This video reports a novel low-cost method for assessing gait asymmetries in rats, using a precise locomotor limb-positioning task. The phase durations over a range of walking speeds of the quadrupedal gait are used to calculate an index to score the animals'abilities. Our rationale was to develop a behavioral task that quantitatively assesses the changes in precise control for symmetrical locomotor behaviors.
This task offers an opportunity to discern the contributions of a descending pathway to the locomotor phase regulation. For example, simultaneous recordings or stimulation in the motor cortex would report as contribution to the modulation of muscle phases in this task. Build the apparatus as an open-topped plastic box with aluminum supports at each corner.
Include a frame formed of grooved aluminium railing to provide flexible opportunities for peg placement. The placement of the pegs defines the imposed stride length on the animal. In each of the four corners, put a 20-centimeter square platform.
The space between the platforms should accommodate the distance traversed by at least one full step cycle. To create a gait condition, attach aluminum pegs to the rails. These function as footholds for the rat's stride.
The pegs should be pre-bent to two point five centimeters from the tip, and should be one centimeter in diameter. Secure the pegs with sliding brackets which can be adjusted with a screwdriver, and, thus, precisely position the footholds. After positioning the footholds, make sure the distances are correct, and that they are on the same horizontal level.
Standardize the peg positions for three stepping tasks. First, make a symmetric motor task with 15-centimeter stride lengths by setting the left and right interstride lengths to seven point five centimeters. Second, create a symmetric condition with an interstride challenge by changing those lengths to six centimeters, and, thus, changing the stride to 12 centimeters.
Thirdly, create an asymmetric task with a stride length of 15 centimeters. Adjust the position between the pegs to make the interstride length uneven by 20%The task has two configurations with either the left or right interstride being shorter. Thus, each walkway side can represent a separate gait condition.
Now, to the side of the apparatus, position an HD camera that can record 60 frames per second. Adjust the field of view to cover about seven steps, and not include the steps closest to the platforms. First, prepare the training environment.
Set the interstride lengths to just one centimeter, and place a reward on each platform. Now, let the rat freely explore the new environment for about five minutes. Next, guide the animal across the peg arrangement by presenting it with the reward.
When the rat reaches a platform, in addition to the food reward, provide a verbal reward, and pet it on the back. Come right here, right here. You're so close.
After five training runs, increase the space between the pegs by one or two centimeters for the next five runs. The goal is to gradually extend the peg distance until the symmetric 15-centimeter stride is accomplished. If the rat is having trouble learning the task, it will be evident from inconsistent stepping with frequent stops and crouching posture.
Whenever an increased peg spacing induces anxiety or discomfort, just readjust the pegs to their previous positions and repeat that level of training. It is possible to train and then test the same rat in a single day if the rat is motivated. When the rat has learned the task, its walking will be consistent, without stops or missteps.
Its head-bobbing will be minimal, its back will be arched, and its tail will be raised. Rapid stepping with little speed variability, however, is usually a sign that the rat is using a gallop rather than a walk. This behavior should be preventable by training the rat to move at lower speeds.
From the camera's view, it is essential that each limb be visible at the onset and offset of the stance phase. When these criteria are all met, testing may commence. For the tests, use the four standardized peg configurations with a randomized session design, including breaks, to avoid adaptation to the task.
Details are provided in the text protocol. Save the videos, and use video editing software to cull the walking bouts for analysis. The 60-frame per second rate is essential to the task scoring.
For each walking bout, score the onset and offset of the kinematic phases. This can be done in a variety of software by frame-by-frame basis. In this case, custom software written in MATLAB is employed.
Stance onset is scored when there is a loss of motion blur. Stance offset is scored when there is first evidence of motion blur. Once the swing phases are all counted, plot them as a function of the corresponding step cycle using a MATLAB script.
Data was collected from eight subjects and analyzed as described. Seen here is the asymmetry analysis for one subject. The combined analysis of the eight subjects shows that, generally, the modulation of the forelimb stance phase was lessened for the side to which the locomotion condition was favored, and vice-versa.
ANOVA with a conservative Bonferroni correction showed a significant difference between the two asymmetric conditions in the asymmetry indices, including the anterior horizontal symmetry index. The right vertical asymmetry showed a trend towards difference between the two conditions. Similarly, a significant difference was found in the diagonality index between two asymmetric conditions.
No differences were found in this index between other tasks, based on ANOVA with a post hoc t test, and no alpha correction. Animal models of focal stroke and spinal cord amolesion cause movement deficits akin to those observed clinically, and lesioning of the cortical spinal track in this way impedes this precise stepping behavior. This may result in a delay in the onset of consecutive locomotor phases, which is consistent with an asymmetry in both the step-length ratio and the single limb support time observed in post-stroke patients.
The animals which acquired the task are now prime for locomotor assessment in models of cortical injuries such as middle cerebral artery occlusion and traumatic brain injury.
