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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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.

<table><tbody><tr><td>Acetic acid, &amp;ge;99.8%</td><td>Sigma-Aldrich</td><td>33209-1L</td><td></td></tr><tr><td>Appose ULC skin stapler 35W</td><td>Covidien</td><td>8886803712</td><td></td></tr><tr><td>Beam</td><td>self made</td><td></td><td></td></tr><tr><td>Bepanthen eye cream</td><td>Bayer Vital GmbH</td><td>81552983</td><td></td></tr><tr><td>Box for OFT</td><td>self made</td><td></td><td></td></tr><tr><td>Camcorder GC-PX100</td><td>JVC</td><td></td><td></td></tr><tr><td>Catwalk XT</td><td>Noldus</td><td></td><td>&amp;nbsp;setup and software</td></tr><tr><td>Chamber for isofluran</td><td>GT-Labortechnik</td><td></td><td>custom made</td></tr><tr><td>Disposable scalpel No. 11</td><td>Feather</td><td>20.001.30.011</td><td></td></tr><tr><td>Dr&amp;auml;ger Vapor 19.3 isoflurane system</td><td>Dr. Wilfried M&amp;uuml;ller GmbH</td><td></td><td></td></tr><tr><td>Dumont #2 - laminectomy forceps</td><td>Fine Science Tools</td><td>11223-20</td><td></td></tr><tr><td>Dumont #5 forceps</td><td>Fine Science Tools</td><td>11251-30</td><td>super-fine</td></tr><tr><td>Dustless precision pellets 45 mg</td><td>Bio-Serv</td><td>F0021</td><td></td></tr><tr><td>EthoVision XT</td><td>Noldus</td><td></td><td>&amp;nbsp;setup and software</td></tr><tr><td>Forceps 160 mm</td><td>Hartenstein</td><td>PZ09</td><td></td></tr><tr><td>Gas anesthesia mask, rat</td><td>Dr. Wilfried M&amp;uuml;ller GmbH</td><td></td><td></td></tr><tr><td>Goal box for ladder rung walking task apparatus</td><td>self made</td><td></td><td></td></tr><tr><td>Hair clipper Magnum 5000</td><td>Wahl GmbH</td><td></td><td></td></tr><tr><td>Hardened fine scissors</td><td>Fine Science Tools</td><td>14090-11</td><td></td></tr><tr><td>Heating table</td><td>MEDAX</td><td>13801</td><td></td></tr><tr><td>Isofluran CP 1ml/ml, 250 ml</td><td>cp-pharma</td><td>1214</td><td>prescription needed</td></tr><tr><td>Kinovea</td><td>www.kinovea.org</td><td></td><td></td></tr><tr><td>Ladder rung walking task apparatus</td><td>self made</td><td></td><td></td></tr><tr><td>Needleholder</td><td>KLS Martin</td><td>20-526-14-07</td><td></td></tr><tr><td>Octeniderm</td><td>Sch&amp;uuml;lke</td><td>118211</td><td></td></tr><tr><td>Rimadyl 50 mg/ml, injectable</td><td>Zoetis</td><td></td><td>Carprofen, prescription needed</td></tr><tr><td>Rubber band retractors</td><td>self made</td><td></td><td></td></tr><tr><td>Spacer for beam</td><td>self made</td><td></td><td></td></tr><tr><td>Spacer for ladder rung walking task apparatus</td><td>self made</td><td></td><td></td></tr><tr><td>Suture Silkam 4/0 DS 19</td><td>B. Braun</td><td>C0762202</td><td></td></tr><tr><td>Ultra fine hemostats (non-serrated clamp)</td><td>Fine Science Tools</td><td>13020-12</td><td></td></tr></tbody></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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Research

JoVE Journal

Neuroscience

7.2K Views.  University Hospital of Würzburg. Het algemene doel van dit protocol is om de procedures te beschrijven die betrokken zijn bij het uitvoeren van onherproduceerbare ischiaszenuwverplettering en om een protocol te bieden voor de beoordeling van motorisch gedrag na perifeer trauma. De selectie van een krachtige en betrouwbare gedragstestbatterij is essentieel om een breed scala aan gedragsaspecten te dekken en hangt af van het gebruikte diermodel en de vraag naar wetenschappelijk onderzoek. Dit protocol beschrijft in detail hoe ...

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

Assessing Forelimb Function after Unilateral Cervical SCI using Novel Tasks: Limb Step-alternation, Postural Instability and Pasta Handling

Cervical spinal cord injury (cSCI) can cause devastating neurological deficits, including impairment or loss of upper limb and hand function. A majority of the spinal cord injuries in humans occur at the cervical levels. Therefore, developing cervical injury models and developing relevant and sensitive behavioral tests is of great importance. Here we describe the use of a newly developed forelimb step-alternation test after cervical spinal cord injury in rats. In addition, we describe two behavioral tests that have not been used after spinal cord injury: a postural instability test (PIT), and a pasta-handling test. All three behavioral tests are highly sensitive to injury and are easy to use. Therefore, we feel that these behavioral tests can be instrumental in investigating therapeutic strategies after cSCI.

Three new behavioral tests (forelimb step-alternation, postural instability test, pasta handling test) for evaluating forelimb function after cervical spinal cord injury in rodents are described.

Cervical spinal cord injury (cSCI) can cause devastating neurological deficits, including impairment or loss of upper limb and hand function. A majority ...

Behavior

A Novel Method for Assessing Proximal and Distal Forelimb Function in the Rat: the Irvine, Beatties and Bresnahan (IBB) Forelimb Scale

Several experimental models of cervical spinal cord injury (SCI) have been developed recently to assess the consequences of damage to this level of the spinal cord (Pearse et al., 2005, Gensel et al., 2006, Anderson et al., 2009), as the majority of human SCI occur here (Young, 2010; www.sci-info-pages.com). Behavioral deficits include loss of forelimb function due to damage to the white matter affecting both descending motor and ascending sensory systems, and to the gray matter containing the segmental circuitry for processing sensory input and motor output for the forelimb. Additionally, a key priority for human patients with cervical SCI is restoration of hand/arm function (Anderson, 2004). Thus, outcome measures that assess both proximal and distal forelimb function are needed. Although there are several behavioral assays that are sensitive to different aspects of forelimb recovery in experimental models of cervical SCI (Girgis et al., 2007, Gensel et al., 2006, Ballerman et al., 2001, Metz and Whishaw, 2000, Bertelli and Mira, 1993, Montoya et al., 1991, Whishaw and Pellis, 1990), few techniques provide detailed information on the recovery of fine motor control and digit movement.
The current measurement technique, the Irvine, Beatties and Bresnahan forelimb scale (IBB), can detect recovery of both proximal and distal forelimb function including digit movements during a naturally occurring behavior that does not require extensive training or deprivation to enhance motivation. The IBB was generated by observing recovery after a unilateral C6 SCI, and involves video recording of animals eating two differently shaped cereals (spherical and doughnut) of a consistent size. These videos were then used to assess features of forelimb use, such as joint position, object support, digit movement and grasping technique.
The IBB, like other forelimb behavioral tasks, shows a consistent pattern of recovery that is sensitive to injury severity. Furthermore, the IBB scale could be used to assess recovery following other types of injury that impact normal forelimb function.

A Novel Method for Assessing Proximal and Distal Forelimb Function in the Rat: the Irvine, Beatties and Bresnahan IBB Forelimb Scale

Here we will describe a rodent behavioral assay that can detect recovery of both proximal and distal forelimb function including digit movements during a naturally occurring behavior that does not require extensive training or deprivation to enhance motivation.

Several experimental models of cervical spinal cord injury (SCI) have been developed recently to assess the consequences of damage to this level of the ...

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 ...

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 ...

Automated Gait Analysis in Mice with Chronic Constriction Injury

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some disadvantages such as subjective data and the requirement of a skilled, experienced experimenter. To date, a variety of modifications have improved the von Frey method, but it still has a few limitations. Recent reports have suggested that gait analysis produces more accurate and objective data from the neuropathic animals. This protocol demonstrates how to perform the automated gait analysis to determine the degree of neuropathic pain in mice. After several days of acclimation, the mice were allowed to walk freely on the glass floor to illuminate footprints. Then, quantification of the footprints and gait were performed through video clips with automatic analysis of various walking parameters, such as area of paw print, swing time, angle of paw, etc.
The main purpose of this study is to describe the methodology of automated gait analysis and briefly compare it with data from the classical sensory test using von Frey filament.

The precise assessment of pain response in a neuropathic animal model is critical to investigate the pathophysiology of pain diseases and develop new analgesics. We present a sensitive and objective method to determine the sensory function of the rodent hind paw by an automated gait analysis system.

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...

Spinal Cord Lateral Hemisection and Asymmetric Behavioral Assessments in Adult Rats

Incomplete spinal cord injury (SCI) often leads to impairments of sensorimotor functions and is clinically the most frequent type of SCI. Human Brown-S&#233;quard syndrome&#160;is a common type of incomplete SCI caused by a lesion to one half of the spinal cord which results in paralysis and loss of&#160;proprioception&#160;on the same (or ipsilesional) side as the injury, and loss of pain and temperature sensation on the opposite (or contralesional) side. Adequate methodologies for producing a spinal cord lateral hemisection (HX) and assessing neurological impairments are essential to establish a reliable animal model of Brown-S&#233;quard syndrome. Although lateral hemisection model plays a pivotal role in basic and translational research, standardized protocols for creating such a hemisection and assessing unilateralized function are lacking. The goal of this study is to describe step-by-step procedures to produce a rat spinal lateral HX at the 9th thoracic (T9) vertebral level. We, then, describe a combined behavior scale for HX (CBS-HX) that provides a simple and sensitive assessment of asymmetric neurological performance for unilateral SCI. The CBS-HX, ranging from 0 to 18, is composed of 4 individual assessments which include unilateral hindlimb stepping (UHS), coupling, contact placing, and grid walking. For CBS-HX, the ipsilateral and contralateral hindlimbs are assessed separately. We found that, after a T9 HX, the ipsilateral hindlimb showed impaired behavior function whereas the contralateral hindlimb showed substantial recovery. The CBS-HX effectively discriminated behavioral functions between ipsilateral and contralateral hindlimbs and detected temporal progression of recovery of the ipsilateral hindlimb. The CBS-HX components can be analyzed separately or in combination with other measures when needed. Although we only provided visual descriptions of the surgical procedures and behavioral assessments of a thoracic HX, the principle may be applied to other incomplete SCIs and at other levels of the injury.

Here we describe surgical procedures to produce a reliable spinal cord lateral hemisection (HX) at the 9th thoracic level in adult rats and neurobehavioral assessments designed for detecting asymmetric deficits after such a unilateral injury.

Incomplete spinal cord injury (SCI) often leads to impairments of sensorimotor functions and is clinically the most frequent type of SCI. Human ...

Medicine

Thoracic Spinal Cord Hemisection Surgery and Open-Field Locomotor Assessment in the Rat

Spinal cord injury (SCI) causes disturbances in motor, sensory, and autonomic function below the level of the lesion. Experimental animal models are valuable tools to understand the neural mechanisms involved in locomotor recovery after SCI and to design therapies for clinical populations. There are several experimental SCI models including contusion, compression, and transection injuries that are used in a wide variety of species. A hemisection involves the unilateral transection of the spinal cord and disrupts all ascending and descending tracts on one side only. Spinal hemisection produces a highly selective and reproducible injury in comparison to contusion or compression techniques that is useful for investigating neural plasticity in spared and damaged pathways associated with functional recovery. We present a detailed step-by-step protocol for performing a thoracic hemisection at the T8 vertebral level in the rat that results in an initial paralysis of the hindlimb on the side of the lesion with graded spontaneous recovery of locomotor function over several weeks. We also provide a locomotor scoring protocol to assess functional recovery in the open-field. The locomotor assessment provides a linear recovery profile and can be performed both early and repeatedly after injury in order to accurately screen animals for appropriate time points in which to conduct more specialized behavioral testing. The hemisection technique presented can be readily adapted to other transection models and species, and the locomotor assessment can be used in a variety of SCI and other injury models to score locomotor function.

The rat thoracic spinal hemisection is a valuable and reproducible model of unilateral spinal cord injury to investigate the neural mechanisms of locomotor recovery and treatment efficacy. This article includes a detailed step-by-step guide to perform the hemisection procedure and to assess locomotor performance in an open-field arena.

Spinal cord injury (SCI) causes disturbances in motor, sensory, and autonomic function below the level of the lesion. Experimental animal models are ...

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 ...

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 ...

Maximum Isometric Tetanic Force Measurement of the Tibialis Anterior Muscle in the Rat

Traumatic nerve injuries result in substantial functional loss and segmental nerve defects often necessitate the use of autologous interposition nerve grafts. Due to their limited availability and associated donor side morbidity, many studies in the field of nerve regeneration focus on alternative techniques to bridge a segmental nerve gap. In order to investigate the outcomes of surgical or pharmacological experimental treatment options, the rat sciatic nerve model is often used as a bioassay. There are a variety of outcome measurements used in rat models to determine the extent of nerve regeneration. The maximum output force of the target muscle remains the most relevant outcome for clinical translation of experimental therapies. Isometric force measurement of tetanic muscle contraction has previously been described as a reproducible and valid technique for evaluating motor recovery after nerve injury or repair in both rat and rabbit models. In this video, we will provide a step-by-step instruction of this invaluable procedure for assessment of functional recovery of the tibialis anterior muscle in a rat sciatic nerve defect model using optimized parameters. We will describe the necessary pre-surgical preparations in addition to the surgical approach and dissection of the common peroneal nerve and tibialis anterior muscle tendon. The isometric tetanic force measurement technique will be detailed. Determining the optimal muscle length and stimulus pulse frequency is explained and measuring the maximum tetanic muscle contraction is demonstrated.

Evaluation of motor recovery remains the benchmark outcome measure in experimental peripheral nerve studies. The isometric tetanic force measurement of the tibialis anterior muscle in the rat is an invaluable tool to assess functional outcomes after reconstruction of sciatic nerve defects. The methods and nuances are detailed in this article.

Traumatic nerve injuries result in substantial functional loss and segmental nerve defects often necessitate the use of autologous interposition nerve ...

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

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