).
Materials
| 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 |
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