Published: July 31st, 2021
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 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.
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'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 naïve 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.
All animal experiments were approved by the local authorities at the Regierung von Unterfranken (Würzburg, Germany) and performed according to applicable international, national, and/or institutional guidelines for care and use of animals.
1. Sciatic nerve crush injury
NOTE: Maintain a sterile environment during the whole surgical procedure. Set up the surgery table with the necessary equipment.
2. Open field test (OFT)
NOTE: Locomotor activity as well as behavioral activity can be analyzed by the OFT.
3. CatWalk XT gait analysis
NOTE: A gait analysis via the CatWalk XT system can help to assess many different parameters concerning the footprints, stance and gait of animal models. A glass walkway is illuminated with green light and the light scattered by the footprints of the animals is captured with a high-speed video camera, which is located underneath the walkway. The signals can be analyzed with the CatWalk XT software.
4. Beam walking task
NOTE: Gait deficits can be determined by the beam walking task. The focus of the beam walking task in this specific research topic will be the analysis of motor coordination, defined as the ability to coordinate muscle activation from multiple body parts, and not the assessment of motor balance, defined as the ability for postural control during body movements.
5. Ladder rung walking task
NOTE: The ladder rung walking task can assess motor function, placement of both frontlimbs and hindlimbs, and interlimb coordination.
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 naïve 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 "Interactive Footprint Measurements" 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 naïve rats. In addition, the paw angle body axis and the paw angle movement vector significantly differ when comparing nerve-injured wt rats with wt naïve 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 naïve 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 naïve 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.
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 naïve rats and wt rats five weeks after nerve crush injury. Data are shown as mean ± SEM. Statistical analysis was performed using the unpaired t test of the normally distributed data. Please click here to view a larger version of this figure.
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 naïve 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 naïve rats and wt rats five weeks after nerve crush injury. (D) Data of selected parameters from the "standard" classification comparing wt naïve rats and wt rats five weeks after nerve crush injury. (E) Data of selected parameters from the "Interactive Footprint Measurements module" comparing wt naïve rats and wt rats five weeks after nerve crush injury. Data are shown as mean ± SEM. Statistical analysis was performed using the unpaired t test of the normally distributed data, unpaired t test with Welch's correction of normally distributed data with unequal variance and Mann-Whitney U test of the non-normal distributed data. P-value < 0.05 was defined as statistically significant labeled as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Please click here to view a larger version of this figure.
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 naïve 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 ± SEM. Statistical analysis was performed using the Mann-Whitney U test of the non-normal distributed data. P-value < 0.05 was defined as statistically significant labeled as *p < 0.05, **p < 0.01. Please click here to view a larger version of this figure.
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 naïve 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 naïve rats and wt rats five weeks after nerve crush injury. Data are shown as mean ± 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. Please click here to view a larger version of this figure.
Figure 5: Exemplary representation of each category according to the 7-category scale from Metz et al. during the ladder rung walking task. Representative 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. Please click here to view a larger version of this figure.
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 naïve 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 & 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 naïve wt rats. Furthermore, the latency time of traversing the ladder rung apparatus did not differ between nerve-injured wt rats and wt naïve 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.
The authors have nothing to disclose.
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 Würzburg (N-362 to C.W.I.; Z2-CSP3 to L.R.). In addition, this project has received funding from the European Union's Horizon 2020 research and innovation programme under the EJP RD COFUND-EJP N° 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 Deutsche Stiftung Neurologie and ParkinsonFonds. L.R. is additionally supported by the Dystonia Medical Reseach Foundation.
The authors thank Keali Röhm, Veronika Senger, Heike Menzel and Louisa Frieß for their technical assistance as well as Helga Brünner for the animal care.
|Acetic acid, ≥99.8%
|Appose ULC skin stapler 35W
|Bepanthen eye cream
|Bayer Vital GmbH
|Box for OFT
|setup and software
|Chamber for isofluran
|Disposable scalpel No. 11
|Dräger Vapor 19.3 isoflurane system
|Dr. Wilfried Müller GmbH
|Dumont #2 - laminectomy forceps
|Fine Science Tools
|Dumont #5 forceps
|Fine Science Tools
|Dustless precision pellets 45 mg
|setup and software
|Forceps 160 mm
|Gas anesthesia mask, rat
|Dr. Wilfried Müller GmbH
|Goal box for ladder rung walking task apparatus
|Hair clipper Magnum 5000
|Hardened fine scissors
|Fine Science Tools
|Isofluran CP 1ml/ml, 250 ml
|Ladder rung walking task apparatus
|Rimadyl 50 mg/ml, injectable
|Carprofen, prescription needed
|Rubber band retractors
|Spacer for beam
|Spacer for ladder rung walking task apparatus
|Suture Silkam 4/0 DS 19
|Ultra fine hemostats (non-serrated clamp)
|Fine Science Tools
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