Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease

Podsumowanie

Here we describe the RatWalker system, built by redesigning the MouseWalker apparatus to accommodate for the increased size and weight of rats. This system uses frustrated total internal reflection (FTIR), high-speed video capture, and open-access analysis software to track and quantify gait parameters.

Streszczenie

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. Gait abnormalities, including decreased arm swing, slower walking speed, and shorter steps are common in PD patients and appear early in the course of disease. Thus, the quantification of motor patterns in animal models of PD will be important for phenotypic characterization during disease course and upon therapeutic treatment. Most cases of PD are idiopathic; however, the identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in Pink1 and Parkin, two proteins involved in mitochondrial quality control that could be harnessed to create animal models. While mice are resistant to neurodegeneration upon loss of Pink1 and Parkin (single and combined deletion), in rats, Pink1 but not Parkin deficiency leads to nigral DA neuron loss and motor impairment. Here, we report the utility of FTIR imaging to uncover gait changes in freely walking young (2 months of age) male rats with combined loss of Pink1 and Parkin prior to the development of gross visually apparent motor abnormality as these rats age (observed at 4-6 months), characterized by hindlimb dragging as previously reported in Pink1 knockout (KO) rats.

Wprowadzenie

PD, the most common age-related neurodegenerative movement disorder, is caused by the loss of DA neurons in the substantia nigra pars compacta. This loss of nigral DA neurons and the DA inputs into the striatum lead to the observed motor function impairments seen in patients with PD^1,2. The defining motor characteristics of patients with PD, known collectively as Parkinsonism, include rigidity, resting tremor, bradykinesia, postural instability, and micrographia³. Furthermore, gait disturbances, which are common in PD patients, appear early in the course of disease^1,4,5. While certain lifestyles are suggested to help slow the progression of PD, such as healthy eating and regular exercise, there is currently no cure for PD, only medications to manage the symptoms. This leaves room for the need of further investigation in hopes of improved therapeutics. Thus, characterization of the gait pattern in PD animal models is a crucial tool to characterize the relevance of the model as well as how therapeutic treatments aimed at controlling PD are preventing or improving motor impairments.

There are various PD animal models that have been used to test therapeutic treatments, however each one has their limitations. For example, animal models treated with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) have yielded a great wealth of information about processes important for nigral DA neuron loss and subsequent striatal adaptations, and pointed to the role of mitochondria in PD pathogenesis; however, the pathogenetic background of the MPTP model is of a toxic nature rather than a neurodegenerative process as in human PD⁶. Additional chemically inducible models include 6-hydroxydopamine (6-OHDA) and rotenone. 6-OHDA was the first agent used to induce PD by selective accumulation of the drug in the DA neurons, which eventually kills the neurons and leads to PD like symptoms. This model was first used for the tracking of DA depletion by examining the behavior in response to amphetamine and apomorphine⁷. This method of PD induction has proved to be useful for the screening of pharmacological agents that impact DA and its receptors⁸. While the 6-OHDA model is a great model for tracking quantifiable motor deficits, this model does not show how the gradual loss of neurons and formation of Lewy bodies impact the animal. The other method of induction, rotenone, has been shown to have progressive degeneration of nigrostriatal neurons with the loss of tyrosine hydroxylase and DA transporter, allowing for a better model to track loss of neurons over time⁹. The rotenone treated rats showed bradykinesia, postural instability, and unsteady gait¹⁰. However, this method has been found to be widely variable between different strains of rats, which has provoked questioning whether or not rotenone is a reliable PD model^11,12,13. While gait analysis has been shown to be impacted by the induction of PD in rats, to date, genetically induced PD rat models have not been readily used for gait analysis by freely walking down a runway.

One way to analyze motor impairment in freely walking rodents is kinematic gait analysis, which can be performed by utilizing FTIR imaging. This established method uses an optical touch sensor based on FTIR, which records and tracks the footprints of the rodents as they move down the runway^14,15,16. As compared to other methods, FTIR does not depend on any markers on the animal's body that could interfere with the paw prints. Generation of the video data produces digital paw prints of all four limbs that can be combined to create a dynamic and reproducible walking pattern for various rodent models. The principle of imaging-based gait analysis is to take each individual paw and measure the contact area over time as the rodent walks down the runway. Each stance is represented by an increase in paw area (in the braking phase) and a decrease in paw area (in the propulsion phase). This is proceeded by the swing phase, which is when there is no paw signal detected. After evaluation of the video, several parameters are generated that can be used to compare wild-type (WT) versus PD model. Some examples of the parameters are step length (distance the paw covers in one step), swing duration (duration of time the paw is not in contact with the runway), swing speed (step length as a function of swing duration), and step pattern (diagonal steps, lateral steps, or girdle steps).

To demonstrate the utility of FTIR to uncover early gait pattern changes in rats, we used a genetic rat model of PD. While most cases of PD are idiopathic; the identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in Pink1 and Parkin, two proteins involved in mitochondrial quality control¹⁷, that could be harnessed to create animal models¹⁸. Unfortunately, mice are resistant to neurodegeneration upon loss of these proteins (single and combined)^19,20,21. In rats, Pink1 but not Parkin deficiency leads to nigral DA neuron loss and motor impairments²², but without complete penetrance. Therefore, we generated a combined Pink1/Parkin double knockout (DKO) rat model, which displays the overt visually apparent hindlimb dragging phenotype reported in male Pink1 KO rats²² but now at a higher rate: 100% versus 30-50% of males between 4-6 months.

While this method works well for analyzing motor deficits in mice¹⁴, FTIR imaging gait system specifications to accommodate the size and weight of rats was previously unavailable noncommercially. Here we explain how to build the RatWalker, a modified FTIR gait imaging system modeled after the MouseWalker¹⁴, except adapted for the size and weight of rats. This system utilizes an optical effect, FTIR, to provide a method to visualize and subsequently record animal footprints for analysis. Contact of an animal's foot with the optical waveguide (platform) causes disruption in the light path resulting in a visible scattering effect, which is captured using domestic-grade, high-speed videography and processing using open-source software. This study demonstrates the power of FTIR imaging in studying gait changes in genetic rat models of PD. For example, while overt visually apparent motor changes (i.e. hindlimb dragging) are observed in male DKO rats at 4 months at the earliest, using FTIR we are able to uncover gate abnormalities in male DKO rats at 2 months of age.

Protokół

All animal studies were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee (IACUC).

1. Gait apparatus

NOTE: Modeled from the MouseWalker¹⁴, the RatWalker was designed with dimensions in proportion to the difference in step length between rats and mice. It consists of a side illumination backlight, walkway enclosure, optical waveguide walkway, mirror, and camera (Figure S1). LED strips, oriented in a staggered position, were used on each side of the walkway and backlight waveguides to accommodate the extra material. The materials needed to build the modified gait apparatus can be found in Table S1.

1. Use a backlight (Figure S2) to create a silhouette of the animal which is utilized by the software to assign position, direction of movement, and morphometric qualities. Construction is comprised of a layered panel of an acrylic diffuser, optical waveguide, reflector, and LED light strips assembled within a stock aluminum frame (Table S1).
2. Use a walkway enclosure (Figure S3) to guide the animal along the platform and direct the animal to the home-cage. Construction consists of clear acrylic sheets solvent welded with dichloromethane (Table S2).
3. Use the walkway (Figure S4) to provide the medium to generate lit footprints. The walkway is constructed from clear acrylic, which is side-lit with strip LEDs and housed in aluminum angle (Table S3).
4. Place a mirror (Figure S5) directly under the walkway at a 45-degree angle to reflect the underside of the walkway for videography. It is constructed from a 1/4" thick glass mirror supported by acrylic, and angled brackets arranged in a row (Table S4).
5. Perform videography using a tripod-mounted, domestic-quality, high-speed action camera.

2. Equipment Setup

1. Align the backlight, walkway, and mirror according to Figure S1, on top of a countertop, workbench, or stable cart. Ensure each component is centered with respect to the walkway.
2. Using a level, make certain that the components are horizontally plumb.
3. Place the walkway enclosure on top of the walkway.
4. Clean all contact surface areas with 70% ethanol. Make sure to use a nonabrasive towel to prevent scratching of the walkway.
5. Mount the high-speed camera onto a 57-inch tripod and place it mid-line to the mirror, spaced far enough to capture the entire walkway inside of the field of view. From the video settings menu, ensure that the high-speed camera is set to linear acquisition in 1080p mode at 120 frames per second (fps) with any type of auto-adjust or optimizations turned off.
6. Plug in and turn on the LED strip lights for the backlight and walkway. It may be necessary to dim the backlight to reduce background capture.

3. Animal Acclimation

NOTE: One week prior to the first experiment, run the animals through the modified gait apparatus.

1. Position a home-cage at the terminus of the walkway.
2. With the enclosure installed and the lights off, place the rat at the end of walkway opposite the home-cage and allow it to walk across the walkway in an unforced manner.
3. Run each rat through the modified gait apparatus several times, until they can smoothly cross the entire walkway.
4. Repeat the process two days before the experiment.

4. Gait Procedure

1. Place a home-cage at the end of the walkway before the start of each run to serve as a positive cue for the rat to traverse the walkway.
2. Turn off the room lights, power on the camera, and start recording several seconds before the rat is placed on the platform.
   NOTE: Be certain to use a memory card that is officially recommended by the camera manufacturer. An unlisted memory card may still work but is not guaranteed to capture at the purported frame rate.
3. With the enclosure installed, place the rat at the end of the walkway opposite the home-cage and allow it to walk across the walkway in an unforced manner.
4. Stop recording once the animal reaches the terminus of the walkway.
5. Clean the walkway using 70% ethanol and a nonabrasive towel in between runs and after an animal urinates or defecates, then allow ethanol to evaporate before introducing another animal.
6. Run the rats through the walkway a total of 7 times during each observation period, taking the first three runs that score as passing for analysis.
7. Score a run as passing if the animal makes four or more consecutive steps in the direction of the home-cage without interruption due to grooming, pausing, or errant movements.
   ​NOTE: It is good practice to record the mass of the animals before each round of measures. For our study, WT (n = 7) and DKO (n = 8) weighed 200.3 ± 21.67 g and 296.6 ± 3.85 g, respectively (p = 0.004, Unpaired t test with Welch's correction). We do not see an issue with rats of any age or size.

5. Video preprocessing

NOTE: The videos captured by the high-speed camera are rendered in mp4 format at 120 fps and a resolution of 1080p. To ease the burden on the analytical software downstream, first trim unnecessary footage and strip the audio from each video using LosslessCut software (version 3.23.7, https://github.com/mifi/lossless-cut), then convert the mp4 video stream into a png image sequence using the open-source software FFmpeg (version 4.2, http://ffmpeg.org/). Note: other Lossless formats such as tiff can be utilized in place of png.

1. Create a directory for the videos on a PC running Windows 7 or higher, then transfer the videos from the high-speed camera's storage device to the newly created directory. In addition, copy ffmpeg.exe to the same location.
2. In LosslessCut, drag the videos to the interface to open. Discard the audio, set the start and end cut points to include only the analytically relevant portion of the video, set the capture frame format to png, and export. Once the video is exported, rename the video file using any naming convention followed by "_trimmed".
3. To batch convert the videos to image sequences, open a command prompt, set the working directory to the location of the videos with "cd [path to directory]", and run the following commands:
   for %i in (*) do mkdir "%~ni_cropped"
   for %i in (*) do mkdir "%~ni_trimmed"
   for /f "tokens=1 delims=." %a in ('dir /B *_trimmed.MP4') do ffmpeg -i "%a.MP4" "%a/%a_%04d.png"
4.  After the batch process completes, open each image sequence in ImageJ Fiji²³ and crop the sequence to th e region of interest (ROI) encompassing the area of the floor within which the rat is observed.
5. To reduce background from the walkway illumination, increase the color balance minimum of the cyan channel to 76.
6. Save as image sequence and change the "_trimmed" suffix to "_cropped", saving the files in their respective "_cropped" folder.

6. Gait Processing

NOTE: Gait data is processed and quantified using the freely available software, MouseWalker (http://biooptics.markalab.org/MouseWalker/)¹⁴.

1. Unpack and install the MouseWalker software onto a PC running a 64-bit windows environment with Microsoft Excel installed.
2. After launching MouseWalker.exe, perform an initial scale calibration for each set of runs. Load an image sequence and using landmarks or a ruler captured in the video, measure two points of known distance. Calculate the number of pixels per centimeter in the video frame and enter this value into the parameters section of the settings form along with the video acquisition frame rate.
3. Similarly, measure the head, tail, and feet of the rat to determine head length, maximum tail width and area, minimum and maximum foot area, and other features necessary to complete the tracking parameters section of the MouseWalker settings form. See http://biooptics.markalab.org/MouseWalker/ for the user manual and other documentation.
4. To obtain the body area values, open the same image sequence in ImageJ, draw a selection outlining the rat, and perform a region of interest (ROI) pixel count.
5. Parameters and settings used for this publication (Figure S6).
   ​NOTE: Parameters are provided for illustration and are dependent on the scale of the video, acquisition hardware, and conditions. Software calibration and adjustment is required each time the camera or equipment is re-positioned. Capturing a measuring device within the acquisition improves accuracy and facilitates calibration.
6. Following calibration, load each image sequence. Selecting auto will start the autonomous assignment of footprints.
7. Scroll through each frame of the sequence, manually correcting miss-assigned footprints. Save once this step is complete.
8. Lastly, select evaluate to process the footprint position and pressure data. A series of graphs, images, and a spreadsheet with quantitative gait metrics will export into a results folder.

7. Data Analysis

1. Use the spreadsheet exported at the end of each evaluation that contains quantitative gait data for each run. Concatenate data from each run and average per rat. Plot the mean data and test for significance using GraphPad Prism version 7.0a.

Wyniki

Rat Colony Maintenance
The generation and characterization of Pink1 and Parkin single KO rats has been described previously²². The Pink1 and Parkin single KO rats were obtained from SAGE Labs (and now available from Envigo). DKO rats were generated by crossing Pink1^-/- rats with Parkin^-/- rats to obtain Pink1^+/-/Parkin^+/- rats, which were interbred to obtain Pink1^-/-/Parkin^-/- ...

Dyskusje

Gait disturbances, including decreased arm swing, slower walking speed, and shorter steps, are a defining feature of PD, and occur early during disease course^1,5. Several methods have been developed over the years to observe and record footfalls for gait analysis in rodent models of PD, with manual techniques for quantifying footfall position leading to automated approaches that are more sensitive and capable of capturing dynamic parameters. Some static approache...

Materiały

Name	Company	Catalog Number	Comments
Aluminum
1.5” Aluminum Angle (1/8” - 6063)			Dimensions: 8' Qty: 8
1” Aluminum Square Tube (1/16” - 6063)			Dimensions: 8' Qty: 4
32 Gauge Aluminum Sheet			Dimensions: 10' Qty: 1
1” Aluminum Tube (1/8” - 6063)			Dimensions: 8' Qty: 1
Acrylic
7/32” Clear Acrylic Sheet			Dimensions: 4'x8' Qty: 2
1/8” White Acrylic Sheet 55% (2447)			Dimensions: 4'x8' Qty: 1
Mirror
7/32” Glass Mirror			Dimensions: 60"x12" Qty: 1
LED
5050 LED Tape Light (Green)			Dimensions: 16.4' Qty: 1
5050 LED Tape Light (Red)			Dimensions: 16.4' Qty: 1
Camera
GoPro Hero 6 Black			Qty: 1
Tripod			Dimensions: 57" Qty: 1