Name: 3d kinematic analysis for the functional evaluation in the rat model of sciatic nerve crush injury
Uploaded: 2020-02-12T12:00:00
Description: 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

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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M., Hsieh, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+functional+recovery+from+sciatic+nerve+crush+injury+through+a+combined+treatment+of+cold-water+swimming+and+mesenchymal+stem+cell+transplantation.">Enhanced functional recovery from sciatic nerve crush injury through a combined treatment of cold-water swimming and mesenchymal stem cell transplantation.</a> Neurological Research. 37 (90), 816-826 (2015).</li><li>Jiang, W., 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=Low-intensity+pulsed+ultrasound+treatment+improved+the+rate+of+autograft+peripheral+nerve+regeneration+in+rat.">Low-intensity pulsed ultrasound treatment improved the rate of autograft peripheral nerve regeneration in rat.</a> Scientific Reports. 6, 22773 (2016).</li><li>Ni, X. 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=The+Effect+of+Low-Intensity+Ultrasound+on+Brain-Derived+Neurotropic+Factor+Expression+in+a+Rat+Sciatic+Nerve+Crushed+Injury+Model.">The Effect of Low-Intensity Ultrasound on Brain-Derived Neurotropic Factor Expression in a Rat Sciatic Nerve Crushed Injury Model.</a> Ultrasound in Medicine &amp; Biology. 43 (2), 461-468 (2017).</li><li>Weber, R. A., Proctor, W. H., Warner, M. R., Verheyden, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autotomy+and+the+sciatic+functional+index.">Autotomy and the sciatic functional index.</a> Microsurgery. 14 (5), 323-327 (1993).</li><li>Dellon, A. L., Mackinnon, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sciatic+nerve+regeneration+in+the+rat.+Validity+of+walking+track+assessment+in+the+presence+of+chronic+contractures.">Sciatic nerve regeneration in the rat. 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>

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.

3d-kinematic-analysis-for-functional-evaluation-rat-model-sciatic

Research

JoVE Journal

Neuroscience

Axoplasm Isolation from Rat Sciatic Nerve

Isolation of pure axonal cytoplasm (axoplasm) from peripheral nerve is crucial for biochemical studies of many biological processes. In this article, we demonstrate and describe a protocol for axoplasm isolation from adult rat sciatic nerve based on the following steps: (1) dissection of nerve fascicles and separation of connective tissue; (2) incubation of short segments of nerve fascicles in hypotonic medium to release myelin and lyse non-axonal structures; and (3) extraction of the remaining axon-enriched material. Proteomic and biochemical characterization of this preparation has confirmed a high degree of enrichment for axonal components.

We demonstrate a protocol for axoplasm isolation from adult rat sciatic nerve based on dissection of nerve fascicles and incubation in hypotonic medium to release myelin and lyse non-axonal structures, followed by extraction of the remaining axon-enriched material.

Isolation of pure axonal cytoplasm (axoplasm) from peripheral nerve is crucial for biochemical studies of many biological processes. In this article, we ...

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.
The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.
Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.

This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Facial Nerve Surgery in the Rat Model to Study Axonal Inhibition and Regeneration

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial nerve can be manipulated along its entire length, from its intracranial segment to its extratemporal course. There are three primary types of nerve injury used for the experimental study of regenerative properties: nerve crush, transection, and nerve gap. The range of possible interventions is vast, including surgical manipulation of the nerve, delivery of neuroactive reagents or cells, and either central or end-organ manipulations. Advantages of this model for studying nerve regeneration include simplicity, reproducibility, interspecies consistency, reliable survival rates of the rat, and an increased anatomic size relative to murine models. Its limitations involve a more limited genetic manipulation versus the mouse model and the superlative regenerative capability of the rat, such that the facial nerve scientist must carefully assess time points for recovery and whether to translate results to higher animals and human studies. The rat model for facial nerve injury allows for functional, electrophysiological, and histomorphometric parameters for the interpretation and comparison of nerve regeneration. It thereby boasts tremendous potential toward furthering the understanding and treatment of the devastating consequences of facial nerve injury in human patients.

This protocol describes a reproducible approach to facial nerve surgery in the rat model, including descriptions of various inducible patterns of injury.

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial ...

Automated Gait Analysis to Assess Functional Recovery in Rodents with Peripheral Nerve or Spinal Cord Contusion Injury

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and a lot of comparative data has been published in the literature. This includes a multitude of assessment methods to study functional recovery following nerve injury and repair. Besides evaluation of nerve regeneration by means of histology, electrophysiology, and other in vivo and in vitro assessment techniques, functional recovery is the most important criterion to determine the degree of neural regeneration. Automated gait analysis allows recording of a vast quantity of gait-related parameters such as Paw Print Area and Paw Swing Speed as well as measures of inter-limb coordination. Additionally, the method provides digital data of the rats&#8217; paws after neuronal damage and during nerve regeneration, adding to our understanding of how peripheral and central nervous injuries affect their locomotor behavior. Besides the predominantly used sciatic nerve injury model, other models of peripheral nerve injury such as the femoral nerve can be studied by means of this method. In addition to injuries of the peripheral nervous systems, lesions of the central nervous system, e.g., spinal cord contusion can be evaluated. Valid and reproducible data assessment is strongly dependent on meticulous adjustment of the hard- and software settings prior to data acquisition. Additionally, proper training of the experimental animals is of crucial importance. This work aims to illustrate the use of computerized automated gait analysis to assess functional recovery in different animal models of peripheral nerve injury as well as spinal cord contusion injury. It also emphasizes the method&#8217;s limitations, e.g., evaluation of nerve regeneration in rats with sciatic nerve neurotmesis due to limited functional recovery. Therefore, this protocol is thought to help researchers interested in peripheral and central nervous injuries to assess functional recovery in rodent models.

Automated gait analysis is a feasible tool to evaluate functional recovery in rodent models of peripheral nerve injury and spinal cord contusion injury. While it requires only one setup to assess locomotor function in various experimental models, meticulous hard- and soft-ware adjustment and training of the animals is highly important.

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and ...

Multifactorial Assessment of Motor Behavior in Rats after Unilateral Sciatic Nerve Crush Injury

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In addition, in the research field of movement disorders, sciatic crush injury has been employed to trigger a dystonia-like phenotype in genetically predisposed DYT-TOR1A rodent models of dystonia. To achieve consistent, reproducible and comparable results after a sciatic nerve crush injury, a standardized method for inducing the nerve crush is essential, in addition to a standardized phenotypical characterization. Attention must be paid not only to the specific assortment of behavioral tests, but also to the technical requirements, the correct execution and consecutive data analysis. This protocol describes in detail how to perform a sciatic nerve crush injury and provides a behavioral test battery for the assessment of motor deficits in rats that includes the open field test, the CatWalk XT gait analysis, the beam walking task, and the ladder rung walking task.

We provide a protocol for the assessment of motor behavior via a behavioral test battery in rats after sciatic nerve crush injury.

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In ...

Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology

Ex vivo preparations enable the study of many neurophysiological processes in isolation from the rest of the body while preserving local tissue structure. This work describes the preparation of rat sciatic nerves for ex vivo neurophysiology, including buffer preparation, animal procedures, equipment setup and neurophysiological recording. This work provides an overview of the different types of experiments possible with this method. The outlined method aims to provide 6 h of stimulation and recording on extracted peripheral nerve tissue in tightly controlled conditions for optimal consistency in results. Results obtained using this method are A-fibre compound action potentials (CAP) with peak-to-peak amplitudes in the millivolt range over the entire duration of the experiment. CAP amplitudes and shapes are consistent and reliable, making them useful to test and compare new electrodes to existing models, or the effects of interventions on the tissue, such as the use of chemicals, surgical alterations, or neuromodulatory stimulation techniques. Both conventional commercially available cuff electrodes with platinum-iridium contacts and custom-made conductive elastomer electrodes were tested and gave similar results in terms of nerve stimulus strength-duration response.

Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology

This protocol describes the preparation of rat whole sciatic nerve tissue for ex vivo electrophysiological stimulation and recording in an environmentally-regulated, two-compartment, perfused saline bath.

Ex vivo preparations enable the study of many neurophysiological processes in isolation from the rest of the body while preserving local tissue structure. ...