Paw-Print Analysis of Contrast-Enhanced Recordings (PrAnCER): A Low-Cost, Open-Access Automated Gait Analysis System for Assessing Motor Deficits

We are presenting a new gait analysis system that is a user friendly, affordable, and accurate alternative to commercial gait systems. The use of a semitransparent floor in correct lighting creates high contrast paw prints while obscuring the animal's body. Our system can be used to assess motor function in a wide range of injury and disease models, such as spinal cord or nerve damage, neurodegenerative diseases, and neuromuscular disorders.

Both our gait apparatus and automated program PrAnCER can be easily adapted to fit different setups or animal models. To set up a gait analysis walkway, place a clear plexiglass enclosed walkway on a clear plexiglass floor and cover the floor with a piece of 16-pound drafting vellum cut to the same width as the walkway. Then, place a camera with at least 30 frames per second frame rate directly below the walkway to capture the middle of the track and secure a strip of 12-volt light emitting diode lights with 18 light emitting diodes per foot approximately two inches away from and one inch above the floor of the walkway to eliminate the track.

To habituate the animals to the walkway, place the rat home cage at the surface level at the end of the gait walkway to serve as a goal box and allow the rat to walk from the hand down the length of the walkway to the home cage. Rats will often stop at the end of the walkway to look around before jumping down into the home cage. If a rat takes longer than one minute to exit the walkway, encourage the animal to enter the home cage with a gentle push.

If the rat turns around, use a small piece of plexiglass to block the start end of the walkway. When the rats are comfortable crossing the walkway at a steady pace without freezing, adjust the settings on the webcam software to achieve the clearest picture of the paw prints and record each run separately, labeling the runs appropriately for use with the automated analysis program. After confirming that there are no spots or debris on the vellum, begin recording a few seconds before the rat enters the walkway and stop once the rat exits the walkway and enters the home cage.

It is important to make sure that the recorded videos capture at least four consecutive steps that occur without pause or acceleration to accurately reflect the animal's motor function. Wipe down the walkway with ethanol after each rat, replacing the vellum as needed. For analysis of the gait recordings, place all of the videos to be analyzed in a single folder and run the paw print analysis of contrast-enhanced recordings, or PrAnCER, Python script.

In the pop-up menu, click choose a folder to select the folder of videos and select the appropriate custom options for the analysis as desired. Click continue when finished. To define a region of interest on the image of the walkway, left click to define a top edge and right click to define a bottom edge.

If the box that appears is correct, press N to continue. The program will run automatically. After PrAnCER is complete, click enter in the terminal to terminate the program.

Then, manually review the images output by PrAnCER by running the Python script, Gait Editor Graphic User Interface and selecting the appropriate MP4 file for each video. Correct any misidentified or merged prints as necessary. To extract spatial and temporal gait parameters, run the Python script Parameter Analyzer, and select the number of hind prints to analyze and the folder of videos to analyze.

Then, click continue to output a csv file for each video containing a number of common gait parameters. While not identical to manual scoring, PrAnCER performs with high accuracy and generates reliable measures of gait. Results from a group of control videos are shown here for comparison.

PrAnCER analysis of stride length was not significantly different from manual scoring, but there was a significant difference observed in the base of support measurement. This is likely due to variances in centering selection rather than detection errors. In animals treated with high-dose haloperidol, a significant increase in stride length and maximum contact area is observed.

Animals given low-dose haloperidol also demonstrate a significant increase in stance duration and maximum contact area. Additionally, there is a significant difference between the high and low-dose haloperidol conditions in the base of support, maximum contact area, and interlimb distance gait parameters. While PrAnCER already analyzes many common spatiotemporal gait parameters, the code can be modified to assess any additional gait characteristics the user desires.

We hope that our system will make gait analysis more accessible to researchers who would like a simple and affordable way to measure motor function.