Automated gait analysis is a widely used method for evaluating functional recovery enrollment models of peripheral nerve injury, repair, and regeneration. The method can be used to study changes in gait after peripheral nerve injury, relating both to motor and sensory nerve function. Researchers new to the method might experience difficulties in acquiring high quality data.
Typical hurdles are adequate animal training and accurate hardware and software calibration. During training sessions and on the test day, turn off all of the light sources in the behavioral test room and face the computer screen for data acquisition away from the camera to prevent its light from interfering with the camera. Confirm that the device is installed in a stable position in a way that prevents any form of vibration, and bring the rats into the behavioral test room in their own home cage for at least 30 minutes before the test, On the first day of training, gently grasp an animal under its trunk and carefully place it in the walkway entrance.
Let the rat explore the opening of the corridor without any interference, and wait until the animal voluntarily crosses the walkway to reach its home cage without any external stimulus or motivation. On the second day of training, allow the animals to become accustomed to entering the walkway and returning to their home cage without hesitation. On the third day of training, confirm that the animals have learned to cross the walkway at a uniform speed without hesitation, sniffing, or other explorative movements.
On the fourth and fifth, repeat the training to reinforce the testing procedure. On the day of the experiment, use commercial glass cleaner and a squeegee to clean the top and bottom of the walkway taking care to remove all of the fluid from the ends of the walkway. Place both the lightest and heaviest rats onto the walkway and select a registered camera from the setup tab to allow the camera gain, red ceiling light, green walkway light, and green intensity threshold to be adjusted to ensure an optimal paw print detection for all of the animals in the analysis.
Next, click open acquisition and acquire a snapshot of the empty cleaned walkway to be used as a reference throughout the data acquisition procedure. The status will change from waiting for snapshot to ready for acquisition. Click start acquisition.
The status will change from ready for acquisition to waiting for run to start. When the camera is ready, place a rat onto the walkway and follow the animal's movement on the computer screen. Note, the status change from waiting for run to start to recording run.
At the end of the analysis, click classify in the experimental explorer tab of the trial to be classified, and play the acquired run at normal speed to determine whether the data conforms to the classification requirements. Click auto classify for automatic classification of the paw prints by the software. For correct calculation of the normal step sequence patterns, make sure that the classifying algorithm is not confused by non-visible paw prints, leading to flawed NSSP, and that only paw prints that are detectable while the contralateral paw is also visible for NSSP calculations are included.
For statistical analysis of the data, click view run statistics to obtain a comprehensive overview of the run statistics. Then select file and export, to export the run or trial statistics into a spreadsheet software. Following sciatic nerve injury, rats use the heel of the paw for weight support only and the limb is moved in a sweeping circumductory movement.
Therefore, locomotor changes assessed via automated gait analysis become apparent by a significantly reduced print area and a significantly increased swing time. Femoral nerve resection results in denervation of the quadriceps muscle of the thigh, impairing knee extension and inducing hyperflection of the ankle joint with consecutive lifting of the corresponding heel. Starting from post-operative week four, reenervation of the quadriceps by the regenerating femoral nerve leads to reversal of these changes.
As the quadriceps muscle of the thigh also plays a role in the swing phase of the respective paw, the swing time is greatly prolonged in rats with femoral nerve injury. Gait analysis reveals markedly altered paw prints after thoracic spinal cord contusion. Spinal cord contusion at the thoracic 11 level also results in a decrease of the print area ratio, an increment of the swing time ratio.
The coordination related regularity index also decreases at post-operative week two, while the base of support of the hind paws shows a marked increase. When using automated gate analysis to evaluate functional recovery in rodents, be sure to carefully train the animals and to be meticulous when making any software or hardware adjustments. By using automated gait analysis, injury and regeneration of the central and peripheral nervous system can be evaluated in various rodent models, including yet unstudied formal nerve models, such as the median nerve.
