Name: a novel single animal motor function tracking system using simple, readily available software
Uploaded: 2018-08-31T13:30:00
Description: Watch this Scientific Journal Video about A Novel Single Animal Motor Function Tracking System Using Simple, Readily Available Software at JoVE.com

This study was supported by the Merit Review Award # B1495-R (to Jeffrey R. Capadona) and the Presidential Early Career Award for Scientist and Engineers (PECASE) (to Jeffrey R. Capadona) from the United States Department of Veterans Affairs Rehabilitation Research and Development Service. Additionally, this work was supported in part by the Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Medical Research Program under Award No. W81XWH-15-1-0608. The authors acknowledge SOURCE for its summer research funding support. The contents do not represent the views of the United States Department of Veterans Affairs or the United States Government. The authors would like to thank Hiroyuki Arakawa in the CWRU Rodent Behavior Core for his guidance in designing and testing rodent behavioral protocols. The authors would also like to thank James Drake and Kevin Talbot from the CWRU Department of Mechanical and Aerospace Engineering for their help in designing and manufacturing the rodent ladder test.

All procedures and animal care practices were approved by and performed in accordance with the Louis Stokes Cleveland Department of Veterans Affairs Medical Center Institutional Animal Care and Use Committee. The behavioral testing protocol closely follows previously published work12,13.
1. Behavioral Testing: Filming the Tasks
NOTE: Here, the animals were tested for 8 weeks to detect any chronic behavioral ch...

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The most critical portion of the protocol to ensure a strong analysis is the consistent filming. If the videos are well lit and filmed at the correct position as discussed in the first section of the protocol, the system will be able to do a precise analysis. As with any image-processing problem, the work done in preprocessing will make the post-processing more accurate and simple. As such, making sure that the apparatus and animals are well-lit during the testing and any shadows or other motion in the frame is kept to a...

There are many established methods to assess both functional and behavioral motor and cognitive impairments1,2,3. Some of the more commonly employed methods include testing fine motor function via paw placement, stepping, and limb coordination on a ladder test4, testing gross motor function and stress behavior via the open field grid test5,6, and testing for fear, depression, and despair via the forced swim test7,8 or rotor rod9. However, many of these methods rely on human researchers to &#34;score&#34; the animal or to judge its performance subjectively. The need for a subjective human assessment can slow the generation and analysis of the data, as well as present the opportunity for an intentional or unintentional influence of research bias in the study10. Further, subjective assessment of the data also presents the risk of inaccurate data representation, be it through forgetfulness, poor motivation, improper training, or negligence11.
We have recently reported the use of both an open field grid test and a ladder test in rats implanted with intracortical microelectrodes12,13. Due to the novelty of the findings in those studies, we immediately began employing those and additional functional testing in many ongoing studies in the laboratory. In anticipation of unintentional human-generated variability resulting from an increase in the number of subjective evaluators, and to improve the analysis throughput, we set out to create an automated, computer-assisted program to score behavioral testing, and greatly limit the potential for error.
Here, we report on the development of the BVAS. The BVAS uses computer analysis to score an open field grid test and a ladder test as metrics of gross and fine motor function, respectively. The results can be used to elucidate possible motor function deficits caused by injury or disease, regardless of the injury or disease model. The analysis codes can be adapted to account for changes in behavioral testing equipment or to score various metrics of motor function. Therefore, the BVAS can be implemented in many applications, beyond our intended use or the intended use of those currently employed by other laboratories.
Note that the open field grid and ladder tests require video recording. Therefore, each test will require a video camera [1080 p, minimum 15 frames per second (fps)], a laptop, and a room to store the video data. For both tests, place the camera in a centered position, allowing for the whole apparatus to be seen on the frame. Anchor the camera on a tripod or scaffolding so that it does not move during the testing. Keep the edges of the video frame as close to parallel with the edges of the testing apparatus as possible. Be sure the same personnel complete all testing and the room is well-lit with a temperature-controlled system. Use the same room for all animals throughout the course of the testing, with minimal changes to the room. Cereals or banana chips make good rewards to encourage the animals to complete the behavior tests.

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			<td height="40" style="height:40px;width:216px;">Sprague Dawley rats, male, 201-225g</td>
			<td style="width:197px;">Charles River</td>
			<td style="width:163px;">CD</td>
			<td style="width:267px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Webcam HD Pro c920</td>
			<td style="width:197px;">Logitec</td>
			<td style="width:163px;">960-000764</td>
			<td style="width:267px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Excel</td>
			<td style="width:197px;">Microsoft</td>
			<td style="width:163px;">N/A</td>
			<td style="width:267px;"></td>
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			<td height="40" style="height:40px;width:216px;">Matalb 2017a, Computer Vision System Toolbox</td>
			<td style="width:197px;">Mathworks</td>
			<td style="width:163px;">N/A</td>
			<td style="width:267px;"></td>
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			<td height="40" style="height:40px;width:216px;">Open field grid test</td>
			<td style="width:197px;">Made in-house at Case Western Reserve University</td>
			<td style="width:163px;">N/A</td>
			<td style="width:267px;"></td>
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			<td height="40" style="height:40px;width:216px;">Ladder test</td>
			<td style="width:197px;">Made in-house at Case Western Reserve University</td>
			<td style="width:163px;">N/A</td>
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a novel single animal motor function tracking system using simple, readily available software

We have recently demonstrated that implanting intracortical microelectrodes in the motor corteces of rats&#160;results in immediate and lasting motor deficits. Motor impairments were manually quantified through an open field grid test to measure the gross motor function and through a ladder test to measure the fine motor function. Here, we discuss a technique for the automated quantification of the video-recorded tests using our custom Capadona Behavioral Video Analysis System: Grid and Ladder Test, or BVAS. Leveraging simple and readily available coding software (see the Table of Materials), this program allows for the tracking of a single animal on both the open field grid and the ladder tests. In open field grid tracking, the code thresholds the video for intensity, tracks the position of the rat over the 3 min duration of the grid test, and analyzes the path. It then computes and returns measurements for the total distance traveled, the maximum velocity achieved, the number of left- and right-handed turns, and the total number of grid lines crossed by the rat. In ladder tracking, the code again thresholds the video for intensity, tracks the movement of the rat across the ladder, and returns calculated measurements including the time it took the rat to cross the ladder, the number of paw slips occurring below the plane of the ladder rungs, and the incidence of failures due to stagnation or reversals. We envision that the BVAS developed here can be employed for the analysis of motor function in a variety of applications, including many injury or disease models.

Following the methods presented here, rats completed the open field grid and ladder tests 2x per week. The data were analyzed both by using BVAS and manually with a stop-watch by trained and novice reviewers. The results presented are an average of the raw weekly scores from a single non-implanted control animal over an 8 week study, where week &#34;0&#34; corresponds to the baseline testing. Note there was no testing during week 1 as this was a rest week for the surgery animals. Because ...

Watch this Scientific Journal Video about A Novel Single Animal Motor Function Tracking System Using Simple, Readily Available Software at JoVE.com

A Novel Single Animal Motor Function Tracking System Using Simple, Readily Available Software

The current study aimed to automate the quantification of motor deficits in rats. The initial evaluation model assesses motor loss resulting from an intracortical microelectrode implantation in the motor cortex. We report on the development and use of a tracking algorithm using easily adaptable, simple, and readily available coding software.

We have recently demonstrated that implanting intracortical microelectrodes in the motor corteces of rats&#160;results in immediate and lasting motor ...

This method can help improve consistency in small animal Behavioral Testing by creating objective Motor Function Metrics. The advantage of this technique is that unlike conventional methods, the results are quantified by a computer system which cuts down on human error, effort, and time. Demonstrating this procedure will be Marina Yu and Sydney Lindner, Undergraduate Researchers from the Capadona Lab.Begin filming and place the animal in the center of the grid, facing away from the researcher to begin the Grid Test. Allow the animal to run freely for three minutes while being video recorded from above. Stop filming when the three-minute time period has ended and return the animal to its home cage.Clean the grid with a Chlorine Dioxide-based Sterilant, taking care to fully dry the surfaces before testing. Next, for the Ladder Test, place the animal in a clean temporary cage. Set the Ladder Apparatus up to span two cages, where the Start Cage is a clean cage and the End Cage is the animal's living cage, an incentive to complete the run.Place the camera on a tripod, centered on the length of the ladder. Extend to the tripod so the lens of the camera is at the height of the ladder rung. Position the camera so that the rungs are exactly aligned with the lens, as this is important for the Slip Detection Algorithm in the BVAS Code.Start the video recording and allow the animal to begin the run by holding their front paws over the first rung of the ladder. Allow the animal to move onto the ladder unassisted. Allow the animal to move from the start line to the finish line at their own pace while filming.Finally, end the video recording and remove the animal from the ladder once the animal has completed the run. Begin by naming each video with the animal's designation for all Grid Testing videos. Take the videos now named after each animal and place the files in their own storage folder.Then, create a new empty spreadsheet file to store the data for each study and testing mode. Next, install the latest BVAS version by double-clicking on the Installer icon. After installation is complete, double-click on the BVAS Program icon to bring up the BVAS Main Menu, which allows the following options:Grid Analysis, Ladder Analysis, Ladder Review, or Quit.Within the BVAS Program, begin by selecting Grid Analysis from the top menu screen. Then, use the Ellipses button next to the upper input box to browse the file system to find the file of videos to analyze. Select the Ellipses button next to the lower input box to browse the file system to select the spreadsheet file in which to store the data.Click go to start the Analysis Process. Examine the images of each video on the Reviewer screen. Select the Edit button if there are no green lines or the green line does not appear to be in the correct square around the testing area.To edit the corners, select the Edit Corners button. Follow the instructions to the right of the representative image to select the Testing Area and hit Enter to complete the selection process. Lastly, select Done to save the selection and return to the Reviewer screen.Then select the center Done button to start the Complete Analysis process, once the Grid Area for each video is selected correctly. Allow the program to run and upon completion, a Success prompt will appear. Next, select a Ladder Analysis from the top menu to open the Ladder Analysis Menu.Select the upper input Ellipses button to select the folder with Ladder Videos named after each animal. Then, select the lower Ellipses button to select a Directory to save a folder with data from the Ladder Videos to review later. Select Go to begin the Analysis Process, and repeat the same steps for the Open Field Test Analysis.Allow the program to run and upon competition, a Success prompt will appear. Within the BVAS Program, select a Ladder Image Review from the top menu to pull up the Ladder Review file select screen. Select the top Ellipses button to select the folder of data files, and select the bottom Ellipses button to select the spreadsheet file created for the study.Then, press Go to start the Manual Review Process. Next, view the automatically opened Ladder Data Reviewer menu. If a Slip is detected, use the next frame and last frame buttons to check any consecutively recorded frames, if available.Use the Slip Toggle to denote what foot of the rat has slipped. Leave the toggle on No Slip if the detection is a false positive. Click Save and Continue to move on to the next detection.In the case of failure, use the toggle to denote whether the failure is due to Reversal or Stagnation. Finally, click Save and Continue to move on to the next detection. On the last detection, click Save and Continue to complete the analysis and save the data to the spreadsheet file chosen earlier.Dismiss the Completion notice. In this study, the results for the Open Field Grid Test showed that Expert Reviewers were more consistent than Novice Reviewers were, but when using the BVAS, there was zero variance for both Expert and Novice Users. Similarly, the results for the Ladder Test showed that Expert Reviewers were more consistent than Novice Reviewers were, but when using the BVAS, there was, again, zero variance for both Expert and Novice Users.For the Open Field Grid Test, trends were seen over the course of the study of increased Maximum Velocity achieved, increased Total Distance Traveled, and increased number of Gridlines crossed. The percentage of Right Turns largely hovered between 40 and 50%Lastly, for the Ladder Test, there was a decreasing trend in the time to cross the ladder, followed by an increase in time, starting around Week four. The software automatically detects and tabulates the number of Paw Slips during a Ladder Crossing.And in Video Review Process, the user can sort the detected Slips into Left versus Right Paw Slips as shown in the bar graph. Once mastered, this technique can be completed in 30 to 40 minutes per animal per testing session. While attempting this procedure, it's important to film in a well-lit environment on the correct plane and with a high-contrast between the animal and the background.Following this procedure, additional Behavior Tasks can be performed in order to answer specific questions in Disease or Injury Models. While we use this method to provide insight into Motor Deficit caused by Device Implantation in the brain, this analysis technique could be used in many other Disease or Injury Models. After watching this video, you should have a good understanding of how to film and analyze Behavioral videos using the Capadona Lab's BVAS Software, which provides a repeatable Behavior Quantification Technique.

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