This work was supported by the Canadian Institutes for Health Research (CIHR; MOP-142288) to M.M. M.M. was supported by a salary award from Fonds de Recherche Qu&#233;bec Sant&#233; (FRQS), and A.R.B was supported by a fellowship from FRQS.

The experiments described in this article were performed in compliance with the guidelines of the Canadian Council on Animal Care and were approved by the ethics committee at the Universit&#233; de Montr&#233;al.
1. Thoracic hemisection surgery
<ol>
	<li>Wear appropriate protective equipment (gloves, mask, and gown) to maintain an aseptic environment for surgery. Clean the surgical area with alcohol wipes, and place sterile surgical drapes over the surgical field. Sterilize surgical tools an...

A major strength of the hemisection technique is the selectivity and reproducibility of the lesion which leads to reduced variability in histological and behavioral phenotypes between animals25. In order to ensure a unilateral lesion at the appropriate spinal level, accurate identification of both the proper vertebral segment and spinal cord midline is critical. As there can be a tendency for the spinal cord to rotate in the direction of the cut during the hemisection procedure, it can be benefici...

Spinal cord injury (SCI) is associated with severe disturbances in motor, sensory, and autonomic function. Experimental animal models of SCI are valuable tools to understand the anatomical and physiological events involved in SCI pathology, to investigate the neural mechanisms in repair and recovery, and to screen for efficacy and safety of potential therapeutic interventions. The rat is the most commonly used species in SCI research1. Rat models are low cost, easy to reproduce, and a large battery of behavioral tests are available to assess functional outcomes2. Despite some differences in tract locations, the rat spinal cord shares overall similar sensorimotor functions with larger mammals, including primates3,4. Rats also share analogous physiological and behavioral consequences to SCI that relate to humans5. Non-human primate and large animal models can provide a closer approximation of human SCI6 and are essential to prove treatment safety and efficacy prior to human experimentation, but are less commonly used due to ethical and animal welfare considerations, expenses, and regulatory requirements7.
Rat transection SCI models are performed by the targeted interruption of the spinal cord with a selective lesion using a dissection knife or iridectomy scissors after a laminectomy. Compared to a complete transection, partial transection in the rat results in a less severe injury, easier postoperative animal care, spontaneous locomotor recovery, and more closely models SCI in humans which is predominately incomplete with partial sparing of tissue connecting the spinal cord and supraspinal structures8. A unilateral hemisection disrupts all ascending and descending tracts on one side only, and produces quantifiable and highly reproducible locomotor deficits, enhancing exploration of the underlying biological mechanisms. The most prominent functional consequence of the hemisection is an initial limb paralysis on the same side and below the level of the lesion with graded spontaneous recovery of locomotor function over several weeks9,10,11,12. The hemisection model is particularly useful to investigate neural plasticity of damaged and residual tracts and circuits associated with functional recovery9,11,12,13,14,15,16,17,18. Specifically, hemisection performed at the thoracic level, i.e., above the spinal circuits that control hindlimb locomotion, is particularly useful for investigating changes in locomotor control. As a non-linear relationship exists between lesion severity and locomotor recovery after SCI19, appropriate behavioral testing to assess functional outcomes is paramount in experimental models.
A comprehensive battery of behavioral tests are available to assess specific aspects of functional locomotor recovery in the rat2,20. Many locomotor tests do not provide reliable measures early after SCI as rats are too disabled to support their body weight. A measure of spontaneous locomotor performance that is sensitive to deficits early after injury, and does not require preoperative training or specialized equipment, is beneficial in order to monitor locomotor recovery for appropriate time points in which to supplement specialized behavioral testing. The Martinez open-field assessment score10, originally developed for evaluating locomotor performance after cervical SCI in the rat, is a 20-point ordinal score assessing global locomotor performance during spontaneous overground locomotion in an open-field. Scoring is conducted separately for each limb using a rubric that evaluates specific parameters of a range of locomotor measures including articular limb movement, weight support, digit position, stepping abilities, forelimb-hindlimb coordination, and tail position. The assessment score is derived from the Basso, Beattie and Bresnahan (BBB) open-field rating scale designed to evaluate locomotor performance after thoracic contusion21. It is adapted to accurately and reliably evaluate both forelimb and hindlimb locomotor function, allows for independent assessment of the different scoring parameters that is not amenable with the hierarchical scoring of the BBB, and provides a linear recovery profile10. Additionally, in comparison to the BBB, the assessment score is sensitive and reliable in more severe injury models10,11,20,22. The assessment score has been used to assess locomotor impairment in the rat following cervical10,12 and thoracic9 SCI alone and in combination with traumatic brain injury23.
We present here a detailed step-by-step protocol for performing a thoracic hemisection SCI at the T8 vertebral level in the female Long-Evans rat, and for assessing hindlimb locomotor recovery in the open-field.

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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.

Reproducible lesions with a high degree of consistency can be generated with the hemisection technique. To assess and compare lesions sizes between experimental groups, the maximal area of the lesion as a percentage of the total cross-section of the spinal cord can be readily calculated with histological staining of spinal cord sections. Figure 1 shows a representative lesion of the left hemicord and an overlay of the proportion of maximal lesion area shared between rats with a mean lesion s...

Watch this Scientific Journal Video about Thoracic Spinal Cord Hemisection Surgery and Open-Field Locomotor Assessment in the Rat at JoVE.com

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

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 ...

Spinal hemisection produces quantifiable and ideal reproducible locomotor deficits that can be captured with the locomotive scale that we have developed. The main advantage of this technique is the selectivity and reproducibility of the lesion, which leads to a reduced variability in behavioral phenotypes between animals. After confirming a lack of response to toe pinch in an anesthetized adult rat, make a 2.5 centimeter incision in the shaved skin overlying the T-6 to T-10 vertebrae and use blunt scissors to retract the skin and superficial fat.Use blunt dissection scissors and a self-retaining retractor to separate the paravertebral muscles, inserting on the dorsal aspect of the T-7 to T-9 vertebrae and use fine forceps and cotton-tipped applicators to debride and clear any remaining tissue to expose the spinous processes and vertebral lamina. Place the rate under a stereo microscope and use delicate bone trimmers to carefully cut the facets bilaterally on the T-7 and T-8 vertebrae. Use a scalpel to make a one-millimeter superficial cut in the dorsal connective tissue between the T-8 and T-9 vertebral laminae, taking care not to injure the underlying cord and use bone trimmers to remove the spinous process of the T-8 vertebra.With curved hemostatic forceps carefully clamped on the T-7 spinous process, rotate the caudal end of the T-8 laminae slightly rostrally approximately 20 degrees and insert the bone trimmers under the T-8 lamina. Make a midline cut extending along the lamina, continuing the laminectomy by repeating the cuts on the left and right side of the vertebral lamina medial to the transverse processes to expose the spinal cord. Drip 100 microliters of 2%lidocaine into the exposed spinal canal and use fine forceps and iridectomy scissors to remove the dura overlaying the T-8 spinal segment.Repeat the lidocaine administration to the exposed cord and identify the midline of the cord by visualization of a center line created between the spinous processes extending between the exposed T-7 to T-9 vertebra. Using fine forceps to stabilize the spinal cord, use a dissecting knife to hemisect the spinal cord from the midline toward one side of the animal, taking care not to cut through the anterior spinal artery on the ventral side. Using iridectomy scissors, carefully cut through any remaining tissue on the lesion side of the spinal cord to ensure the ventral-lateral quadrant is appropriately transected and place a sterile, approximately six by two millimeter saline-soaked hemostatic sponge into the exposed cavity above the spinal cord.Then, use 4-O polyglactin 910 sutures to close muscle layers and the skin around the incision site and place the rat in a warm environment under a heat lamp with monitoring until a full recovery. At the appropriate experimental time point for behavioral testing, after the rats have been habituated to the arena, begin the video recording and place a rat in the center of the arena, under dim light conditions to encourage locomotor activity. Allow the rat to explore for at least four minutes, moving the rat back to the center of the arena when it remains stationary for longer than 20 seconds to promote locomotion.Then, score the locomotor performance of the recorded testing session. For articular limb movements, score the hindlimb joint movements during spontaneous locomotion separately for the ankle, knee, and hip, as either normal, slight, or absent. For weight support, evaluate the ability of the hindlimb extensor muscles to contract and support the loaded body weight when the limb is on the ground for when the rat is stationary, as well as during active locomotion.For the digit position, evaluate the position of the hindlimb digits while the rat is stationary and during locomotion. Complete the stepping parameter only if the rat can support its body weight during stepping by rating the orientation of the hindlimb paw placement at the time of the initial contact and at lift off from the ground in addition to the fluidity of the swing phase during stepping. Complete the forelimb/hindlimb coordination parameter only if four consecutive steps occur during testing and if the limbs can actively support body weight.Evaluate the tail position during locomotion as either up or down. Then add the individual scores from each parameter to provide a total for each hindlimb to a maximum of 20 points. To assess and compare lesion sizes between experimental groups, the maximal area of the lesion as a percentage of the total cross-section of the spinal cord can be readily calculated with histological staining of the spinal cord sections.For example, this representative lesion of the left hemicord with an overlay of the proportion of maximal lesion area shared between rats demonstrated the mean lesion size of approximately 47%of the cross-sectional cord area. These representative changes in locomotor performance in the intact state over the first five weeks after left side hemisection demonstrate a significant impairment of locomotion in the left hindlimb of the animal during the first three weeks after the surgery. An improvement to the level of locomotion is observed in the right hindlimb by two weeks post-procedure.The most important things to remember are that an accurate identification of spinal midline for the hemisection and proper post-surgical animal care and monitoring are essential for a successful protocol. This behavioral assessment provides an ideal protocol to screen for upper paretrecorvial indices for supplementing with specialized testing to address specific question of interest on locomotor recovery.

thoracic-spinal-cord-hemisection-surgery-open-field-locomotor

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JoVE Journal

Neuroscience

9.3K Views.  Université de Montréal. Spinal hemisection produces quantifiable and ideal reproducible locomotor deficits that can be captured with the locomotive scale that we have developed. The main advantage of this technique is the selectivity and reproducibility of the lesion, which leads to a reduced variability in behavioral phenotypes between animals. After confirming a lack of response to toe pinch in an anesthetized adult rat, make a 2.5 centimeter incision in the shaved skin overlying the T-6 to T-10 vertebrae and use ...

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

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...

a minimally invasive, fast spinal cord lateral hemisection technique for modeling open spinal cord injuries in rats

A Minimally Invasive, Fast Spinal Cord Lateral Hemisection Technique for Modeling Open Spinal Cord Injuries in Rats

Here, we describe a new, fast technique modeling open spinal cord injury in rats that eliminates laminectomy. Lateral hemisection is performed while viewing through a microscope. The technique is versatile and can also be used in the cervical, thoracic, and lumbar regions of the spinal cord of other...

spinal cord lateral hemisection and asymmetric behavioral assessments in adult rats

Spinal Cord Lateral Hemisection and Asymmetric Behavioral Assessments in Adult Rats

Here we describe surgical procedures to produce a reliable spinal cord lateral hemisection (HX) at the 9th thoracic level in adult rats and neurobehavioral assessments designed for detecting asymmetric deficits after such a unilateral...

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

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

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

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Diffusion Imaging in the Rat Cervical Spinal Cord

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Author Spotlight: Neuromotor Control and Recovery of Diaphragm Function Following Cervical Spinal Hemisection in Rats

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Assessing Functional Recovery of Eupneic Diaphragm Activity Following Unilateral Cervical Spinal Cord Hemisection in Rats

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Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury

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Standardized Evaluation of DIAm EMG Post-C2SH in Rats to Minimize Variability

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combining peripheral nerve grafting and matrix modulation to repair the injured rat spinal cord

Combining Peripheral Nerve Grafting and Matrix Modulation to Repair the Injured Rat Spinal Cord

Traumatic injury to the spinal cord disrupts communication with the brain. To restore lost connectivity we utilize a peripheral nerve graft to provide a substratum for regenerating fibers in combination with neurotrophic factors and matrix-modulating enzymes to remove inhibitory molecules to promote long distance...

using laser doppler imaging and monitoring to analyze spinal cord microcirculation in rat

Using Laser Doppler Imaging and Monitoring to Analyze Spinal Cord Microcirculation in Rat

Here we present a combination of laser Doppler perfusion imaging (LDPI) and laser Doppler perfusion monitoring (LDPM) to measure spinal cord local blood flows and oxygen saturation (SO2), as well as a standardized procedure for introducing spinal cord trauma on...

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Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion

The lack of mechanistic understanding of spinal cord ischemia-reperfusion injury has hindered further adjuncts to prevent paraplegia following high risk aortic operations. Thus, the development of animal models is imperative. This manuscript demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion in a murine model.

a neuronal apoptosis model induced by spinal cord compression in rat

A Neuronal Apoptosis Model induced by Spinal Cord Compression in Rat

Here, we present a protocol to generate a rat spinal cord compression model, assess its behavioral score, and observe the compressed spinal cord region. The behavioral assessments showed decreased monitor motor disability. Hematoxylin and eosin staining and immunostaining revealed considerable neuronal apoptosis in the compressed region of the spinal...

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Spinal Cord Transection in the Larval Zebrafish

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days...

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Disruption of the Blood-Spinal Cord Barrier Using Low-Intensity Focused Ultrasound in a Rat Model

Disruption of the blood-spinal cord barrier (BSCB) can be successfully achieved with the intravenous administration of microbubbles and the application of low-intensity focused ultrasound (LIFU). This protocol details the opening of the BSCB using LIFU in a rodent model, including equipment setup, microbubble injection, target localization, and BSCB disruption visualization.

direct injection of a lentiviral vector highlights multiple motor pathways in the rat spinal cord

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

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Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

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 ...

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a diagnostic and prognostic tool in cases of disease or injury. Diffusion weighted imaging (DWI), is sensitive to the thermal motion of water molecules and allows for inferences of tissue microstructure. This report describes a protocol to acquire and analyze DWI of the rat cervical spinal cord on a small-bore animal system. It demonstrates an imaging setup for the live anesthetized animal and recommends a DWI acquisition protocol for high-quality imaging, which includes stabilization of the cord and control of respiratory motion. Measurements with diffusion weighting along different directions and magnitudes (b-values) are used. Finally, several mathematical models of the resulting signal are used to derive maps of the diffusion processes within the spinal cord tissue that provide insight into the normal cord and can be used to monitor injury or disease processes noninvasively.

The goal of this protocol is to obtain high-quality diffusion weighted magnetic resonance imaging (DWI) of the rat spinal cord for noninvasive characterization of tissue microstructure. This protocol describes optimizations of the MRI sequence, radiofrequency coil, and analysis methods to enable DWI images free from artifacts.

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

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 ...

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 ...

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

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 ...