3D Kinematic Gait Analysis for Preclinical Studies in Rodents

3D kinematics of movement and locomotion are being increasingly used. But the complexity of most systems limits your use in pre-clinical research. What we're doing in this work is providing a detailed and simple method of collecting 3D data during quadrupedal locomotion in adult rats.

The described system provides an in-depth qualitative and quantitative data analysis, without the need for complex algorithms. My favorite part of the system is its versatility of use. We have been able to use it for locomotion, as well as reaching and grasping function.

Here, I will demonstrate its use for treadmill walking. Mount six cameras in the wall, two meters away from the treadmill, using finely adjustable geared heads. Slightly angle down below the horizon for maximum coverage of markers.

Equip each camera with a ring light for the visualization of retro-reflective markers. Define the desired markers for the experiment. Use markers for both the forelimbs and the hindlimbs to assess bilateral quadrupedal locomotion.

Here, we used 22 markers. However, this can be adjusted according to ones experimental design. Calibrate the motion capture system using the wand system, which consists of the L-frame and the wand.

Place the L-frame orthogonally on the treadmill with the long leg of the L-frame pointing towards the direction that the rat will be walking in. Open the motion capture software. Select Record"to capture the calibration video.

Move the wand calibration frame throughout the treadmill area in space so as to cover all areas that the rat will be walking in. Record a minimum of one minute of footage to ensure adequate wand data points are present for accurate calibration. Save the videos as a 3D calibration file.

Right click the camera group and select 3D tracking"after recording the calibration videos. Select 3D calibration videos"and select All calibration cameras"Track the L-frame markers on all six videos using fixed point function. Define all points and then select the Search Automatically"button.

After tracking, exit the window and select Automatic 3D Wand Tracking"Select Options"and deselect Detect L-Frame"Begin tracking. After the software has finished tracking, click Assign Markers"Assign Wand Short"Wand Mid"and Wand Long"markers for all six cameras. After the wand and the L-frame have been tracked, right click Camera Calibration Group"and select New Wand Calibration Group"Select All Cameras"and hold down the control key while selecting OK"Change wand length, L-frame height, and the number of cameras according to what was used during calibration.

Accept calibrations with a wand length standard deviation less than three millimeters and a calibration residual of 0.004 or less. This system is commercially available. And in this report, we provide a thorough guide to the research here and effectively using it.

What I like best about this particular protocol and the system per se, is that there are a variety of more outcomes and one can select predefined outcomes of interest that are specific to the condition. Acclimatize rats to the treadmill for 5 min prior to every training session. Train the rats to walk with full weight bearing on their limbs at various speeds.

Train all rats until they are capable of consistently walking on the treadmill. Prior to data collection, shave the rats in the regions where markers will be placed. Palpate the skin for bony landmark to accurately place markers.

Use pen markers for joints distal to the elbow and the knee. Select the red camera button on the top bar of the motion capture software to record a trial. Allow the rat to walk for approximately 30 seconds or a minimum of 10 continuous steps.

Create a new camera group for each trial after saving the recorded video. The software presented in this protocol can be utilized by students, staff, and researchers without the need of technical expertise. This process is user friendly and can be easily mastered with little practice.

This protocol can be implemented in approximately 45 minutes to one hour, depending on the quantity of data desired. Right-click the camera group for motion tracking. Select 2D tracking"Select seven to 10 best continuous and consistent steps for tracking.

Right click the marker of interest and select Automatic Tracking"which will detect bright circular spots created by retro-reflective markers. Alternatively, track markers using pattern matching, which will use an algorithm built into the software to track markers based on size and color. Track black markers using advanced image processing by inverting black markers to bright spots for automatic tracking.

Manually track and correct undetectable markers or errors in tracking. Right-click Phases"and select Edit phase model"Customize gait cycle phases for each limb according to the deficits one chooses to study. Assign phases of the gait cycle for each limb within the software using the Add phase"button or the F11 shortcut key.

Perform 3D calculations after tracking all six cameras. Right click the camera group and select New 3D calculations"A new folder will appear. Generate data of interest, such as joint height or velocity diagrams with data points by dragging out marker of interest to view side-by-side with the assigned gait phases.

Click 3D diagram"to generate a 3-dimensional figure of the trial. This figure shows the elbow angle profile in a representative healthy rat walking on the treadmill. Note that healthy rates are able to continually step for longer durations.

The smooth single peaks represent gait cycles with complete range of motion. The alternating stance phase and swing phase durations, with consistent timing at each step is indicative of normal intralimb coordination. In contrast, continuous stepping is less common after an injury.

The elbow angle profile of a representative spinal cored injured rat demonstrates multiple distorted peaks, which are less consistent and of smaller range of motion. In addition, lengthened stance phase and shortened swing phase durations suggests deficiency in intralimb coordination for the right forelimb. This figure shows representative data plotted for intralimb coordination between two limb pairs.

Representative healthy rat demonstrates well defined alternating rhythmic coordination, which is seen as an L-shaped pattern in a scatter line plot. In contrast, representative cervical spinal cord injured rat demonstrates poor non-alternating, non-rhythmic coordination between the two forelimbs. After watching this video, you should have a good understanding on how to set up and calibrate a multi-camera motion capture system, how to prepare a rat for motion capture, how to record treadmill locomotion, and how to obtain and analyze the outputted 3D kinematic data.

The ultimate goal for researchers then, is to employ these highly standardized motor assessment tools and do our research on a routine basis to decipher effects of interventions on motor recovery after neurotrauma.