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

Summary

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Abstract

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

Introduction

The Sciatic Functional Index (SFI) is the benchmark method for carrying out functional sciatic nerve evaluations¹. The SFI has been widely adopted and is frequently used within various functional evaluation studies on rat sciatic nerve injuries^2,3,4,5,6. In spite of its popularity, there are several problems with SFI, including automutilation⁷, joint contracture risk, and smearing of the footprints⁸. These problems seriously affect its prognostic value⁹. Therefore, an alternative, less error-prone method is required as a substitute for the SFI.

One such alternative method is kinematic analysis. This includes comprehensive gait analysis using tracking markers attached to bony landmarks or joints. Kinematic analysis is increasingly used for functional evaluations⁹. This method is progressively being recognized as a reliable and sensitive tool for functional evaluation¹⁰ without the shortcomings attributed to the SFI^11,12.

In this protocol, we describe a series of kinematic analyses that use a 3D motion capture apparatus consisting of a treadmill, four 120 Hz charged coupled device (CCD) cameras, and data processing software (see Table of Materials). This kinematic analysis method differs from general video walking or gait analysis^13,14. Two cameras are positioned in different directions to record posterior limb movements from a single side. Subsequently, a 3D digital model of the posterior limb is constructed using computer graphics⁹. We can calculate designated joint angles, such as hip, knee, ankle, and toe joint, by closely recapitulating the actual limb dimensions. Additionally, we can determine various parameters such as stride/step length and the ratio of the stance phase to the swing phase. These reconstructions are based on a completely reconstructed 3D digital model of the posterior limbs, generated from data transmitted by two sets of cameras. Even the imaginary center of gravity (CoG) trajectory can be calculated automatically.

We used this 3D motion capture apparatus to introduce and assess multiple kinematic parameters that reveal functional changes over time within the context of the rat sciatic nerve crush injury model.

Protocol

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.

Results

We selected four parameters to investigate functional changes over time in a rat sciatic nerve crush injury model. These were the ratio of the stance-to-swing phase, center of gravity (CoG) trajectory, ankle angles, and toe angles in the 'toe off' phase⁹. Twenty-four rats were randomly assigned to one of four groups: the control group (C), rats at the first (1w), third (3w), and sixth (6w) week following left sciatic nerve crush injury.

By means of 3D kinematic ...

Discussion

In this protocol, a stable and continuously walking rat is the most vital component of kinematic analysis. The treadmill speed was set to 20 cm/s. This walking speed is by no means considered "high" if rats move without space constraints¹⁶. Nevertheless, this speed is too fast for untrained rats to stably walk on the treadmill and would likely result in an abnormal gait and nonuniform movements. These events may seriously affect data reliability and authenticity. However, treadmill speeds ...

Materials

Name	Company	Catalog Number	Comments
9-0 nylon suture	Bear Medic Corporation.	T06A09N20-25
Anesthetic Apparatus for Small Animals	SHINANO MFG CO.,LTD.	SN-487-0T
ISOFLURANE Inhalation Solution	Pfizer Japan Inc.	(01)14987114133400
Kine Analyzer	KISSEI COMTEC CO.,LTD.	N.A.	A analysis software
Liquid adhesive	KANBO PRAS CORPORATION	PT-B180
Micro forceps	BRC CO.	16171080
Motion Recorder	KISSEI COMTEC CO.,LTD.	N.A.	A recording software
Standard surgical hemostat	Fine Science Tools, Inc.	12501-13
Surgical blade No.10	FEATHER Safety Razor CO., LTD	100D
Surgical hemostat	World Precision Instruments	503740
Three-dimensional motion capture apparatus (KinemaTracer for Animal)	KISSEI COMTEC CO.,LTD.	N.A.	A 3D motion analysis system that consists of cameras
Three-dimensional(3D) Calculator	KISSEI COMTEC CO.,LTD.	N.A.	A marker tracing software
Treadmill	MUROMACHI KIKAI CO.,LTD	MK-685	a treadmill with affialiated the electrical schocker, transparent sheats and a speed control apparatus