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 Jo&#227;o Lobo Antunes. The authors want to acknowledge financial support from Pr&#233;mios Santa Casa Neuroci&#234;ncias - Prize Melo e Castro for Spinal Cord Injury Research (MC-36/2020) to L.S. and C.S.M. This work was supported by Funda&#231;&#227;o para a Ci&#234;ncia 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.CEECIND). A.F.I. was supported by a doctoral fellowship from FCT (2020.08168.BD). A.M.M. was supported by a doctoral fellowship from FCT (PD/BD/128445/2017). I.M. was supported by a post-doctoral fellowship from FCT (SFRH/BPD/118051/2016). D.N.S. was supported by a doctoral fellowship from FCT (SFRH/BD/138636/2018).

Quantifying Locomotor Dysfunction in Spinal Cord Injury Mice Using MouseWalker

<ol>
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The authors declare that they have no competing financial interests.

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 highly dimensional locomotor process, a PCA was employed; indeed, PCA has been widely used in other kinematic datasets similar to this one27,35,36 (Figure 3). This dimensionality reduction technique is a simple and robust method with minimal assumptions that allows for quantitatively identifying kinematic profiles and quickly distinguishing them from control or baseline conditions. In addition, heatmaps were generated to promptly identify the parameters that were statistically different from baseline (Figure 4), which could later be analyzed individually (Figure&#160;5, Figure 6, and Figure 7).
Subsequently, it was shown how specific parameters that can be extracted from the MW illustrate the robust locomotor deficits seen in SCI animals, such as coordination. Coordination is the ability to use each limb in an organized manner to perform a task. Often, coordination is measured indirectly by the number of mistakes/positive events on a ladder16,17 or the time spent on the rotarod15. Distinctly, the MW computes both the variability in paw positioning during the step cycle (footprint pattern)&#160;and the &#34;stance straightness&#34; index27. It was shown that both parameters were significantly affected by the injury (Figure 4).
Moreover, the MW allows the user to extract parameters related to temporal dynamics. A significant disruption of the swing/stance duration in SCI animals was seen (Figure 4), and it was found that the SCI mice compensated for the loss of support on their hindlimbs by accelerating their forelimb step cycle, thus reducing the forelimb step length and average swing time (Figure 5). This effect could relate to the shift in the center of gravity, which forces the forepaws to endure more body weight1. Likewise, it could be attributed to the loss of the long ascending propriospinal pathways, which are responsible for the synergetic interaction between the forelimb and hindlimb CPGs1,9,32, ultimately leading to forelimb-hindlimb rhythm dissociation.
The characteristics of stepping gaits can illustrate the modular organization of locomotor networks1,34. Although stepping patterns are not a direct proxy for the gait strategies adopted in this case23, some important observations can still be drawn. Important alterations were seen in the stepping patterns of SCI mice (Figure 6). The injured animals started to adopt lateral swings (pacing-like gaits), and there was also a decrease in diagonal swings. These adaptations seem to be associated with an attempt to coordinate the forelimbs and hindlimbs, again demonstrating the dissociation between the spinal tracks below and above the injury, which does not recover1,9,32.
The limited recovery after SCI was also verified by a decrease in muscle vigor in the hindlimbs, as measured by the average pressure (Figure 7). Consequently, the forelimbs are forced to support more body weight, which increases the pressure. While many parameters related to muscle dynamics were not analyzed here (i.e., flexor and extensor muscle coordination7,21), this quantitative measurement can provide a direct read-out of the amount of limb support.
This MW analysis can also be partnered with the standard BMS test, which measures limb rotation, paw placement, tail position, and stepping mistakes. Generally, SCI mice achieve a maximum score of 4-5, with a maximum subscore of 3, meaning they can perform occasional to frequent steps but mostly rotate at the paw lift-off and/or touchdown14 (Supplementary&#160;Figure 2). It becomes clear why the AEP values for the hindpaws were significantly altered in the SCI group, as the non-parallel positioning of the limbs during the swing phase may affect the paw coordinates. In addition, in the BMS, the trunk is usually classified as severe due to the presence of instances or events that prevent stepping, such as butt down and hopping14. Although it was not possible to quantify butt down behavior or instances that prevented stepping, restricted hindpaw stepping in the MW was detected, with higher swing duration (Figure 5F), increased three leg-swing (Figure 6E), and lower pressure (Figure 7B-D). In addition, a significant increase in fore/hind swings was observed (Figure 6D). There could be two complementary explanations for this behavioral manifestation. First, the hopping-like gait was measured, which could be already observed in the BMS test. However, this behavior could also be related to a lack of correct plantar stepping in both hindlimbs, resulting in foot drag or dorsal placement. Overall, these observations support the claim that trunk stability is severely damaged after SCI. Finally, one of the requirements to score higher than 5 in the BMS test is coordination14, and for this, at least three accessible passes must be observed by the raters during the test. Of those, two out of the three or more accessible passes need to be classified as coordinated (i.e., forelimbs and hindlimbs alternating). From the MW data, one can extract objective measurements, the stance straightness index, and footprint clustering, which directly quantify coordination independently of animal speed or accessible pass (Figure 4). In addition, the alternation between limbs can be quantified objectively by measuring the stepping strategies (Figure 6). These parameters clearly illustrate that SCI mice consistently cannot walk in straight lines and misplace their hindlimbs.
While the MW toolbox is a helpful strategy for studying locomotor defects after SCI, one should consider some of its limitations. First, it is critical to remain consistent with the acquisition parameters (i.e., camera position, focus, light intensity) so that the image features between time points are maintained. Secondly, it was noticed that repetition led to habituation, which, in turn, led to a decreased speed. This also contributed to increased rearing/grooming behaviors and stops mid-run.&#160;For injured animals, there was also a tendency to lean on the walkway walls and an increased number of stops mid-run. As these behaviors are not part of the locomotor assessment, they must not be considered. Preferentially, researchers should choose videos in which the animals walk continuously with their heads pointing straight. To counteract the influence of these behaviors on the results of this study, mice were recorded continuously for at least four or five runs and allowed to run in either direction along the walkway. Afterward, the three best recorded runs were selected and adjusted to the same direction on ImageJ/FIJI24 (180&#176; rotation). Thus, each animal was represented by a mean of three runs pooled together per time point. A larger number of animals per condition could also decrease the expected variability. Moreover, it should be noted that this locomotor test is only recommended after plantar stepping is achieved, as the tracking system was designed for fine-tuned locomotor assessment. In this study, it was noted that before 15 dpi, the MW test was not advantageous for locomotor assessment due to the increased foot drags and incorrect plantar placement, which could affect the tracking (data not shown). Lastly, some parameters (such as footprint clustering) are very sensitive to the extrapolations made by the regression model generated. Therefore, the code was adjusted accordingly (see the documentation in the script in the GitHub link).
Overall, it was shown that the MW could strongly discriminate SCI animals from sham-injured controls, and MW could also be partnered with established tests as a valuable method to study locomotor defects after SCI. Moreover, it was shown that quantitative outputs could be easily generated from the MW dataset using the supplied Python scripts. These tools provide an efficient experimental pipeline to generate a rich and diverse set of quantitative and graphical outputs that complement the original MW output files and can be changed to fulfill the researcher&#39;s goals in terms of the type of analysis and graphical representation.
This toolbox is a valuable method for studying other motor-related diseases or motor dysfunctions, not only those related to spinal cord injury. Skarlatou et al.10 already demonstrated that a specific mutation in Afadin, an important scaffold protein during development, caused an aberrant phenotype in the spinal cord by generating two central canals. This defect resulted in the loss of right-left limb alternation and a higher prevalence of limb synchronization, typical of a hopping gait. Stauch et al.10 also illustrated that this type of system can be adapted according to user&#39;s needs. In this case, it was applied to study specific behavioral deficits in a rat Parkinson&#39;s disease model. Thus, this toolbox has a broad range of applications in different models in which motor disturbances are expected and can be combined with already established behavior protocols in the field.

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&#160;and should be considered when analyzing therapeutic options for motor disorders, such as spinal cord injury (SCI)6,7,8.
In order to walk, several spinal connections from the forelimbs and hindlimbs need to be properly wired and rhythmically activated, which requires inputs from the brain and feedback from the somatosensory system2,9,10. These connections culminate in the central pattern generators (CPGs), which are situated at a cervical and lumbar level for the forelimbs and hindlimbs, respectively1,9,10. Often, after SCI, the disruption of neuronal connectivity and the formation of an inhibitory glial scar12 limit the recovery of locomotor function, with outcomes varying from total paralysis to restricted function of a group of limbs depending on the injury severity. Tools to precisely quantify locomotor function after SCI are critical for monitoring recovery and evaluating the effects of treatments or other clinical interventions6.
The standard metric assay for mouse contusion models of SCI is the Basso mouse scale (BMS)13,14, a non-parametric score that considers trunk stability, tail position, plantar stepping, and forelimb-hindlimb coordination in an open field arena. Even though the BMS is extremely reliable for most cases, it requires at least two experienced raters to observe all the angles of animal movement in order to account for natural variability and reduce bias.
Other assays have also been developed to assess motor performance after SCI quantitatively. These include the rotarod test, which measures time spent on a rotating cylinder15; the horizontal ladder, which measures the number of missed railings and positive ladder grabs16,17; and the beam walking test, which measures the time an animal takes and the number of failures it makes when crossing a narrow beam18. Despite reflecting a combination of motor deficits, none of these tests produce direct locomotor information about forelimb-hindlimb coordination.
To specifically and more thoroughly analyze walking behavior, other assays have been developed to reconstruct step cycles and gaiting strategies. One example is the footprint test, where the inked paws of an animal draw a pattern over a sheet of white paper19. Although simple in its execution, extracting kinematic parameters such as stride length is cumbersome and inaccurate. Moreover, the lack of dynamic parameters, such as the duration of the step cycle or leg-timed coordination, limits its applications; indeed, these dynamic parameters can only be acquired by analyzing frame-by-frame videos of rodents walking through a transparent surface. For SCI studies, researchers have analyzed walking behavior from a lateral view using a treadmill, including reconstructing the step cycle and measuring the angular variations of each leg joint4,20,21. Even though this approach can be extremely informative6, it remains focused on a specific set of limbs and lacks additional gait features, such as coordination.
To fill these gaps, Hamers and colleagues developed a quantitative test based on an optical touch sensor using frustrated total internal reflection (fTIR)22. In this method, light propagates through glass via internal reflection, becomes scattered upon paw pressing, and, finally, is captured by a high-speed camera. More recently, an open-source version of this method, called MouseWalker, was made available, and this approach combines an fTIR walkway with a tracking and quantification software package23. Using this method, the user can extract a large set of quantitative parameters, including step, spatial, and gait patterns, footprint positioning, and forelimb-hindlimb coordination, as well as visual outputs, such as footprint patterns (mimicking the inked paw assay6) or stance phases relative to the body axis. Importantly, due to its open-source nature, new parameters can be extracted by updating the MATLAB script package.
Here, the previously published assembly of the MouseWalker23 system is updated. A description of how to set it up is provided, with all the steps required to achieve the best video quality, tracking conditions, and parameter acquisition. Additional post-quantification tools are also shared to enhance the analysis of the MouseWalker (MW) output dataset. Finally, the usefulness of this tool is demonstrated by obtaining quantifiable values for general locomotor performance, specifically step cycles and forelimb-hindlimb coordination, in a spinal cord injury (SCI) context.

<table><tbody><tr><td>&lt;strong&gt;45&amp;ordm; Mirror&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2 aluminum extrusion (2 x 2 cm), 16 cm height, 1 on each side</td><td>Misumi</td><td></td><td></td></tr><tr><td>2 aluminum extrusion (2 x 2 cm), 23 cm, @ 45&amp;deg; , 1 on each side</td><td>Misumi</td><td></td><td></td></tr><tr><td>1 aluminum extrusion (2 x 2 cm), 83 cm long</td><td>Misumi</td><td></td><td></td></tr><tr><td>87 x 23 cm mirror</td><td>General glass supplier&amp;nbsp;</td><td></td><td></td></tr><tr><td>black cardboard filler&amp;nbsp;</td><td>General stationery supplier</td><td></td><td>We used 2, one with 69 x 6 cm and another with 69 x 3cm to limit the reflection on the mirror</td></tr><tr><td>&lt;strong&gt;Background backlight&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>109 x 23 cm plexiglass (0.9525 cm thick)</td><td>General hardware supplier</td><td></td><td></td></tr><tr><td>2 lateral aluminum extrusion (4 x 4 cm), 20 cm long, 1 on each side</td><td>Misumi</td><td></td><td></td></tr><tr><td>multicolor LED strip</td><td>General hardware supplier</td><td></td><td></td></tr><tr><td>white opaque paper to cover the plexyglass</td><td>General stationery supplier</td><td></td><td></td></tr><tr><td>&lt;strong&gt;fTIR Support base and posts&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2 aluminum extrusion (4 x 4 cm), 100 cm height</td><td>Misumi</td><td></td><td></td></tr><tr><td>60 x 30 cm metric breadboard</td><td>Edmund Optics&amp;nbsp;</td><td>#54-641</td><td></td></tr><tr><td>M6 12 mm screws</td><td>Edmund Optics&amp;nbsp;</td><td></td><td></td></tr><tr><td>M6 hex nuts and wahers</td><td>Edmund Optics&amp;nbsp;</td><td></td><td></td></tr><tr><td>&lt;strong&gt;fTIR Walkway&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>109 x 8.5 cm plexyglass (1.2 cm thick)</td><td>General hardware supplier</td><td></td><td>109 x 8.5 cm plexyglass (1.2 cm thick)</td></tr><tr><td>109 cm long Base-U-channel aluminum with 1.6 cm height x 1.9 cm depth thick folds (to hold the plexyglass)</td><td>General hardware supplier</td><td></td><td></td></tr><tr><td>2 lateral aluminum extrusion (4 x 4 cm) 20 cm length, 1 on each side</td><td>Misumi</td><td></td><td></td></tr><tr><td>black cardboard filler&amp;nbsp;</td><td>General stationery supplier</td><td></td><td>we used 2 fillers on each side to cover the limits of the plexyglass, avoiding bright edges</td></tr><tr><td>M6 12 mm screws</td><td>Edmund Optics&amp;nbsp;</td><td></td><td></td></tr><tr><td>&lt;strong&gt;High speed camera (on a tripod)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Blackfly S USB3</td><td>FLIR</td><td>#BFS-U3-32S4M-C</td><td>This is a reccomendation. The requirement is to record at least 100 frames per second</td></tr><tr><td>&lt;strong&gt;Infinite Horizon Impactor&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Infinite Horizon Impactor&amp;nbsp;</td><td>Precision Systems and Instrumentation, LLC.</td><td>#0400</td><td></td></tr><tr><td>&lt;strong&gt;Lens&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Nikon AF Zoom-Nikkor 24-85mm</td><td>Nikon</td><td>#1929</td><td>This 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</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MATLAB R2022b</td><td>MathWorks</td><td></td><td></td></tr><tr><td>Python 3.9.13&amp;nbsp;</td><td>Python Software Foundation</td><td></td><td></td></tr><tr><td>Anaconda Navigator 2.1.4</td><td>Anaconda, Inc.</td><td></td><td></td></tr><tr><td>Spyder 5.1.5&amp;nbsp;</td><td>Spyder Project Contributors</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Walkway wall&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2 large rectagular acrilics with 100 x 15 cm</td><td>Any bricolage convenience store</td><td></td><td></td></tr><tr><td>2 Trapezian acrilic laterals with 6-10 length x 15 cm height</td><td>Any bricolage convenience store</td><td></td><td></td></tr><tr><td>&lt;strong&gt;GitHub Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Folder name&lt;/strong&gt;</td><td>&lt;strong&gt;URL&lt;/strong&gt;</td><td></td><td></td></tr><tr><td>Boxplots</td><td>https://github.com/NeurogeneLocomotion/MouseWalker/tree/main/Boxplots</td><td></td><td>Script to create Boxplots</td></tr><tr><td>Docs</td><td>https://github.com/NeurogeneLocomotion/MouseWalker/tree/main/Docs</td><td></td><td>Additional documents</td></tr><tr><td>Heatmap</td><td>https://github.com/NeurogeneLocomotion/MouseWalker/tree/main/Heatmaps</td><td></td><td>Script to create heatmap</td></tr><tr><td>Matlat script</td><td>https://github.com/NeurogeneLocomotion/MouseWalker/tree/main/Matlab%20Script</td><td></td><td>MouseWalker matlab script</td></tr><tr><td>PCA</td><td>https://github.com/NeurogeneLocomotion/MouseWalker/tree/main/PCA%20plots</td><td></td><td>Script to perform Principal Component Analysis</td></tr><tr><td>Raw data Plots</td><td>https://github.com/NeurogeneLocomotion/MouseWalker/tree/main/Rawdata%20Plots</td><td></td><td>Script to create Raw data plots</td></tr><tr><td>Residual Analysis</td><td>https://github.com/NeurogeneLocomotion/MouseWalker/tree/main/Residual_Analysis</td><td></td><td>Code to compute residuals from Raw data</td></tr></tbody></table>

using the mousewalker to quantify locomotor dysfunction in a mouse model of spinal cord injury

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.

Watch this Scientific Journal Video about Using the MouseWalker to Quantify Locomotor Dysfunction in a Mouse Model of Spinal Cord Injury at JoVE.com

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

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.

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

The execution of complex and highly coordinated motor programs, such as walking and running, is dependent on the rhythmic activation of spinal and ...

using-mousewalker-to-quantify-locomotor-dysfunction-mouse-model

Nanyang Technological University

Research

JoVE Journal

Neuroscience

2.6K Views.  Faculdade de Medicina da Universidade de Lisboa. In deze studie gebruiken we een muismodel van ruggenmergletsel om manieren te ontdekken om herstel van het ruggenmerg te bevorderen die kunnen leiden tot effectief locomotorisch en functioneel sensorisch herstel. Met een muiswandelaar kunnen we een stap vooruit zetten naar een meer kwantitatieve analyse van ruggenmergherstel door verschillende grafische outputs en kinematische parameters te combineren met een reeks postkwantificeringstools. Videocamera's van hoge kwaliteit en geavanceerde sof...

Video: Auteur in de schijnwerpers: de MouseWalker gebruiken om locomotorische disfunctie te kwantificeren in een muismodel van ruggenmergletsel  

Asymmetric Walkway: A Novel Behavioral Assay for Studying Asymmetric Locomotion

Behavioral assays are commonly used for the assessment of sensorimotor impairment in the central nervous system (CNS). The most sophisticated methods for quantifying locomotor deficits in rodents is to measure minute disturbances of unconstrained gait overground (e.g., manual BBB score or automated CatWalk). However, cortical inputs are not required for the generation of basic locomotion produced by the spinal central pattern generator (CPG). Thus, unconstrained walking tasks test locomotor deficits due to motor cortical impairment only indirectly. In this study, we propose a novel, precise foot-placement locomotor task that evaluates cortical inputs to the spinal CPG. An instrumented peg-way was used to impose symmetrical and asymmetrical locomotor tasks mimicking lateralized movement deficits. We demonstrate that shifts from equidistant inter-stride lengths of 20% produce changes in the forelimb stance phase characteristics during locomotion with preferred stride length. Furthermore, we propose that the asymmetric walkway allows for measurements of behavioral outcomes produced by cortical control signals. These measures are relevant for the assessment of impairment after cortical damage.

Here, we present a protocol to quantify precise stepping in rodents. Cortical and the spinal central pattern generator signals are required for precise foot-placement during obstructed locomotion. We report here the novel constrained walking task that directly examines precise stepping behavior.

Behavioral assays are commonly used for the assessment of sensorimotor impairment in the central nervous system (CNS). The most sophisticated methods for ...

Behavior

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation in mouse models is a non-invasive and reproducible activity assay that is directly analogous to measures of patient ambulation such as the 6-minute walk test and related mobility scores. Many common methods for testing mouse ambulation speed and distance are based on the open field test, where an animal&#39;s free movement within an arena is measured over time. One major downside to this approach is that commercial software and equipment for high-resolution motion tracking is expensive and may require transferring mice to specialized facilities for testing. Here, we describe a low-cost, video-based system for measuring mouse ambulation that utilizes free and open-source software. Using this protocol, we demonstrate that voluntary ambulation in the dystrophin-null mdx mouse model for Duchenne muscular dystrophy (DMD) is decreased relative to wild-type mouse activity. In mdx mice expressing the utrophin transgene, these activity deficits are not observed and the total distance traveled is indistinguishable from wild-type mice. This method is effective for measuring changes in voluntary ambulation associated with dystrophic pathology, and provides a versatile platform that can be readily adapted to diverse research settings.

This protocol describes a flexible, low-cost system for measuring mouse ambulation in an open field activity assay. We show that a 6-minute ambulation assay based on this system detects a decrease in voluntary movement in mdx mice, and accurately distinguishes improvement in a muscle-specific rescue of these animals.

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation ...

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

Biology

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 ...

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 ...

Sagittal Plane Kinematic Gait Analysis in C57BL/6 Mice Subjected to MOG35-55 Induced Experimental Autoimmune Encephalomyelitis

Kinematic gait analysis in the sagittal plane has frequently been used to characterize motor deficits in multiple sclerosis (MS). We describe the application of these techniques to identify gait deficits in a mouse model of MS, known as experimental autoimmune encephalomyelitis (EAE). Paralysis and motor deficits in mice subjected to EAE are typically assessed using a clinical scoring scale. However, this scale yields only ordinal data that provides little information about the precise nature of the motor deficits. EAE disease severity has also been assessed by rotarod performance, which provides a measure of general motor coordination. By contrast, kinematic gait analysis of the hind limb in the sagittal plane generates highly precise information about how movement is impaired. To perform this procedure, reflective markers are placed on a hind limb to detect joint movement while a mouse is walking on a treadmill. Motion analysis software is used to measure movement of the markers during walking. Kinematic gait parameters are then derived from the resultant data. We show how these gait parameters can be used to quantify impaired movements of the hip, knee, and ankle joints in EAE. These techniques may be used to better understand disease mechanisms and identify potential treatments for MS and other neurodegenerative disorders that impair mobility.

Kinematic gait analysis in the sagittal plane yields highly precise information about how movement is executed. We describe the application of these techniques to identify gait deficits for mice subjected to autoimmune-mediated demyelination. These methods may also be used to characterize gait deficits for other mouse models featuring impaired locomotion.

Kinematic gait analysis in the sagittal plane has frequently been used to characterize motor deficits in multiple sclerosis (MS). We describe the ...

Thoracic Spinal Cord Hemisection Surgery and Open-Field Locomotor Assessment in the Rat

Spinal cord injury (SCI) causes disturbances in motor, sensory, and autonomic function below the level of the lesion. Experimental animal models are valuable tools to understand the neural mechanisms involved in locomotor recovery after SCI and to design therapies for clinical populations. There are several experimental SCI models including contusion, compression, and transection injuries that are used in a wide variety of species. A hemisection involves the unilateral transection of the spinal cord and disrupts all ascending and descending tracts on one side only. Spinal hemisection produces a highly selective and reproducible injury in comparison to contusion or compression techniques that is useful for investigating neural plasticity in spared and damaged pathways associated with functional recovery. We present a detailed step-by-step protocol for performing a thoracic hemisection at the T8 vertebral level in the rat that results in an initial paralysis of the hindlimb on the side of the lesion with graded spontaneous recovery of locomotor function over several weeks. We also provide a locomotor scoring protocol to assess functional recovery in the open-field. The locomotor assessment provides a linear recovery profile and can be performed both early and repeatedly after injury in order to accurately screen animals for appropriate time points in which to conduct more specialized behavioral testing. The hemisection technique presented can be readily adapted to other transection models and species, and the locomotor assessment can be used in a variety of SCI and other injury models to score locomotor function.

The rat thoracic spinal hemisection is a valuable and reproducible model of unilateral spinal cord injury to investigate the neural mechanisms of locomotor recovery and treatment efficacy. This article includes a detailed step-by-step guide to perform the hemisection procedure and to assess locomotor performance in an open-field arena.

Spinal cord injury (SCI) causes disturbances in motor, sensory, and autonomic function below the level of the lesion. Experimental animal models are ...

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 ...

A Spinal Cord Injury Contusion Device for Accurate Mouse Models

Automated Impactor for Contusive Spinal Cord Injury Model in Mice

Spinal cord injury (SCI) due to traumatic injuries such as car accidents and falls is associated with permanent spinal cord dysfunction. Creation of contusion models of spinal cord injury by impacting the spinal cord results in similar pathologies to most spinal cord injuries in clinical practice. Accurate, reproducible, and convenient animal models of spinal cord injury are essential for studying spinal cord injury. We present a novel automated spinal cord injury contusion device for mice, the Guangzhou Jinan University smart spinal cord injury system, that can produce spinal cord injury contusion models with accuracy, reproducibility, and convenience. The system accurately produces models of varying degrees of spinal cord injury via laser distance sensors combined with an automated mobile platform and advanced software. We used this system to create three levels of spinal cord injury mice models, determined their Basso mouse scale (BMS) scores, and performed behavioral as well as staining assays to demonstrate its accuracy and reproducibility. We show each step of the development of the injury models using this device, forming a standardized procedure. This method produces reproducible spinal cord injury contusion mice models and reduces human manipulation factors via convenient handling procedures. The developed animal model is reliable for studying spinal cord injury mechanisms and associated treatment approaches.

Author Spotlight: Insight Into Innovations in Spinal Cord Injury Research

Presented here is a novel automated spinal cord injury contusion device for mice, which can accurately produce spinal cord injury contusion models with varying degrees.

Spinal cord injury (SCI) due to traumatic injuries such as car accidents and falls is associated with permanent spinal cord dysfunction. Creation of ...

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

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