This work was supported by the German Federal Ministry of Education and Research (BMBF DysTract to C.W.I.) and by the Interdisciplinary Center for Clinical Research (IZKF) at the University of W&#252;rzburg (N-362 to C.W.I.; Z2-CSP3 to L.R.). In addition, this project has received funding from the European Union&#39;s Horizon 2020 research and innovation programme under the EJP RD COFUND-EJP N&#176; 825575 (EurDyscover to J.V.), and from the VERUM Foundation. Moreover C.W.I. is funded by the Deutsche Forschungsgemeinschaft (DFG, German Reseach Foundation) Project-ID 424778381-TRR 295, by the&#160;Deutsche Stiftung Neurologie and ParkinsonFonds. L.R. is additionally supported by the Dystonia Medical Reseach Foundation.
The authors thank Keali R&#246;hm, Veronika Senger, Heike Menzel and Louisa Frie&#223; for their technical assistance as well as Helga Br&#252;nner for the animal care.

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducible+mouse+sciatic+nerve+crush+and+subsequent+assessment+of+regeneration+by+whole+mount+muscle+analysis.">Reproducible mouse sciatic nerve crush and subsequent assessment of regeneration by whole mount muscle analysis.</a> Journal of Visualized Experiments. (60), e3606 (2012).</li><li>Savastano, L. E., 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=Sciatic+nerve+injury:+a+simple+and+subtle+model+for+investigating+many+aspects+of+nervous+system+damage+and+recovery.">Sciatic nerve injury: a simple and subtle model for investigating many aspects of nervous system damage and recovery.</a> Journal of Neuroscience Methods. 227, 166-180 (2014).</li><li>Menorca, R. M., Fussell, T. S., Elfar, J. 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The authors have nothing to disclose.

This behavioral assessment protocol provides an overview of advantages and disadvantages as well as possible readouts of the selected behavioral test battery in a rodent model after sciatic nerve crush injury. 
To obtain a comparative outcome of the sciatic nerve crush injury, a consistent crush technique is mandatory. The use of a non-serrated clamp (Ultra Fine Hemostat) instead of forceps can improve the consistency of the crush. Use the same clamp as well as the same crush position to guarantee equal nerve compression. The exclusive use of the clamp for the crush injury and handling of the clamp with care improves consistency. Also, perform the procedure of the crush injury with care. Additional damage to the nerve during surgery such as unwanted traction of the nerve can lead to undesired side effects like automutilation. Therefore, a careful nerve preparation as well as an administration of a pain reliever for a minimum of two days is recommended. 
Multifactorial assessment of motor behavior can characterize the phenotype after nerve crush injury in rats at various levels. We used the OFT, CatWalk XT gait analysis, beam walking task and ladder rung walking task. A blinded experimental procedure and data analysis to experimental groups is essential for these experiments. Before behavior assessment, animals were acclimated in the testing room under testing conditions for at least 30 minutes. All the behavioral tests applied herein have the advantage that food or water deprivation are not required. The same group set of animals were used in all described behavioral tests. A maximum of two different behavioral tests per day were performed for each animal. If behavioral tests are performed in regular intervals, pay attention of a comparable procedure, like performing the test in the same animal order and at the same time of the day. A further important aspect for behavioral analysis is the day-night cycle of rats. Consider a reversed day-night cycle to obtain more natural and higher levels of activity at the day cycle (dark cycle). This has to be considered especially for measurement of spontaneous behavior, like the OFT. In this experiment, a reversed day-night cycle could not be implemented, but an adequate acclimatization to the testing conditions was ensured. A perfect illumination is essential for high-resolution videos for the beam walking task and the ladder rung walking task. This high video quality cannot be reached when performing experiments in the dark. 
The assessment of gait requires a continuous task performance. The first important aspect of a continuous task performance is to convince the animals to cross the setup. To increase the motivation, place small food pellets (45 mg) at the end of the setup. In order for animals to get familiarized with the food pellets, the pellets should be fed to them prior to testing. Also, a goal box at the end of the setup can be helpful. The setup of the CatWalk already includes a goal box, but rats sometimes hesitate to enter the goal box. Alternatively, you can add a small cage into the goal box, but the home cage from rats does not fit into the goal box. Let the rat habituate in the cage for a few minutes before acquisition. Additionally, another rat from the same home cage may be placed into the goal box or into the cage inside the goal box. Make sure that the second rat remains in the box and does not block the entrance to the goal box. Furthermore, it is also possible to remove the goal box from the CatWalk system and to place the rat home cage at the end of the walkway, which allows the rat to enter their "home territory" after each run. For the setup of the beam walking task and the ladder rung walking task, we recommend to add a goal box or the home cage at the end of the setup. To ensure consistency, the CatWalk, the beam walking task, and the ladder rung walking task should be performed at least once a week with six to ten runs.
Although not every analysis yielded significant differences in this study, consider that an inclusion of genetically modified animals or treatment groups could produce valuable data that can distinguish between groups from the same behavioral tests. 
The nerve crush injury had no effect on the locomotor activity of the rat, which was measured in a five minutes OFT. Catwalk XT gait analysis is a more objective and sensitive tool to analyze gait, paw and toe placement. After an intensive training, the rats learn to cross the walkway of the CatWalk XT apparatus to the default settings. The nerve injury does not reduce the ability of the rats to cross the walkway. The automatic computation of various parameters presents the data objectively. Additional information can be gained by using the "Interactive Footprint Measurements" module and indeed, these analyzes yielded significant differences in various parameters of toe spread, print length and paw angle to body axis comparing rats with and without nerve injury.
Rats can be trained easily for the beam walking task. Differences in the latency time to cross the beam and in the number of foot slips per step of the nerve-injured hindlimb were detected by comparing na&#239;ve with crush-injured rats. A disadvantage of analyzing nerve-injured rats with the beam walking task is the size of the beam. Within the first two weeks after the sciatic nerve crush injury, the rats need assistance to cross the beam as their balance is impaired. Although some rats may be capable of crossing the beam, the risk of injuries caused by a fall is high. Nerve-crushed animals should therefore be assisted to cross the beam for the first two weeks after sciatic nerve crush injury or longer, if necessary. However, it is difficult to compare runs with and without assistance. Also, the motor balance is an important parameter assessed by the beam walking task. We considered this parameter not to be relevant to our nerve crush rat model. Therefore, scores described by Ohwatashi et al. and Johansson &amp; Ohlsson could not be used and runs with an incomplete beam traverse were excluded for data analysis18,19.
The 7-category scale from Metz et al. can analyze both fore- and hindlimbs and distinguish between different severity levels of errors of all limbs during the ladder rung walking task16,17. By analyzing the most prominent errors, which include the categories from 0 to 2, no differences of errors per step could be detected in the hindlimb when comparing nerve-injured wt rats with na&#239;ve wt rats. Furthermore, the latency time of traversing the ladder rung apparatus did not differ between nerve-injured wt rats and wt na&#239;ve rats. Deep learning models could improve and speed up data analysis of the ladder rung walking task through an automated approach. 
It is important to mention that the nerve crush injury as well as all described behavioral tests can easily translated to mice, by adapting the settings and sizes of the setups. The use of mice as a model organism has the beneficial effect that transgenic models for many human diseases exist.

Rodents are excellent model organisms to deepen the understanding of human diseases1,2 by testing hypotheses on multiple biological levels. One fundamental biological level for the characterization of rodent models is the phenotype level, measured by behavioral assessments. Depending on the animal model and the scientific research question, the selection of a powerful and reliable behavioral test battery is essential to cover a wide range of behavioral aspects such as for animal models of Parkinson&#39;s disease and dystonia3,4,5,6.
The sciatic nerve is the largest nerve in the human body with motor as well as sensory fibers. Injuries of the sciatic nerve can result easily from a variety of events such as traffic accidents and surgeries7,8. Therefore, research activities using rodent models with sciatic nerve injuries, are of translationally relevant value. Even though the translational aspect of nerve regeneration from rat to human has to be regarded critically9, the sciatic nerve crush injury (axonotmesis) in rodent models is a commonly used method to analyze degeneration and regeneration processes of peripheral nerves10,11. In case of a crush injury the nerve is not completely transected. It damages the axon, resulting in conduction block directly after crush injury followed by regenerative processes 4,12,13.
Moreover, in dystonia research, the unilateral sciatic nerve crush injury is an established method to trigger dystonia-like movements (DLM) in genetically predisposed dystonia rodent models, which do not show DLM per se4,14. It is assumed that the peripheral nerve trauma disturbs the sensorimotor integration by affecting the sciatic nerve fibers, which are responsible for motor and sensory functions15.
We here provide a detailed description for a standardized crush injury of the sciatic nerve and a battery of motor behavior assessments that is composed of the open field test (OFT), CatWalk XT gait analysis, beam walking task and ladder rung walking task in na&#239;ve wild type (wt) rats (n= 8-9) and wt rats five weeks after unilateral sciatic nerve crush injury (n= 10). The OFT provides information about the general locomotor activity, while a detailed gait analysis is achieved by the automated gait analysis system CatWalk XT. The beam walking task is used to assess the motor coordination by evaluating the time to cross the beam and the number of foot placement errors. For gait performance analysis the ladder rung walking task gives information about foot or paw placement and errors on a horizontal ladder rung apparatus with a constant but irregular rung pattern.

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			<td>Bayer Vital GmbH</td>
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			<td>self made</td>
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			<td>JVC</td>
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			<td>Noldus</td>
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			<td>&#160;setup and software</td>
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			<td>Dr. Wilfried M&#252;ller GmbH</td>
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			<td>Fine Science Tools</td>
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			<td>Dumont #5 forceps</td>
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			<td>Noldus</td>
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			<td>Forceps 160 mm</td>
			<td>Hartenstein</td>
			<td>PZ09</td>
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			<td>Dr. Wilfried M&#252;ller GmbH</td>
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			<td>Goal box for ladder rung walking task apparatus</td>
			<td>self made</td>
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			<td>Wahl GmbH</td>
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			<td>self made</td>
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			<td>self made</td>
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			<td>Spacer for ladder rung walking task apparatus</td>
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			<td>Suture Silkam 4/0 DS 19</td>
			<td>B. Braun</td>
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			<td>Ultra fine hemostats (non-serrated clamp)</td>
			<td>Fine Science Tools</td>
			<td>13020-12</td>
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</table>

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.

The representative results of the five minutes OFT show that the nerve crush injury five weeks post surgery has no effect on the locomotor activity (Figure 1).
Gait analysis with the CatWalk XT system (Figure 2) generates many different parameters. Selective parameters were statistically analyzed by comparing wt na&#239;ve rats with nerve-injured wt rats five weeks after the nerve crush (Figure 2D). Significant alterations could be detected for the run average speed, the stride length and the print area of the nerve-injured (right) hind paw. A more detailed analysis of the nerve-injured hind paw was performed with the &#34;Interactive Footprint Measurements&#34; module. A significant reduction of the parameters toe spread, intermediate toe spread and print length were observed in nerve-injured wt rats compared to wt na&#239;ve rats. In addition, the paw angle body axis and the paw angle movement vector significantly differ when comparing nerve-injured wt rats with wt na&#239;ve rats (Figure 2E).
Figure 3 presents data of motor coordination obtained through beam walking task assessment. Nerve-injured wt rats showed a significantly increased latency time to cross the beam compared to wt na&#239;ve rats five weeks post injury (Figure 3B). As an additional read out of the beam walking task, full slips and half slips of the nerve-injured hindlimb were counted and considered as an error for statistical analysis. The percentage of errors per step of the nerve-injured (right) hindlimb was significantly increased in nerve-injured wt rats compared to wt na&#239;ve rats.
Representative data of the ladder rung walking task (Figure 4) does not show significant alterations in the latency time to cross the walkway of the ladder rung apparatus (Figure 4C) or in the percentage of errors per step of the nerve-injured (right) hindlimb (Figure 4D). The analysis of the error percentage per step of the nerve-injured hindlimb considered only the score from 0 to 2 of the 7-category scale from Metz et al. The distribution of all score categories per step from the 7-category scale of the nerve-injured hindlimb and the non-nerve-injured (left) hindlimb is illustrated in Figure 4E.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62606/62606fig01.jpg" /> 
Figure 1: Assessment of locomotor activity during open field test. (A) Picture of the open field test setup. Selected picture subtracted from a recorded video during open field test showing a rat in the open field arena without (B) and with (C) tracking. (D) The velocity during a five minutes open field test recording was investigated in wt na&#239;ve rats and wt rats five weeks after nerve crush injury. Data are shown as mean &#177; SEM. Statistical analysis was performed using the unpaired t test of the normally distributed data. <a href="https://www.jove.com/files/ftp_upload/62606/62606fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62606/62606fig02.jpg" /> 
Figure 2: Gait analysis with the CatWalk XT system. (A) Picture of the CatWalk XT apparatus. (B) Examples of the print view showing the labeled paw prints in false color mode and examples of the timing view showing time-based gait diagram of wt na&#239;ve rats and wt rats five weeks after nerve crush injury. (C) Examples of the toe classification showing the toe spread (TS), intermediate toe spread (ITS) and print length (PL) as well as examples of the body axis view showing the body axis (white line) and the movement vector (red line) of wt na&#239;ve rats and wt rats five weeks after nerve crush injury. (D) Data of selected parameters from the &#34;standard&#34; classification comparing wt na&#239;ve rats and wt rats five weeks after nerve crush injury. (E) Data of selected parameters from the &#34;Interactive Footprint Measurements module&#34; comparing wt na&#239;ve rats and wt rats five weeks after nerve crush injury. Data are shown as mean &#177; SEM. Statistical analysis was performed using the unpaired t test of the normally distributed data, unpaired t test with Welch&#39;s correction of normally distributed data with unequal variance and Mann-Whitney U test of the non-normal distributed data. P-value &#60; 0.05 was defined as statistically significant labeled as *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001, ****p &#60; 0.0001. <a href="https://www.jove.com/files/ftp_upload/62606/62606fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62606/62606fig03.jpg" /> 
Figure 3: Gait analysis with the beam walking task. (A) Picture and schematic drawings of the beam walking task setup. The latency time to cross the beam (B) and the percentage foot slip errors per step of the nerve-injured hindlimb during the beam walking task (C) was analyzed in wt na&#239;ve rats and wt rats five weeks after nerve crush injury. Representative picture for the start time position (D) and the end time position (E) of the beam walking task. Representative image sequence of a full slip error (F) and a half slip error (G) of the beam walking task. Data are shown as mean &#177; SEM. Statistical analysis was performed using the Mann-Whitney U test of the non-normal distributed data. P-value &#60; 0.05 was defined as statistically significant labeled as *p &#60; 0.05, **p &#60; 0.01. <a href="https://www.jove.com/files/ftp_upload/62606/62606fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62606/62606fig04.jpg" /> 
Figure 4: Gait analysis using the ladder rung walking task. Picture (A) and schematic drawings (B) of the ladder rung walking task setup. The latency time of traversing the ladder rung apparatus (C) and the percentage foot slip errors per step of the nerve-injured hindlimb during the ladder rung walking task (D) was assessed in wt na&#239;ve rats and wt rats five weeks after nerve crush injury. (E) The percentage distribution of the score category per step according to the 7-category scale from Metz et al. for the left and right hindlimb of wt na&#239;ve rats and wt rats five weeks after nerve crush injury. Data are shown as mean &#177; SEM. Statistical analysis was performed using the unpaired t test of the normally distributed data and Mann-Whitney U test of the non-normal distributed data. <a href="https://www.jove.com/files/ftp_upload/62606/62606fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62606/62606fig05.jpg" /> 
Figure 5: Exemplary representation of each category according to the 7-category scale from Metz et al. during the ladder rung walking task. Representative&#160;image sequence from the right hindlimb of category 0 - total miss, category 1 - deep slip, category 2 - slight slip, category 3 - replacement, category 4 - correction, during the ladder rung walking task. Representative pictures for category 5 - partial placement and category 6 - correct placement. <a href="https://www.jove.com/files/ftp_upload/62606/62606fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Multifactorial Assessment of Motor Behavior in Rats after Unilateral Sciatic Nerve Crush Injury at JoVE.com

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

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 ...

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7.0K Views.  University Hospital of Würzburg. We provide a protocol for the assessment of motor behavior via a behavioral test battery in rats after sciatic nerve crush injury.

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

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 ...

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling of auditory axons within the VIIIth cranial nerve would enhance the study of central auditory circuit development. This approach is challenging because multiple sensory organs of the inner ear contribute to the VIIIth nerve 1. Moreover, markers that reliably distinguish auditory versus vestibular groups of axons within the avian VIIIth nerve have yet to be identified. Auditory and vestibular pathways cannot be distinguished functionally in early embryos, as sensory-evoked responses are not present before the circuits are formed. Centrally projecting VIIIth nerve axons have been traced in some studies, but auditory axon labeling was accompanied by labeling from other VIIIth nerve components 2,3. Here, we describe a method for anterograde tracing from the acoustic ganglion to selectively label auditory axons within the developing VIIIth nerve. First, after partial dissection of the anterior cephalic region of an 8-day chick embryo immersed in oxygenated artificial cerebrospinal fluid, the cochlear duct is identified by anatomical landmarks. Next, a fine pulled glass micropipette is positioned to inject a small amount of rhodamine dextran amine into the duct and adjacent deep region where the acoustic ganglion cells are located. Within thirty minutes following the injection, auditory axons are traced centrally into the hindbrain and can later be visualized following histologic preparation. This method provides a useful tool for developmental studies of peripheral to central auditory circuit formation.

Here we describe a microdissection technique followed by fluorescent dye injection into the acoustic ganglion of early chick embryos for selective tracing of auditory axon fibers in the nerve and hindbrain.

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

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 ...

Unilateral Pyramidotomy of the Corticospinal Tract in Rats for Assessment of Neuroplasticity-inducing Therapies

The corticospinal tract (CST) can be completely severed unilaterally in the medullary pyramids of the rodent brainstem. The CST is a motor tract that has great importance for distal muscle control in humans and, to a lesser extent, in rodents. A unilateral cut of one pyramid results in loss of CST innervation of the spinal cord mainly on the contralateral side of the spinal cord leading to transient motor disability in the forelimbs and sustained loss of dexterity. Ipsilateral projections of the corticospinal tract are minor. We have refined our surgical method to increase the chances of lesion completeness. We describe postsurgical care. Deficits on the Montoya staircase pellet reaching test and the horizontal ladder test shown here are detected up to 8 weeks postinjury. Deficits on the cylinder rearing test are only detected transiently. Therefore, the cylinder test may only be suitable for detection of short term recovery. We show how, electrophysiologically and anatomically, one may assess lesions and plastic changes. We also describe how to analyse fibers from the uninjured CST sprouting across the midline into the deprived areas. It is challenging to obtain &#62;90% complete lesions consistently due to the proximity to the basilar artery in the medulla oblongata and survival rates can be low. Alternative surgical approaches and behavioural testing are described in this protocol. The pyramidotomy model is a good tool for assessing neuroplasticity-inducing treatments, which increase sprouting of intact fibers after injury.

The corticospinal tract, one of the major sensorimotor tracts, can be lesioned unilaterally in the rodent brainstem in order to test neuroplasticity-inducing therapies for the central nervous system. This surgical procedure (&#8220;pyramidotomy&#8221;) and postoperative assessments are described in this protocol.

The corticospinal tract (CST) can be completely severed unilaterally in the medullary pyramids of the rodent brainstem. The CST is a motor tract that has ...

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 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 ...

Laser-Induced Brain Injury in the Motor Cortex of Rats

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) occlusion of the middle cerebral artery (MCA) using a catheter. This generally accepted technique, however, has some limitations, thereby limiting its extensive use. Stroke induction by this method is often characterized by high variability in the localization and size of the ischemic area, periodical occurrences of hemorrhage, and high death rates. Also, the successful completion of any of the transient or permanent procedures requires expertise and often lasts for about 30 minutes. In this protocol, a laser irradiation technique is presented that can serve as an alternative method for inducing and studying brain injury in rodent models.
When compared to rats in the control and MCAO groups, the brain injury by laser induction showed reduced variability in body temperature, infarct volume, brain edema, intracranial hemorrhage, and mortality. Furthermore, the use of a laser-induced injury caused damage to the brain tissues only in the motor cortex unlike in the MCAO experiments where destruction of both the motor cortex and striatal tissues is observed.
Findings from this investigation suggest that laser irradiation could serve as an alternative and effective technique for inducing brain injury in the motor cortex. The method also shortens the time for completing the procedure and does not require expert handlers.

The protocol presented here shows a technique to create a rodent model of brain injury. The method described here uses laser irradiation and targets motor cortex.

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) ...

Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and motor phenotypes involved in different patterns of neurotrophic factor gene expression and other biological processes, affecting nerve regeneration. The present study has established a protocol to obtain highly purified rat sensory and motor SCs and fibroblasts more rapidly. The ventral root (motor nerve) and the dorsal root (sensory nerve) of neonatal rats (7-days-old) were dissociated and the cells were cultured for 4-5 days, followed by isolation of sensory and motor fibroblasts and SCs by combining differential digestion and differential adherence methods sequentially. The results of immunocytochemistry and flow cytometry analyses showed that the purity of the sensory and motor SCs and fibroblasts were &#62;90%. This protocol can be used to obtain a large number of sensory and motor fibroblasts/SCs more rapidly, contributing to the exploration of sensory and motor nerve regeneration.

Here, we present a method to purify fibroblasts and Schwann cells from sensory and motor nerves in vitro.

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and ...

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. ...