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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W., Gautier, M., Sofroniew, M. V., Courtine, G., Anderson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+spinal+cord+repair.">Engineering spinal cord repair.</a> Current Opinion in Biotechnology. 72, 48-53 (2021).</li><li>Wenger, N., 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=Spatiotemporal+neuromodulation+therapies+engaging+muscle+synergies+improve+motor+control+after+spinal+cord+injury.">Spatiotemporal neuromodulation therapies engaging muscle synergies improve motor control after spinal cord injury.</a> Nature Medicine. 22 (2), 138-145 (2016).</li><li>Grillner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spinal+locomotor+CPG:+A+target+after+spinal+cord+injury.">The spinal locomotor CPG: A target after spinal cord injury.</a> Progress in Brain Research. 137, 97-108 (2002).</li><li>Boulain, M., 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=Synergistic+interaction+between+sensory+inputs+and+propriospinal+signalling+underlying+quadrupedal+locomotion.">Synergistic interaction between sensory inputs and propriospinal signalling underlying quadrupedal locomotion.</a> Journal of Physiology. 599 (19), 4477-4496 (2021).</li><li>Skarlatou, 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=Afadin+signaling+at+the+spinal+neuroepithelium+regulates+central+canal+formation+and+gait+selection.">Afadin signaling at the spinal neuroepithelium regulates central canal formation and gait selection.</a> Cell Reports. 31 (10), 107741 (2020).</li><li>Viala, D., Vidal, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+distinct+spinal+locomotion+generators+supplying+respectively+fore-and+hindlimbs+in+the+rabbit.">Evidence for distinct spinal locomotion generators supplying respectively fore-and hindlimbs in the rabbit.</a> Brain Research. 155 (1), 182-186 (1978).</li><li>Silver, J., Miller, J. 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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 hi...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>45&#186; Mirror&#160;</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&#176; , 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&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>black cardboard filler&#160;</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>Background backlight</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>fTIR Support base and posts</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&#160;</td>
			<td>#54-641</td>
			<td></td>
		</tr>
		<tr>
			<td>M6 12 mm screws</td>
			<td>Edmund Optics&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>M6 hex nuts and wahers</td>
			<td>Edmund Optics&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>fTIR Walkway&#160;</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&#160;</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>12 mm screws</td>
			<td>Edmund Optics&#160;</td>
			<td>M6</td>
			<td></td>
		</tr>
		<tr>
			<td>High speed camera (on a tripod)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blackfly S USB3</td>
			<td>Blackfly</td>
			<td>USB3</td>
			<td>This is a reccomendation. The requirement is to record at least 100 frames per second</td>
		</tr>
		<tr>
			<td>Infinite Horizon Impactor&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Infinite Horizon Impactor&#160;</td>
			<td>Precision Systems and Instrumentation, LLC.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lens</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikkon AF Zoom-Nikkor 24-85mm</td>
			<td>Nikkon&#160;</td>
			<td>2.8-4D IF</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>Software</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&#160;</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&#160;</td>
			<td>Spyder Project Contributors</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Walkway wall&#160;</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>GitHub Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Folder name</td>
			<td>URL</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.

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...

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

Research

JoVE Journal

Neuroscience

2.5K Views.  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.

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

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 ...

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site &#8212; demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) on a treadmill is widely used as a rehabilitation tool in the SCI population with many benefits and improvements to daily life. We utilize this method of activity-based task-specific training (ABT) in rodents after SCI to both elucidate the mechanisms behind such improvements and to enhance and improve upon existing clinical rehabilitation protocols. Our current goal is to determine the mechanisms underlying ABT-induced improvements in urinary, bowel, and sexual function in SCI rats after a moderate to severe level of contusion. After securing each individual animal in a custom-made adjustable vest, they are secured to a versatile body weight support mechanism, lowered to a modified three-lane treadmill and assisted in step-training for 58 minutes, once a day for 10 weeks. This setup allows for the training of both quadrupedal and forelimb-only animals, alongside two different non-trained groups. Quadrupedal-trained animals with body weight support are aided by a technician present to assist in stepping with proper hind limb placement as necessary, while forelimb-only trained animals are raised at the caudal end to ensure no hind limb contact with the treadmill and no weight-bearing. One non-trained SCI group of animals is placed in a harness and rests next to the treadmill, while the other control SCI group remains in its home cage in the training room nearby. This paradigm allows for the training of multiple SCI animals at once, thus making it more time-efficient in addition to ensuring that our pre-clinical animal model mimics the clinical representation as close as possible, particularly with respect to the body weight support with manual assistance.

This protocol demonstrates our model of activity-based locomotor treadmill training for rats with spinal cord injury (SCI). Included is both quadrupedal and forelimb-only groups, in addition to two distinct types of non-trained control groups. Investigators are able to assess training effects on SCI rats using this protocol.

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) ...

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 ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...