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

The overall goal of this protocol is to describe the procedures involved in performing irreproducible sciatic nerve crush, and to provide a protocol for the assessment of motor behavior after peripheral trauma. The selection of a powerful and reliable behavioral test battery is essential to cover a wide range of behavioral aspects, and depends on the animal model used and scientific research question. 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 gait analysis, the beam walking task, and the ladder rung walking task.

Animal experiments were performed in accordance with applicable national and institutional guidelines for care and use of animals. Maintain a sterile environment during the whole surgical procedure. Set up the surgery table with the necessary equipment.

Deeply anesthetize the rat in a closed cabinet with isoflurane and shave an extensive area of the right hind limb. Next, position the rat into the anesthesia mask and continue deep anesthesia with isoflurane. Fix the torso and both hind limbs of the rat with tape.

Place both hind limbs in a symmetrical and outstretched position and turn each paw flat onto the surgery table. Disinfect the skin of the shaved area with an appropriate antiseptic. Search for the sciatic notch of the ileum.

Make a skin incision from the sciatic notch into the direction of the paw with a scalpel. If the hind limbs are fixed and the skin incision is performed correctly, a cavity in the fascial plane between the gluteus maximus muscle and biceps femoris muscle can be seen that resembles a white line. Insert closed super-fine hemostatic forceps into the cavity and spread the forceps.

The fascial plane should open up without injuring any muscle tissue. Place the rubber band retractors underneath the muscles to keep the skin incision open. Gently remove any surrounding tissue and blood vessels from the sciatic nerve until the nerve is completely exposed.

It is important to not stretch or pull the nerve during the whole procedure. Crush the sciatic nerve with a non-serrated clamp with constant and rapid usable pressure. For this, open the clamp, place the nerve onto the bottom jaw of the clamp, and close the clamp by locking it into the first position for 3 x 10 seconds.

After the crush injury, reopen the clamp carefully. the crush site of the sciatic nerve appears translucent. Remove the rubber band retractors.

Close the fascial plane incision with a resorbable suture. Close the skin incision with body skin staples. The locomotor activity, as well as the behavioral activity of rats can be analyzed by an open field test.

The setup of the open field, which should be located in a dark and quiet environment, consists of the automated video tracking system, EthoVision XT, and an arena with a scratch-resistant, cleanable black surface. Place the arena and the camera into the correct position and set up the EthoVision XT software. Place the rat gently in the middle of the testing arena.

During the recording, the experimenter should stay away from the open field set up, in order to avoid distracting the rat. For the data analysis of the open field test, go to the analysis section in the left sidebar and choose Track Visualization"under the Results"tab. Next, export the needed parameters to XL.Within the software, choose a number of variables from different categories for data analysis.

A gait analysis by 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.

The experiment has to be performed under dark conditions. Start by setting up the CatWalk XT system. Choose the Experimental settings.

Place the camera in position and adjust the focus. Set up the detection settings. Set up the corridor walls of the CatWalk system and make sure the walls remain parallel to the walkway.

Define the length of the walkway and calibrate the walkway. Next, snap a background image. A clean and empty walkway is needed for the background image.

The rat should cross the walkway without any interruption. According to the defined run criteria, three compliant runs per rat are needed for data analysis. Train animals for at least eight days before you start with the actual experiment.

The CatWalk should be performed at least once a week, with 6 to 10 runs to ensure a continuous task performance. For data analysis, only evaluate the compliant runs. Classify paw prints automatically with the CatWalk XT software.

Review the paw print labels manually. Correct wrong paw print labels, add labels of non-detected paw prints, and delete any noise and wrong labels. The numerical results displayed in an XL sheet, showing a number of basic parameters.

For more detailed information about each footprint, classify the toes of the hind paws. This analysis requires the interactive footprint measurements module. Set the markers for the footprint analysis manually.

Analyze every hind paw print in all three compliant runs. Draw a line from the center of the first toe to the center of the fifth toe to measure toe spread. Draw a line from the center of the second toe to the center of the fourth toe to measure intermediate toe spread.

Draw a line from the center of the third toe to the heel of the hind paw to measure manual print length. Review the numerical results of the interactive footprint measurements displayed in a separate sheet. 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 and not the assessment of motor balance. For the beam walking task, a beam, spacer, table, uniform background, and a camcorder are needed. Use a wooden beam of 90 centimeters in length, 1.7 centimeters in width, and 2 centimeters in height.

Train the animals for seven days. Place the rat on the start platform of the beam. The rat should cross the beam three times without any interruption.

Even if the rat reaches three compliant runs within the first three runs, a minimum of 6 to 10 runs should be carried out for continuous task performance. Select the video sequences of three compliant runs from the recording. Define the start time point and the end time point of the selected three compliant runs.

Here, the start time point is labeled by a black line on the beam, and the placement of the first hind limb behind the black line is defined as the start time point of the run. The placement of the first hind limb on the platform at the end of the beam defines the end time point. Next, calculate the time needed for the rat to cross beam.

Score the number of steps and errors from three compliant runs for both hind limbs separately, by using the zoom and slow motion function of the software. Errors include total foot slips and half foot slips. A total foot slip is defined as a foot placement that is followed by deep slip, causing a loss of contact of the affected paw with the beam.

A half slip is defined as the paw sliding off the sidewall of the beam without losing complete contact. Calculate the percentage of foot slips in relation to the number of steps to cross the beam. The ladder rung walking task can assess motor function, placement of both front limbs and hind limbs, and inter-limb coordination.

A ladder rung apparatus, spacer, table, uniform background, and camcorder are needed for this behavioral test. The horizontal letter rung apparatus consists of metal rungs and clear polycarbonate sidewalls. Train the animals for eight days.

Place the rat on the start zone of the ladder rung operators. The rat has to cross the 100 centimeter walkway of the ladder rung apparatus three times without an interruption, in order for it to qualify as a compliant run. Select the video sequences of three compliant runs from the recording.

Define the start time point and the end time point of the selected three compliant runs. The placement of the hind limb behind the first black line on the sidewall of the apparatus defines the start time point of the run. The placement of the first front limb behind the second black line on the sidewall of the apparatus defines the end time point of the run.

Next, calculate the duration of the run across the walkway. Score the three compliant runs with the 7 category scale from Mets and colleagues by using the slow motion or the frame-by-frame function of the software. Determine the number of steps and the number of errors in accordance with the categories of the scale for each limb separately.

The scale distinguishes between the following categories. Total miss. Deep slip.

Slight slip.Replacement.Correction. Partial placement. And correct placement.

Next, calculate the errors per step. The representative results of the five minute open field test show that the nerve crush injury, five weeks post surgery, has no effect on the locomotor activity. Gait analysis with the CatWalk XT system generates many different parameters.

Significant alterations could be detected for the run average speed, the stride length, and the print area of the nerve-injured right hind paw. Nerve-injured wild type rats showed a significantly increased latency time to cross the beam, compared to wild type naive rats five weeks post-injury. As an additional readout of the beam walking task, full slips and half slips of the nerve-injured hind limb were counted and considered as an error for statistical analysis.

The percentage of errors per step of the nerve-injured right hind limb was significantly increased in nerve-injured wild type rats, compared to wild type naive rats. Representative data of the ladder rung walking task does not show significant alterations in the latency time to cross the walkway of the ladder rung apparatus, or in the percentage of errors per step of the nerve-injured right hind limb. The analysis of the error percentage per step of the nerve-injured hind limb considered only the score from 0 to 2 of the 7 category scale from Mets and colleagues.

After watching this video, you should have a good understanding of how to perform a sciatic nerve crush injury, and how to implement a multi-factorial assessment of motor behavior in rats after unilateral sciatic nerve crush. To achieve consistent, reproducible and comparable results after a sciatic nerve crush injury, a standardized method for inducing the nerve crush is essential, as well as a standardized phenotypically characterization. Proper training for specific behavior test provides the advantages of saving time during the experiment assessment and obtaining better results.