Low-Cost Gait Analysis for Behavioral Phenotyping of Mouse Models of Neuromuscular Disease

Footprint analysis is a low cost alternative to digitize gait analysis programs for researchers quantifying movement abnormalities in mice. Because of its speed, simplicity and longitudinal potential, this method is ideal for the behavioral phenotyping of mouse models including models of neurodegenerative disease, neuromuscular disease or stroke. Demonstrating the procedure will be Virginia Wertman, a research technician from my laboratory.

Before beginning the experiment, acclimate mice that have been fully awake and alert for at least five minutes to the assay room for 30 minutes. Position the tunnel over a strip of paper that is slightly wider and longer than the length and width of the tunnel. Mark the paper with the mouse ID and testing date.

Add sunflower seeds to the goal chamber for motivation as necessary and firmly scruff the first mouse to be tested. Using approximately 0.5 centimeter diameter tapered brush tips, paint the entire underside of all of the toes and the center of each forepaw with one nontoxic washable water-based paint color. Coat the hind paws with a contrasting nontoxic washable water-based paint color in a similar fashion.

Use a clean damp cloth to remove any paint that the mouse gets on other parts of its body to prevent smudges that may interfere with the data collection. Then place the mouse at the start of the tunnel. Allow the mouse to walk all the way into the goal chamber before retrieving the animal to gently clean its feet with a water dampened cloth.

Then return the mouse to its home cage and wipe down the testing area and tunnel with a disinfectant before testing the next animal. For accurate scoring, allow the footprints to dry completely before selecting steps that are consistently spaced with clear non-smudged footprints. To generate sufficient scoring data, there must be at least two consecutive steps from each foot.

Do not include the first and last footprints on the paper as they are unlikely to represent a normal gait because the mouse was changing its walking speed. To define the stride length as the distance between two sequential footprints created by the same foot, use a pencil to draw a two to four millimeter circle around the forefoot region of both forelimb footprints in a single stride and use a ruler to draw a line between the circles. Record the distance between two prints from the middle of each circle as right fore one or left fore one as appropriate and repeat the measurement for each of the steps that can be scored.

Then average all of the individual recorded stride distances for each limb averaging individual scores within a cohort together as experimentally appropriate. To define the stride width as the distance between the left and right forelimbs or hindlimbs, draw and measure a line from the circled forefoot region of one hindlimb that intersects perpendicularly with the line for the stride length on the contralateral hindlimb. Repeat this measurement for all of the hindlimb prints that can be scored and average the measurements.

To define the toe spread as the distance between the first and last toes on a single fore or hindlimb footprint, use calipers to measure the distance between the tip of the first toe print and the tip of the last toe print. Then repeat the measurement for all of the hindlimb prints that can be scored and average the measurements. With a sufficient number of animals, this procedure is capable of detecting gait differences between mouse genotypes within the same strain over time.

For example, here representative traces of footprint images collected using a mouse model of x-linked spinal and bulbar muscular atrophy, a neurodegenerative disorder affecting lower motor neurons and skeletal muscle are shown. Gait analysis of pre-symptomatic and post-symptomatic transgenic and litter mate control male mice reveals that prior to disease onset, the transgenic mice display a similar stride length, stride width and toe spread compared to their litter mate non-transgenic controls. After disease onset however, the transgenic animals exhibit significantly shorter stride lengths.

Similar longitudinal analysis revealed no differences in stride width at either age tested. Post-symptomatic transgenic mice also have a significantly narrower hind toe spread than age matched litter mate controls. A proper application of the paint to the paws will yield the best results.

Remember that only steps that are consistently spaced with clear non-smudged footprints should be scored. This technique is sensitive enough to detect minor changes in stride. And due to its non-invasive approach, it allows testing of groups across their lifespan phenotypic presentation and therapeutic interventions.