3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

The protocol was approved by the animal experimentation committee of Kyoto University, and all protocol steps were performed in accordance with the Guidelines of the Animal Experimentation Committee, Kyoto University (approval number: MedKyo17029).

1. Familiarizing rats with treadmill walking

1. Set up two transparent plastic sheets on both sides of the treadmill to let a 12-week-old male Lewis rat walk in a straight, frontward direction, then turn on the electric shock grid.
2. Have each rat walk on the treadmill. Gradually accelerate the treadmill to the desired speed (20 cm/s or 12 m/min) and let the rat walk normally at this speed for 5 min. After each walking session, provide a 1–2 min rest break. Repeat this process 3x per day, 5 days per week, for 1 week.
   NOTE: Start the treadmill walking 1 week before step 2.
3. House rats in groups of three per cage with a 12 h light-dark cycle and feed them commercial rat food and tap water ad libitum.

2. Performing the sciatic nerve crush injury

1. Place the rat in an anesthesia induction chamber and introduce 5% isoflurane inhalation solution.
2. Provide an intraperitoneal injection of a combination anesthetic prepared with 0.15 mg/kg medetomidine hydrochloride, 2 mg/kg midazolam, and 2.5 mg/kg butorphanol tartrate to the rat. Check for the lack of pedal reflexes. Then shave an area from the left greater trochanter to the mid-thigh with an electric shaver.
3. Spread out a piece of aseptic cloth, place the rat on it, and have it lie in the left lateral position. Place sterile surgical instruments on the cloth as well.
4. Create a straight incision from the greater trochanter to the mid-thigh with a surgical no. 10 blade. Then perform a blunt dissection between quadriceps femoris and biceps femoris using a surgical hemostat to expose the sciatic nerve.
5. Detach the sciatic nerve from the surrounding tissue with two pairs of microforceps and crush the sciatic nerve for 10 s, using a standard surgical hemostat, to create a 2 mm long crush injury at the site directly below the gluteal tuberosity.
6. Perform a 9-0 nylon epineural stitch at the proximal end of the injury using a pair of microforceps and then close the muscle and skin with 4-0 nylon sutures.
7. Provide an intraperitoneal injection of an anesthetic antagonist prepared with 0.3 mg/kg atipamezole hydrochloride to the rat, to wake it up within 10 min. After the rat recovers from anesthesia, observe the left toe movements while the rat is suspended by the base of its tail. If the toe does not spread at all, the surgery was successful.
8. House the rats individually after the surgery with a 12 h light-dark cycle and feed them commercial rat food and tap water ad libitum.

3. Attaching the markers

1. Place the trained rat in an anesthesia induction chamber and introduce a 5% isoflurane inhalation solution. Check for the lack of the pedal reflex by pinching the toe.
2. Allow the rat to be continuously anesthetized using an anesthetic mask (2% isoflurane inhalation solution). While the rat receives stable anesthesia, shave an area from the lower back to the bilateral malleoli using an electric shaver.
   CAUTION: To prevent exposing researchers to the leaking isoflurane, make sure that the mask tightly covers the head and face of the rat.
   NOTE: To prevent injury to the rat, shave the hair off as gently as possible.
3. Place the rat in the prone position. Use a black marker pen to mark the following bone landmarks on the shaved skin: A line through the spinous processes from the lumbar to sacral vertebrae, the anterior superior iliac spines, the greater trochanters, the knee joints, the lateral malleoli, the fifth metatarsophalangeal joints, and the tip of the fourth toe.
   NOTE: The line through the spinous processes is used for determining whether the bilateral markers are axially symmetric.
4. Use a liquid adhesive to attach hemispheric markers to these bone landmarks, except for the line through the spinous processes from the lumbar to the sacral vertebrae, and the tip of the fourth toe. Use distinct colors for every other landmark to avoid confusion. The tip of the fourth toe is marked with pink ink.
   CAUTION: Take care not to drip adhesive onto the exposed skin of the operator.
5. After placing all the markers, put the rat back into the cage. Do not put the rat on the treadmill until it fully recovers from anesthesia.
   NOTE: Reduced consciousness may seriously influence normal walking if the rat does not fully recover from anesthesia.

4. Calibration and software setup

1. Set up two transparent plastic sheets on both sides of the treadmill and place the calibration box in the middle of the treadmill. Open the recording software and then click on the Calibration Image icon on the display (Supplementary File 1).
2. Click the Recording icon to record 1–2 s of video from four directions using 120 Hz CCD cameras. Click on the Recording icon again to stop the recording.
   NOTE: The video will be saved automatically once the recording stops.
3. Open the video file in the calculation software. Click and drag the characteristic points of the calibration box 3D models on the bottom right corner of the screen to the corresponding markers on the four pictures, which are automatically transformed from the video in the calibration pattern (Supplementary File 2). Then click on the Save icon.
   NOTE: Do not change the positions of the cameras after the calibration is completed.

5. Recording the walking

1. Take the calibration box out of the treadmill, turn on the electric shock grid, and place the fully awake rat on the treadmill. Open the recording software and input the basic information about the rat, including its serial number, walking speed, and the name of the principal operator.
2. Turn on the treadmill and set the speed to 20 cm/s. After the rat adapts to the speed and is able to walk normally, click the Recording icon on the display to record the walking rat with the four cameras. Once enough steps are recorded (>10), click on the icon again to stop recording, and turn off the treadmill.
   NOTE: The video will be saved automatically once the recording stops.
3. Put the rat back into the anesthesia induction chamber for anesthetization. While the rat is under continuous anesthesia (administered via the anesthetic mask), remove the hemispheric markers.
   NOTE: Remove the markers as gently as possible to avoid causing pain to the rat.
4. At the designated time (e.g., 1 week, 3 weeks, or 6 weeks after surgery), perform the kinematic measurement on the rat by repeating steps 3.1–5.3. Make the kinematic measurement only once, at the beginning of the experiment, for the rats that did not receive surgery (i.e., the control group).

6. Marker tracing

1. Open the calculation software and open the video file on the interface.
2. Click and drag the bilateral control bar on the progress bar of the video to ensure that only a 10-step treadmill walking record is displayed (Supplementary File 3). Click and drag each characteristic point from the 3D model on the bottom right corner of the screen to the corresponding marker on each of the four initial pictures of the videos that were taken by the cameras (Supplementary File 4).
3. Click on the Automatically Tracing icon to start the automatic marker tracing process (Supplementary File 5, Supplementary File 6). If the system does not accurately trace a marker, click on the Manually Digitize icon to switch to the manual tracing mode (Supplementary File 7), click on the tracing characteristic point in the 3D model, and then on the responding marker in the picture.
4. Once the marker is clicked, ensure that the picture switches to the next frame of the video. Now continuously click on the marker until the marker tracing process is completed. Once finished, click on the Save icon.

7. Kinematic analysis

1. Open the analysis software and then open the processed video file on the interface.
2. Click on the Setting icon and select and add designated parameters such as the ankle angle, toe angle, and pelvic shift (X and Z axes) to the Display List in the pop-up window on the right (Supplementary File 8). Click on OK, so that curves representing the value changes in the parameters appear on the interface.
3. Click on the Measurement icon and select Smooth processing in its pull-down menu. Enter 20 Hz in the pop-up window to remove frequencies greater than 20 Hz within the curves (Supplementary File 9).
4. Ensure that there are five panels on the interface: the walking video of the rat, the dynamic 3D model, curves that represent value changes in the parameters from the 10-step cycle, curves that represent mean value changes in parameters, and histograms and schematic diagrams that represent the ratio of the stance and swing phase (Supplementary File 10).
5. Right click the panel for curves representing mean value changes in parameters and select Data Output in the pull-down menu (Supplementary File 11). This will produce the average values of the posterior limb joint angles, including the ankle and toe angles, pelvic shift, and any other desired parameters in 10-step cycle periods.