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Neuroscience

Using the MouseWalker to Quantify Locomotor Dysfunction in a Mouse Model of Spinal Cord Injury

Published: March 24th, 2023

DOI:

10.3791/65207

1Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina da Universidade de Lisboa, 2iNOVA4Health, NOVA Medical School|Faculdade de Ciências Médicas, NMS|FCM, Universidade Nova de Lisboa, 3Instituto de Histologia e Biologia do Desenvolvimento, Faculdade de Medicina da Universidade de Lisboa

An experimental pipeline to quantitatively describe the locomotor pattern of freely walking mice using the MouseWalker (MW) toolbox is provided, ranging from initial video recordings and tracking to post-quantification analysis. A spinal cord contusion injury model in mice is employed to demonstrate the usefulness of the MW system.

The execution of complex and highly coordinated motor programs, such as walking and running, is dependent on the rhythmic activation of spinal and supra-spinal circuits. After a thoracic spinal cord injury, communication with upstream circuits is impaired. This, in turn, leads to a loss of coordination, with limited recovery potential. Hence, to better evaluate the degree of recovery after the administration of drugs or therapies, there is a necessity for new, more detailed, and accurate tools to quantify gait, limb coordination, and other fine aspects of locomotor behavior in animal models of spinal cord injury. Several assays have been developed over the years to quantitatively assess free-walking behavior in rodents; however, they usually lack direct measurements related to stepping gait strategies, footprint patterns, and coordination. To address these shortcomings, an updated version of the MouseWalker, which combines a frustrated total internal reflection (fTIR) walkway with tracking and quantification software, is provided. This open-source system has been adapted to extract several graphical outputs and kinematic parameters, and a set of post-quantification tools can be to analyze the output data provided. This manuscript also demonstrates how this method, allied with already established behavioral tests, quantitatively describes locomotor deficits following spinal cord injury.

The effective coordination of four limbs is not unique to quadruped animals. Forelimb-hindlimb coordination in humans remains important to accomplish several tasks, such as swimming and alterations of speed while walking1. Various limb kinematic2 and motor program1,3,4, as well as proprioceptive feedback circuits5, are conserved between humans and other mammals and should be considered when analyzing therapeutic options for motor disorders, such as spinal cord injury (SCI)6

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All handling, surgical, and post-operative care procedures were approved by Instituto de Medicina Molecular Internal Committee (ORBEA) and the Portuguese Animal Ethics Committee (DGAV) in accordance with the European Community guidelines (Directive 2010/63/EU) and the Portuguese law on animal care (DL 113/2013) under the license 0421/000/000/2022. Female C57Bl/6J mice aged 9 weeks were used for the present study. All efforts were made to minimize the number of animals and to decrease the suffering of the animals used in .......

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The standard BMS system describes the gross motor deficits after SCI14. Due to its subjective nature, other quantitative assays are generally performed alongside the BMS to produce a more detailed and fine assessment of locomotion. However, these tests fail to show specific information about step cycles, stepping patterns, and forelimb-hindlimb coordination, which is extremely important in understanding how the spinal circuitry maintains function and adapts to an incomplete SCI. This section shows.......

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Here, the potential of the MouseWalker method is demonstrated by analyzing locomotor behavior after SCI. It provides new insights into specific alterations in stepping, footprint, and gait patterns that would otherwise be missed by other standard tests. In addition to providing an updated version of the MW package, data analysis tools are also described using the supplied Python scripts (see step 5).

As the MW generates a large dataset and a collection of kinematic parameters that reflect a hi.......

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The authors thank Laura Tucker and Natasa Loncarevic for their comments on the manuscript and the support given by the Rodent Facility of the Instituto de Medicina Molecular João Lobo Antunes. The authors want to acknowledge financial support from Prémios Santa Casa Neurociências - Prize Melo e Castro for Spinal Cord Injury Research (MC-36/2020) to L.S. and C.S.M. This work was supported by Fundação para a Ciência e a Tecnologia (FCT) (PTDC/BIA-COM/0151/2020), iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020), and LS4FUTURE (LA/P/0087/2020) to C.S.M. L.S. was supported by a CEEC Individual Principal Investigator contract (2021.02253.CEEC....

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NameCompanyCatalog NumberComments
45º Mirror 
2 aluminum extrusion (2 x 2 cm), 16 cm height, 1 on each sideMisumi
2 aluminum extrusion (2 x 2 cm), 23 cm, @ 45° , 1 on each sideMisumi
1 aluminum extrusion (2 x 2 cm), 83 cm longMisumi
87 x 23 cm mirrorGeneral glass supplier 
black cardboard filler General stationery supplierWe used 2, one with 69 x 6 cm and another with 69 x 3cm to limit the reflection on the mirror
Background backlight
109 x 23 cm plexiglass (0.9525 cm thick)General hardware supplier
2 lateral aluminum extrusion (4 x 4 cm), 20 cm long, 1 on each sideMisumi
multicolor LED stripGeneral hardware supplier
white opaque paper to cover the plexyglassGeneral stationery supplier
fTIR Support base and posts
2 aluminum extrusion (4 x 4 cm), 100 cm heightMisumi
60 x 30 cm metric breadboardEdmund Optics #54-641
M6 12 mm screwsEdmund Optics 
M6 hex nuts and wahersEdmund Optics 
fTIR Walkway 
109 x 8.5 cm plexyglass (1.2 cm thick)General hardware supplier109 x 8.5 cm plexyglass (1.2 cm thick)
109 cm long Base-U-channel aluminum with 1.6 cm height x 1.9 cm depth thick folds (to hold the plexyglass)General hardware supplier
2 lateral aluminum extrusion (4 x 4 cm) 20 cm length, 1 on each sideMisumi
black cardboard filler General stationery supplierwe used 2 fillers on each side to cover the limits of the plexyglass, avoiding bright edges
12 mm screwsEdmund Optics M6
High speed camera (on a tripod)
Blackfly S USB3BlackflyUSB3This is a reccomendation. The requirement is to record at least 100 frames per second
Infinite Horizon Impactor 
Infinite Horizon Impactor Precision Systems and Instrumentation, LLC.
Lens
Nikkon AF Zoom-Nikkor 24-85mmNikkon 2.8-4D IFThis lens is reccomended, however other lens can be used. Make sure it contains a large aperture (i.e., smaller F-stop values), to capture fTIR signals
Software
MATLAB R2022bMathWorks
Python 3.9.13 Python Software Foundation
Anaconda Navigator 2.1.4Anaconda, Inc.
Spyder 5.1.5 Spyder Project Contributors
Walkway wall 
2 large rectagular acrilics with 100 x 15 cmAny bricolage convenience store
2 Trapezian acrilic laterals with 6-10 length x 15 cm heightAny bricolage convenience store
GitHub Materials
Folder nameURL
Boxplotshttps://github.com/NeurogeneLocomotion/MouseWalker/tree/main/BoxplotsScript to create Boxplots
Docshttps://github.com/NeurogeneLocomotion/MouseWalker/tree/main/DocsAdditional documents
Heatmaphttps://github.com/NeurogeneLocomotion/MouseWalker/tree/main/HeatmapsScript to create heatmap
Matlat scripthttps://github.com/NeurogeneLocomotion/MouseWalker/tree/main/Matlab%20ScriptMouseWalker matlab script
PCAhttps://github.com/NeurogeneLocomotion/MouseWalker/tree/main/PCA%20plotsScript to perform Principal Component Analysis
Raw data Plotshttps://github.com/NeurogeneLocomotion/MouseWalker/tree/main/Rawdata%20PlotsScript to create Raw data plots
Residual Analysishttps://github.com/NeurogeneLocomotion/MouseWalker/tree/main/Residual_AnalysisCode to compute residuals from Raw data

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