Comprehensive Understanding of Inactivity-Induced Gait Alteration in Rodents

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 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.
2. 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.
3. Repeat this process every other day for 1 week or more frequently if needed^15,16,17.
   NOTE: Start the familiarization period 1 week prior to step 2.
4. 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 reports^18,19,20. 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.

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
   1. 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.
   2. 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

1. 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.
2. 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.
3. Open the 3DCalculator app (see Table of Materials) and the video file to be analyzed.
4. 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).
5. 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.
6. 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.
7. 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

1. Open the KineAnalyzer app (see Table of Materials) and load the file.
2. 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.
3. 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.
4. 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.
5. Assign colors to the created lines by selecting the desired color from the Color column.
6. 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.
7. 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.