This study was supported by JSPS KAKENHI Grant Number JP19K19793, JP18H03129, and JP18K19739.

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
<ol>
	<li>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.</li>
	<l...

<ol>
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Validity of walking track assessment in the presence of chronic contractures.</a> Microsurgery. 10 (3), 220-225 (1989).</li><li>Wang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+evaluation+outcomes+correlate+with+histomorphometric+changes+in+the+rat+sciatic+nerve+crush+injury+model+:+A+comparison+between+sciatic+functional+index+and+kinematic+analysis.">Functional evaluation outcomes correlate with histomorphometric changes in the rat sciatic nerve crush injury model : A comparison between sciatic functional index and kinematic analysis.</a> PLoS One. 13 (12), e0208985 (2018).</li><li>de Ruiter, G. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+digital+video+ankle+motion+analysis+for+assessment+of+function+in+the+rat+sciatic+nerve+model.">Two-dimensional digital video ankle motion analysis for assessment of function in the rat sciatic nerve model.</a> Journal of the Peripheral Nervous System. 12 (3), 216-222 (2007).</li><li>Walker, J. L., Evans, J. M., Meade, P., Resig, P., Sisken, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gait-stance+duration+as+a+measure+of+injury+and+recovery+in+the+rat+sciatic+nerve+model.">Gait-stance duration as a measure of injury and recovery in the rat sciatic nerve model.</a> Journal of Neuroscience Methods. 52 (1), 47-52 (1994).</li><li>Dijkstra, J. R., Meek, M. F., Robinson, P. H., Gramsbergen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+evaluate+functional+nerve+recovery+in+adult+rats:+walking+track+analysis,+video+analysis+and+the+withdrawal+reflex.">Methods to evaluate functional nerve recovery in adult rats: walking track analysis, video analysis and the withdrawal reflex.</a> Journal of Neuroscience Methods. 96 (2), 89-96 (2000).</li><li>Lee, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+evaluation+in+the+rat+sciatic+nerve+defect+model:+a+comparison+of+the+sciatic+functional+index,+ankle+angles,+and+isometric+tetanic+force.">Functional evaluation in the rat sciatic nerve defect model: a comparison of the sciatic functional index, ankle angles, and isometric tetanic force.</a> Plastic and Reconstructive Surgery. 132 (5), 1173-1180 (2013).</li><li>Rui, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gait+cycle+analysis:+parameters+sensitive+for+functional+evaluation+of+peripheral+nerve+recovery+in+rat+hind+limbs.">Gait cycle analysis: parameters sensitive for functional evaluation of peripheral nerve recovery in rat hind limbs.</a> Annals of Plastic Surgery. 73 (4), 405-411 (2014).</li><li>Yu, P., Matloub, H. S., Sanger, J. R., Narini, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gait+analysis+in+rats+with+peripheral+nerve+injury.">Gait analysis in rats with peripheral nerve injury.</a> Muscle &amp; Nerve. 24 (2), 231-239 (2001).</li><li>Amado, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sensitivity+of+two-dimensional+hindlimb+joint+kinematics+analysis+in+assessing+functional+recovery+in+rats+after+sciatic+nerve+crush.">The sensitivity of two-dimensional hindlimb joint kinematics analysis in assessing functional recovery in rats after sciatic nerve crush.</a> Behavioural Brain Research. 225 (2), 562-573 (2011).</li><li>Monte-Raso, V. V., Barbieri, C. H., Mazzer, N., Yamasita, A. C., Barbieri, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+the+Sciatic+Functional+Index+always+reliable+and+reproducible?.">Is the Sciatic Functional Index always reliable and reproducible?.</a> Journal of Neuroscience Methods. 170 (2), 255-261 (2008).</li><li>Varejao, A. S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motion+of+the+foot+and+ankle+during+the+stance+phase+in+rats.">Motion of the foot and ankle during the stance phase in rats.</a> Muscle &amp; Nerve. 26 (5), 630-635 (2002).</li><li>Lin, F. M., Pan, Y. C., Hom, C., Sabbahi, M., Shenaq, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ankle+stance+angle:+a+functional+index+for+the+evaluation+of+sciatic+nerve+recovery+after+complete+transection.">Ankle stance angle: a functional index for the evaluation of sciatic nerve recovery after complete transection.</a> Journal of Reconstructive Microsurgery. 12 (3), 173-177 (1996).</li><li>Patel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Video-gait+analysis+of+functional+recovery+of+nerve+repaired+with+chitosan+nerve+guides.">Video-gait analysis of functional recovery of nerve repaired with chitosan nerve guides.</a> Tissue Engineering. 12 (11), 3189-3199 (2006).</li><li>Filipe, V. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+skin+movement+on+the+analysis+of+hindlimb+kinematics+during+treadmill+locomotion+in+rats.">Effect of skin movement on the analysis of hindlimb kinematics during treadmill locomotion in rats.</a> Journal of Neuroscience Methods. 153 (1), 55-61 (2006).</li><li>Tajino, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+motion+analysis+for+comprehensive+understanding+of+gait+characteristics+after+sciatic+nerve+lesion+in+rodents.">Three-dimensional motion analysis for comprehensive understanding of gait characteristics after sciatic nerve lesion in rodents.</a> Scientific Reports. 8 (1), 13585 (2018).</li></ol>

The authors have nothing to disclose.

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 &#34;high&#34; 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 ...

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 injuries2,3,4,5,6. 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 SFI11,12.
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 analysis13,14. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>9-0 nylon suture</td>
			<td>Bear Medic Corporation.</td>
			<td>T06A09N20-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthetic Apparatus for Small Animals</td>
			<td>SHINANO MFG CO.,LTD.</td>
			<td>SN-487-0T</td>
			<td></td>
		</tr>
		<tr>
			<td>ISOFLURANE Inhalation Solution</td>
			<td>Pfizer Japan Inc.</td>
			<td>(01)14987114133400</td>
			<td></td>
		</tr>
		<tr>
			<td>Kine Analyzer</td>
			<td>KISSEI COMTEC CO.,LTD.</td>
			<td>N.A.</td>
			<td>A analysis software</td>
		</tr>
		<tr>
			<td>Liquid adhesive</td>
			<td>KANBO PRAS CORPORATION</td>
			<td>PT-B180</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro forceps</td>
			<td>BRC CO.</td>
			<td>16171080</td>
			<td></td>
		</tr>
		<tr>
			<td>Motion Recorder</td>
			<td>KISSEI COMTEC CO.,LTD.</td>
			<td>N.A.</td>
			<td>A recording software</td>
		</tr>
		<tr>
			<td>Standard surgical hemostat</td>
			<td>Fine Science Tools, Inc.</td>
			<td>12501-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical blade No.10</td>
			<td>FEATHER Safety Razor CO., LTD</td>
			<td>100D</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical hemostat</td>
			<td>World Precision Instruments</td>
			<td>503740</td>
			<td></td>
		</tr>
		<tr>
			<td>Three-dimensional motion capture apparatus (KinemaTracer for Animal)</td>
			<td>KISSEI COMTEC CO.,LTD.</td>
			<td>N.A.</td>
			<td>A 3D motion analysis system that consists of cameras</td>
		</tr>
		<tr>
			<td>Three-dimensional(3D) Calculator</td>
			<td>KISSEI COMTEC CO.,LTD.</td>
			<td>N.A.</td>
			<td>A marker tracing software</td>
		</tr>
		<tr>
			<td>Treadmill</td>
			<td>MUROMACHI KIKAI CO.,LTD</td>
			<td>MK-685</td>
			<td>a treadmill with affialiated the electrical schocker, transparent sheats and a speed control apparatus</td>
		</tr>
	</tbody>
</table>

3d kinematic analysis for the functional evaluation in the rat model of sciatic nerve crush injury

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.

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 &#39;toe off&#39; 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 ...

Watch this Scientific Journal Video about 3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury at JoVE.com

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

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.

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

We provide a precise and DiCharacter method for functional evaluations of laboratory animals. It is sensitive and reliable and devoid of shortcomings attributed to other methods for functional evaluations. For example, the sciatic functional index.To begin, 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. Have each rat walk on the treadmill. Gradually accelerate the treadmill to the desired speed of 20 centimeters per second and let the rat walk normally at this speed for five minutes.After each walking session, provide a one to two-minute rest break. Repeat this process three times per day five days per week for one week. While the rat is receiving stable anesthesia through the anesthetic mask, shave an area from the lower back to the bilateral malleoli using an electric shaver.Place the rat in the prone position. Use a black marker pen to mark the bone landmarks on the shaved skin, a line through the spinous processes from the lumbar to sacral vertebrae. Additionally, use a black marker to mark the anterior/superior iliac spines, the greater trochanters, the knee joints, the lateral malleoli, the fifth metatarsal phalangeal joints, and the tip of the fourth toe.Then 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. 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.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. Click the recording icon to record one to two seconds of video from four directions using 120 hertz CCD cameras.Click on the recording icon again to stop the recording. Open the video files 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.Then click on the save icon. Now 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.Turn on the treadmill and set the speed to 20 centimeters per second. 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 over 10 steps are recorded, click on the icon again to stop recording and turn off the treadmill.Put the rat back into the anesthesia induction chamber for anesthetization. While the rat is under continuous anesthesia via the anesthetic mask, remove the hemispheric markers. Open the calculation software and open the video file on the interface.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. 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. Then click on the automatically tracing icon to start the automatic marker tracing process.If the system does not accurately trace a marker, click on the manually digitize icon to switch to the manual tracing mode, click on the tracing characteristic point in the 3D model and then on the responding marker in the picture. 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. Open the analysis software and then open the processed video file on the interface. Click on the setting icon and select and add designated parameters such as the ankle angle, toe angle, and pelvic shift to the display list in the popup window on the right.Click on OK so that the curves representing the value changes in the parameters appear on the interface. Click on the measurement icon and select smooth processing in its pull down menu. Enter 20 hertz in the popup window to remove frequencies greater than 20 hertz within the curves.By means of 3D kinematic analysis, the mean ratio of the stance or swing phase in the 10-step cycle was automatically calculated and represented on the interface. Each bilateral stance and swing phase in 10-step cycle periods for the control group, one week post-surgery, three weeks post-surgery, and six weeks post-surgery groups are shown here. The normal center of gravity trajectory shape resembles the infinity sign.The center of gravity trajectory shape did not return to a normal shape until six weeks after surgery. The mean value of the ankle or toe angle in the toe off phase suggested that they improved in an upward direction after surgery. Locating the bone landmarks on shaved skin as carefully and correctly as possible is most important thing.analysis on the sciatic nerve can be performed and it will reveal the relationship between functional and histological changes.

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8.4K Views.  Kyoto University. We provide a precise and DiCharacter method for functional evaluations of laboratory animals. It is sensitive and reliable and devoid of shortcomings attributed to other methods for functional evaluations. For example, the sciatic functional index.To begin, 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. Have each rat walk on the treadmill. Gradually ac...