Name: applying the ratwalker system for gait analysis in a genetic rat model of parkinson's disease
Uploaded: 2021-01-18T14:00:00
Description: 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

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 str...

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+gait+parameters+in+freely+walking+rodents.">Quantification of gait parameters in freely walking rodents.</a> BMC Biology. 13, 50 (2015).</li><li>Hamers, F. P., Lankhorst, A. J., van Laar, T. J., Veldhuis, W. B., Gispen, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+quantitative+gait+analysis+during+overground+locomotion+in+the+rat:+its+application+to+spinal+cord+contusion+and+transection+injuries.">Automated quantitative gait analysis during overground locomotion in the rat: its application to spinal cord contusion and transection injuries.</a> Journal of Neurotrauma. 18 (2), 187-201 (2001).</li><li>Hamers, F. P., Koopmans, G. C., Joosten, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CatWalk-assisted+gait+analysis+in+the+assessment+of+spinal+cord+injury.">CatWalk-assisted gait analysis in the assessment of spinal cord injury.</a> Journal of Neurotrauma. 23 (3-4), 537-548 (2006).</li><li>Pickrell, A. M., Youle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+PINK1,+parkin,+and+mitochondrial+fidelity+in+Parkinson's+disease.">The roles of PINK1, parkin, and mitochondrial fidelity in Parkinson's disease.</a> Neuron. 85 (2), 257-273 (2015).</li><li>Dawson, T. M., Ko, H. S., Dawson, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+animal+models+of+Parkinson's+disease.">Genetic animal models of Parkinson's disease.</a> Neuron. 66 (5), 646-661 (2010).</li><li>Gispert, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson+phenotype+in+aged+PINK1-deficient+mice+is+accompanied+by+progressive+mitochondrial+dysfunction+in+absence+of+neurodegeneration.">Parkinson phenotype in aged PINK1-deficient mice is accompanied by progressive mitochondrial dysfunction in absence of neurodegeneration.</a> PLoS One. 4 (6), 5777 (2009).</li><li>Goldberg, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkin-deficient+mice+exhibit+nigrostriatal+deficits+but+not+loss+of+dopaminergic+neurons.">Parkin-deficient mice exhibit nigrostriatal deficits but not loss of dopaminergic neurons.</a> Journal of Biological Chemistry. 278 (44), 43628-43635 (2003).</li><li>Kitada, T., Tong, Y., Gautier, C. A., Shen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+of+nigral+degeneration+in+aged+parkin/DJ-1/PINK1+triple+knockout+mice.">Absence of nigral degeneration in aged parkin/DJ-1/PINK1 triple knockout mice.</a> Journal of Neurochemistry. 111 (3), 696-702 (2009).</li><li>Dave, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+characterization+of+recessive+gene+knockout+rat+models+of+Parkinson's+disease.">Phenotypic characterization of recessive gene knockout rat models of Parkinson's disease.</a> Neurobiology of Disease. 70, 190-203 (2014).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Hruska, R. E., Kennedy, S., Silbergeld, E. 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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 ...

This method makes it possible to reproducibly quantify differences in gait parameters. The gait parameters can be assessed in freely-walking rodents, and the technique does not rely on invasive steps such as dipping the feet with ink. Demonstrating the procedure will be Ben Lamberty.Begin by placing 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. Turn off the room lights. Power on the camera and start recording several seconds before the rat is placed on the platform.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. Stop recording once the animal reaches the terminus of the walkway. Clean the walkway using 70%ethanol and a non-abrasive towel in between runs and after an animal urinates or defecates.Then, allow ethanol to evaporate before introducing another animal. Run the rats through the walkway a total of seven times during each observation period, taking the first three runs that score as passing for analysis. 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.Gait analysis was performed on male wild-type and Parkin DKO rats at two months of age to determine if the use of kinematic gait analysis could uncover subtle motor impairments prior to the appearance of gross motor problems. Despite the increased weight of the DKO rats, the foot pressure applied to the walking surface was unaltered. Upon assessment of several gait parameters as a function of walking speed, the walking speed and step lengths were found to be similar between wild-type and DKO rats.The variation between wild-type and DKO rats became apparent in stance phase and swing duration at slower walking speeds. The fraction of the step cycle where the leg is in the stance phase was the duty factor. More time was spent in the swing phase than in the stance phase as the duty factor decreased.Furthermore, while swing speeds increased with increased speed in wild-type animals, the correlation was blunted in DKO rats. The FTIR gait analysis was also used to create plots of stance phase traces of each leg relative to the body in freely-walking rats. Upon comparing the paw positioning, significant changes in AEP and PEP were observed.Several additional parameters were significantly changed in the DKO rats as compared to wild-type. The swing speed of both the left and right hind limbs was increased in DKO rats, while the swing duration of both left and right hind limbs was decreased. Consistent setup of equipment, including camera position with relation to RatWalker, is important for the successful implementation of this protocol.After performing this protocol, animals are available for any additional behavior or experimental endpoints necessary. For example, gait changes can be correlated with histological alterations. This technique can be used to assess gait in other non-PD rodent models, such as models of anxiety and depression.

Research

JoVE Journal

Neuroscience

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