KS and HF thank the Michael J Fox Foundation for Parkinson&#39;s Research for support of their work on Parkinson&#39;s disease.

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.
<ol>
	<li>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).</li>
	<li>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).</li>
	<li>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).</li>
	<li>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&#34; thick glass mirror supported by acrylic, and angled brackets arranged in a row (Table S4).</li>
	<li>Perform videography using a tripod-mounted, domestic-quality, high-speed action camera.</li>
</ol>
2. Equipment Setup
<ol>
	<li>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.</li>
	<li>Using a level, make certain that the components are horizontally plumb.</li>
	<li>Place the walkway enclosure on top of the walkway.</li>
	<li>Clean all contact surface areas with 70% ethanol. Make sure to use a nonabrasive towel to prevent scratching of the walkway.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Animal Acclimation
NOTE: One week prior to the first experiment, run the animals through the modified gait apparatus.
<ol>
	<li>Position a home-cage at the terminus of the walkway.</li>
	<li>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.</li>
	<li>Run each rat through the modified gait apparatus several times, until they can smoothly cross the entire walkway.</li>
	<li>Repeat the process two days before the experiment.</li>
</ol>
4. Gait Procedure
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Stop recording once the animal reaches the terminus of the walkway.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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. 
	&#8203;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 &#177; 21.67 g and 296.6 &#177; 3.85 g, respectively (p = 0.004, Unpaired t test with Welch&#39;s correction). We do not see an issue with rats of any age or size.</li>
</ol>
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.
<ol>
	<li>Create a directory for the videos on a PC running Windows 7 or higher, then transfer the videos from the high-speed camera&#39;s storage device to the newly created directory. In addition, copy ffmpeg.exe to the same location.</li>
	<li>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 &#34;_trimmed&#34;.</li>
	<li>To batch convert the videos to image sequences, open a command prompt, set the working directory to the location of the videos with &#34;cd [path to directory]&#34;, and run the following commands: 
	for %i in (*) do mkdir &#34;%~ni_cropped&#34; 
	for %i in (*) do mkdir &#34;%~ni_trimmed&#34; 
	for /f &#34;tokens=1 delims=.&#34; %a in (&#39;dir /B *_trimmed.MP4&#39;) do ffmpeg -i &#34;%a.MP4&#34; &#34;%a/%a_%04d.png&#34;</li>
	<li>&#160;After the batch process completes, open each image sequence in ImageJ Fiji23 and crop the sequence to th&#160;e region of interest (ROI) encompassing the area of the floor within which the rat is observed.</li>
	<li>To reduce background from the walkway illumination, increase the color balance minimum of the cyan channel to 76.</li>
	<li>Save as image sequence and change the &#34;_trimmed&#34; suffix to &#34;_cropped&#34;, saving the files in their respective &#34;_cropped&#34; folder.</li>
</ol>
6. Gait Processing
NOTE: Gait data is processed and quantified using the freely available software, MouseWalker (http://biooptics.markalab.org/MouseWalker/)14.
<ol>
	<li>Unpack and install the MouseWalker software onto a PC running a 64-bit windows environment with Microsoft Excel installed.</li>
	<li>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.</li>
	<li>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/&#160;for the user manual and other documentation.</li>
	<li>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.</li>
	<li>Parameters and settings used for this publication (Figure S6). 
	&#8203;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.</li>
	<li>Following calibration, load each image sequence. Selecting auto will start the autonomous assignment of footprints.</li>
	<li>Scroll through each frame of the sequence, manually correcting miss-assigned footprints. Save once this step is complete.</li>
	<li>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.</li>
</ol>
7. Data Analysis
<ol>
	<li>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.</li>
</ol>

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The authors declare no competing financial interests.

Gait disturbances, including decreased arm swing, slower walking speed, and shorter steps, are a defining feature of PD, and occur early during disease course1,5. 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...

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 PD1,2. 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 disease1,4,5. 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 model11,12,13. 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 runway14,15,16. As compared to other methods, FTIR does not depend on any markers on the animal&#39;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&#39;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.

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applying the ratwalker system for gait analysis in a genetic rat model of parkinson's disease

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.

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-/- ...

Watch this Scientific Journal Video about Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease at JoVE.com

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

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.

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars ...

applying-ratwalker-system-for-gait-analysis-genetic-rat-model

Research

JoVE Journal

Neuroscience

2.7K Views.  University of Nebraska Medical Center. 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. 

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

Assessment of Sensorimotor Function in Mouse Models of Parkinson's Disease

Sensitive and reliable behavioral outcome measures are essential to the evaluation of potential therapeutic treatments in preclinical trials for many neurodegenerative diseases. In Parkinson's disease, sensorimotor tests sensitive to varying degrees of nigrostriatal dysfunction are fundamental for testing the efficacy of potential therapeutics. Reliable and quite elegant sensorimotor measures exist for rats, however many of these tests measure sensorimotor asymmetry within the rat and are not entirely suitable for the newer genetic mouse models of PD. We have put together a battery of sensorimotor tests inspired by the sensitive tests in rats and adapted for mice. The test battery highlighted in this study is chosen for a) its sensitivity in a wide variety of mouse models of PD, b) its ease in implementing into a study, and c) its low expense. These tests have proven useful in characterizing novel genetic mouse models of PD as well as in testing potential disease-modifying therapies.

In Parkinson's disease and movement disorders in general, sensitive and reliable behavioral assays are essential for testing novel potential therapeutics. Here, we describe a manageable battery of sensorimotor tests for mice that are sensitive to varying degrees of injury to the nigrostriatal system and useful for preclinical studies.

Sensitive and  reliable behavioral outcome measures are essential to the evaluation of  potential therapeutic treatments in preclinical trials for many  ...

Behavior

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

Biology

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

Gait analysis is used to quantify changes in motor function in many rodent models of disease. Despite the importance of assessing gait and motor function in many areas of research, the available commercial options have several limitations such as high cost and lack of accessible, open code. To address these issues, we developed PrAnCER, Paw-Print Analysis of Contrast-Enhanced Recordings, for automated quantification of gait. The contrast-enhanced recordings are produced by using a translucent floor that obscures objects not in contact with the surface, effectively isolating the rat&#8217;s paw prints as it walks. Using these videos, our simple software program reliably measures a variety of spatiotemporal gait parameters. To demonstrate that PrAnCER can accurately detect changes in motor function, we employed a haloperidol model of Parkinson&#8217;s disease (PD). We tested rats at two doses of haloperidol: high dose (0.30 mg/kg) and low dose (0.15 mg/kg). Haloperidol significantly increased stance duration and hind paw contact area in the low dose condition, as might be expected in a PD model. In the high dose condition, we found a similar increase in contact area but also an unexpected increase in stride length. With further research, we found that this increased stride length is consistent with the bracing-escape phenomenon commonly observed at higher doses of haloperidol. Thus, PrAnCER was able to detect both expected and unexpected changes in rodent gait patterns. Additionally, we confirmed that PrAnCER is consistent and accurate when compared with manual scoring of gait parameters.

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

We describe a novel gait analysis system, Paw-Print Analysis of Contrast-Enhanced Recordings (PrAnCER), an open-access automated system for the quantification of gait characteristics in rats that utilizes a novel semitransparent floor to automatically quantify gait. This system was validated using the haloperidol model of Parkinson&#8217;s Disease.

Gait analysis is used to quantify changes in motor function in many rodent models of disease. Despite the importance of assessing gait and motor function ...

3D Kinematic Gait Analysis for Preclinical Studies in Rodents

The utility of three-dimensional (3D) kinematic motion analysis systems is limited in rodents. Part of the reason for this inadequacy is the use of complex algorithms and mathematical modeling that accompany 3D data collection and analysis procedures. This work provides a simple, user-friendly, step-by-step detailed methodology for 3D kinematic gait analysis during treadmill locomotion in healthy and neurotraumatic rats using a six-camera motion capture system. Also provided are details on 1) calibration of the system in an experimental set-up customized for quadrupedal locomotion, 2) data collection for treadmill locomotion in adult rats using markers positioned on all four limbs, 3) options available for video tracking and processing, and 4) basic 3D kinematic data generation and visualization and quantification of data using the built-in data collection software. Finally, it is suggested that the utility of this motion capture system be expanded to studying a variety of motor behaviors before and after neurotrauma.

Presented here is a protocol to collect and analyze three-dimensional kinematics of quadrupedal locomotion in rodents for preclinical studies.

The utility of three-dimensional (3D) kinematic motion analysis systems is limited in rodents. Part of the reason for this inadequacy is the use of ...

Gait Analysis of Age-dependent Motor Impairments in Mice with Neurodegeneration

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. Specifically, gait analysis allows recapturing disease relevant phenotypes that are observed in human patients, especially in neurodegenerative diseases that affect motor abilities such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease (AD), amyotrophic lateral sclerosis (ALS), and others. In early studies along this line, the measurement of gait parameters was laborious and depended on factors that were hard to control (e.g., running speed, continuous running). The development of ventral plane imaging (VPI) systems made it feasible to perform gait analysis at a large scale, making this method a useful tool for the assessment of motor behavior in rodents. Here, we present an in-depth protocol of how to use kinematic gait analysis to examine the age-dependent progression of motor deficits in mouse models of neurodegeneration; mouse lines with decreased levels of endophilin, in which neurodegenerative damage progressively increases with age, are used as an example.

In this study, we demonstrate the use of kinematic gait analysis based on ventral plane imaging to monitor the subtle changes in motor coordination as well as the progression of neurodegeneration with advancing age in mouse models (e.g., endophilin mutant mouse lines).

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. ...

Developmental Biology

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

Measurement of animal locomotion is a common behavioral tool used to describe the phenotype of a given disease, injury, or drug model. The low-cost method of gait analysis demonstrated here is a simple but effective measure of gait abnormalities in murine models. Footprints are analyzed by painting a mouse&#8217;s feet with non-toxic washable paint and allowing the subject to walk through a tunnel on a sheet of paper. The design of the testing tunnel takes advantage of natural mouse behavior and their affinity for small dark places. The stride length, stride width, and toe spread of each mouse is easily measured using a ruler and a pencil. This is a well-established and reliable method, and it generates several metrics that are analogous to digital systems. This approach is sensitive enough to detect changes in stride early in phenotype presentation, and due to its non-invasive approach, it allows for testing of groups across life-span or phenotypic presentation.

Footprint analysis is a low-cost alternative to digitized gait analysis programs for researchers quantifying movement abnormalities in mice. Because of its speed, simplicity, and longitudinal potential, it is ideal for behavioral phenotyping of mouse models.

Measurement of animal locomotion is a common behavioral tool used to describe the phenotype of a given disease, injury, or drug model. The low-cost method ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Medicine

Automated Gait Analysis to Assess Functional Recovery in Rodents with Peripheral Nerve or Spinal Cord Contusion Injury

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and a lot of comparative data has been published in the literature. This includes a multitude of assessment methods to study functional recovery following nerve injury and repair. Besides evaluation of nerve regeneration by means of histology, electrophysiology, and other in vivo and in vitro assessment techniques, functional recovery is the most important criterion to determine the degree of neural regeneration. Automated gait analysis allows recording of a vast quantity of gait-related parameters such as Paw Print Area and Paw Swing Speed as well as measures of inter-limb coordination. Additionally, the method provides digital data of the rats&#8217; paws after neuronal damage and during nerve regeneration, adding to our understanding of how peripheral and central nervous injuries affect their locomotor behavior. Besides the predominantly used sciatic nerve injury model, other models of peripheral nerve injury such as the femoral nerve can be studied by means of this method. In addition to injuries of the peripheral nervous systems, lesions of the central nervous system, e.g., spinal cord contusion can be evaluated. Valid and reproducible data assessment is strongly dependent on meticulous adjustment of the hard- and software settings prior to data acquisition. Additionally, proper training of the experimental animals is of crucial importance. This work aims to illustrate the use of computerized automated gait analysis to assess functional recovery in different animal models of peripheral nerve injury as well as spinal cord contusion injury. It also emphasizes the method&#8217;s limitations, e.g., evaluation of nerve regeneration in rats with sciatic nerve neurotmesis due to limited functional recovery. Therefore, this protocol is thought to help researchers interested in peripheral and central nervous injuries to assess functional recovery in rodent models.

Automated gait analysis is a feasible tool to evaluate functional recovery in rodent models of peripheral nerve injury and spinal cord contusion injury. While it requires only one setup to assess locomotor function in various experimental models, meticulous hard- and soft-ware adjustment and training of the animals is highly important.

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and ...

Multifactorial Assessment of Motor Behavior in Rats after Unilateral Sciatic Nerve Crush Injury

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In addition, in the research field of movement disorders, sciatic crush injury has been employed to trigger a dystonia-like phenotype in genetically predisposed DYT-TOR1A rodent models of dystonia. To achieve consistent, reproducible and comparable results after a sciatic nerve crush injury, a standardized method for inducing the nerve crush is essential, in addition to a standardized phenotypical characterization. Attention must be paid not only to the specific assortment of behavioral tests, but also to the technical requirements, the correct execution and consecutive data analysis. This protocol describes in detail how to perform a sciatic nerve crush injury and provides a behavioral test battery for the assessment of motor deficits in rats that includes the open field test, the CatWalk XT gait analysis, the beam walking task, and the ladder rung walking task.

We provide a protocol for the assessment of motor behavior via a behavioral test battery in rats after sciatic nerve crush injury.

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In ...

Comprehensive Understanding of Inactivity-Induced Gait Alteration in Rodents

It is well known that disuse affects neural systems and that joint motions become altered; however, which outcomes properly exhibit these characteristics is still unclear. The present study describes a motion analysis approach that utilizes three-dimensional (3D) reconstruction from video captures. Using this technology, disuse-evoked alterations of walking performances were observed in rodents exposed to a simulated microgravity environment by unloading their hindlimb by their tail. After 2 weeks of unloading, the rats walked on a treadmill, and their gait motions were captured with four charge-coupled device (CCD) cameras. 3D motion profiles were reconstructed and compared to those of control subjects using the image processing software. The reconstructed outcome measures successfully portrayed distinct aspects of distorted gait motion: hyperextension of the knee and ankle joints and higher position of the hip joints during the stance phase. Motion analysis is useful for several reasons. First, it enables quantitative behavioral evaluations instead of subjective observations (e.g., pass/fail in certain tasks). Second, multiple parameters can be extracted to fit specific needs once the fundamental datasets are obtained. Despite hurdles for broader application, the disadvantages of this method, including labor intensity and cost, may be alleviated by determining comprehensive measurements and experimental procedures.

The present protocol describes three-dimensional motion tracking/evaluation to depict gait motion alteration of rats after exposure to a simulated disuse environment.

It is well known that disuse affects neural systems and that joint motions become altered; however, which outcomes properly exhibit these characteristics ...