Samuel L. Pfaff é professor no Laboratório de Expressão Gênica do Instituto Salk para Estudos Biológicos e um investigador no Instituto Médico Howard Hughes. Este trabalho foi financiado pelo Christopher Reeve e Dana Foundation. Joe Belcovson, Kent Schnoeker e Mike Sullivan em Recursos Multimídia do Instituto Salk prestou assistência com a fotografia e edição.

Gravações eletrofisiológicas de isolados medula espinhal pode revelar mudanças genéticas e de desenvolvimento de um circuito neural. Nós já demonstraram um método para dissecar os cabos do mouse neonatal da coluna vertebral 2. Aqui apresentamos um método para preparar eletrodos de sucção útil na gravação de locomoção fictícia na medula espinhal isolado 3. A ponta de um eletrodo de tubos de plástico podem ser atraídos para uma ponta muito fina à mão usando uma lâmpada de álcool a baixas temperaturas 3-5. O tubo de plástico (PE90, Clay Adams Intramedic TM) é realizada sobre a chama para suavizar o tubo em uma forma maleável. Como o tubo começa a derreter e se torna mais transparente ele é removido da fonte de calor e as extremidades são delicadamente separados. O momento de retirar o tubo da chama é importante para garantir o plástico não colapso ou dividir, enquanto sendo desenhado. A porção fina do tubo é cortado com uma lâmina de barbear de acordo com o diâmetro desejado interna, como ditado pela idade do espécime ou o nível segmentar da medula. Para facilitar a colocação de um o-ring no tubo, um corte de 45 ° é feita no final de espessura da tubulação com uma lâmina de barbear. A ponteira e o-ring são instalados na extremidade grossa para prender a ponta do eletrodo para o barril do eletrodo. Para permitir especificamente eletrodos de tamanho e evitar as dificuldades de mão-de puxar dicas eletrodo, eletrodos podem ser construídas usando tubos de pequeno inserido em um grosso tubo (PE90). Os comprimentos dos tubos são específicos para os requisitos da configuração de gravação e são variáveis. Nós apresentamos as medidas utilizadas em nossos experimentos 1-3,5. Um comprimento 1cm de PTFE tubo fino (Zeus, peças pequenas) e um comprimento de 10 cm de tubos PE90 são cortados com uma lâmina de barbear. As extremidades do tubo de pequeno porte devem ser examinados para ter certeza de que eles não são esmagados ou fechada. Eles podem ser abertos com um pino de insetos (Ferramentas Ciência Fine). Uma gota de adesivo (JB Weld) é colocado no ponto médio do tubo de PTFE. A tubagem de PTFE e adesivo são atraídos para a tubulação de sucção PE90 usando boca no tubo PE90. Um corte de 45 ° é feita no final de espessura da tubulação e uma ponteira e o-ring são fixos, como anterior. O conector BNC e ponteira são desaparafusado a partir de um eletrodo de sucção comerciais. O conector BNC é embebido em xilol por uma hora para remover o lacre de cera. O conector é então lavado com água e seca. Uma porta lateral inferior em ângulo deve ser feita no lado do barril. Uma haste de metal é aquecido através de um bico de Bunsen e pressionado para dentro do buraco existente na lateral do barril em um ângulo baixo. Um comprimento 15 centímetros de fio de prata 0,010 polegadas (AM Systems) é cortado e embebido em lixívia para 15 minutos. Isto é feito para criar um revestimento de cloreto de prata que ajuda na condução de sinal 6. O fio é lavado com água, secos, e então soldada no conector BNC. O fio conector BNC e prata são inseridos no barril eletrodo de sucção com espaço suficiente para o conector BNC para permanecer ao lado do barril. Tubo de sucção é inserido na porta lateral 2-3cm. Selante de silicone é injetado dentro do cano de aproximadamente 2-3cm. O conector BNC é enfiada para o barril do eletrodo empurrando o selante para a frente. A ponta do eletrodo está agora inserido no front end do eletrodo. Tubulação do psiquiatra também pode ser adicionado para reforçar a linha de sucção da porta lateral. Eletrodos são montados em micromanipuladores que estão ligados ao descanso magnético, e colocado perto do prato de gravação. BNC cabos coaxiais são conectados a um headstage que é ligado a um amplificador. O amplificador é conectado a um adaptador (PolyView) que é conectado a uma placa de analógico para digital (National Instruments) em um computador PC com software de aquisição de dados (PolyView). Ao gravar dados eletrofisiológicos, é necessário eliminar todas as interferências externas. O prato de gravação, amplificador, e palco de cabeça são colocados em uma mesa de ar dentro de uma gaiola de Faraday para evitar interferências elétricas e mecânicas, respectivamente. Resultados representativos: <img src="/files/ftp_upload/2580/2580fig1.jpg" alt="Figura 1" /> Figura 1. Um eletrodo de sucção comercialmente disponíveis podem ser modificadas pela adição de um fio de prata calibre mais pesado e uma ponta de plástico que é desenhado à mão sobre uma lâmpada de álcool, ou construídas com tubos de plástico disponível comercialmente de tamanhos específicos. Além disso, o selante de parafina é substituída com mais duráveis ​​selante de silicone calafetar. <img src="/files/ftp_upload/2580/2580fig2.jpg" alt="Figura 2" /> Figura 2. Um esquema do eletrodo de sucção em relação à mesa de ar (para isolamento mecânico), gaiola de Faraday (elétrica isolation), amplificador e computador. Deve-se notar que há um terreno referenciado para o prato de gravação e um segundo motivo que é referenciado para a mesa de ar e gaiola de Faraday.

<ol>
	<li>Gallarda, B. W., Sharpee, T. O., Pfaff, S. L., Alaynick, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+rhythmic+locomotor+burst+patterns+using+a+continuous+wavelet+transform.">Defining rhythmic locomotor burst patterns using a continuous wavelet transform.</a> Ann N Y Acad Sci. 1198, 133-139 (2010).</li><li>Meyer, A., Gallarda, B. W., Pfaff, S., Alaynick, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+electrophysiology.">Spinal cord electrophysiology.</a> J Vis Exp. , (2010).</li><li>Gallarda, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+of+axial+motor+and+sensory+pathways+via+heterotypic+trans-axonal+signaling.">Segregation of axial motor and sensory pathways via heterotypic trans-axonal signaling.</a> Science. 320, 233-236 (2008).</li><li>Landmesser, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+motor+projection+patterns+in+the+chick+hind+limb.">The development of motor projection patterns in the chick hind limb.</a> J Physiol. 284, 391-414 (1978).</li><li>Myers, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+input+is+required+during+embryonic+development+to+mediate+proper+assembly+of+spinal+locomotor+circuits.">Cholinergic input is required during embryonic development to mediate proper assembly of spinal locomotor circuits.</a> Neuron. 46, 37-49 (2005).</li><li>Chanin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+determination+of+chloride+by+use+of+the+silver-silver+chloride+electrode.">The determination of chloride by use of the silver-silver chloride electrode.</a> Science. 119, 323-324 (1954).</li><li>Goulding, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circuits+controlling+vertebrate+locomotion:+moving+in+a+new+direction.">Circuits controlling vertebrate locomotion: moving in a new direction.</a> Nat Rev Neurosci. 10, 507-518 (2009).</li></ol>

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Desenvolvimento do sistema nervoso podem ser estudadas usando cabos isolados de roedores espinhal. Na presença de neurotransmissores, locomoção fictícia pode ser gerada a partir da medula espinhal na forma de padrões de atividade elétrica 1,3. Estas explosões rítmicas são produzidos em 0,2-0,5 Hz e são estampados em alternâncias esquerda-direita e flexores e extensores. Em diferentes estágios de desenvolvimento, a robustez e os padrões dessa atividade varia 1. Mutações genéticas tam...

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<td>PTFE Sub Lite Wall Tubing (Small tubing)</td>
<td>&nbsp;</td>
<td>Zeus</td>
<td>36AWG</td>
<td>0.005&#x201D;ID x 0.003&#x201D; Wall (Small Parts) Also available in 0.003&#x201D; to 0.006&#x201D;</td>
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<td>Large tubing (0.86mm (0.34&#x201D;))</td>
<td>&nbsp;</td>
<td>Clay Adams Brand Intramedic Becton Dickinson and Company</td>
<td>427420</td>
<td>0.86mm (0.34&#x201D;) O.D. 1.27mm (.050&#x201D;)</td>
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<td>Electrode Barrel</td>
<td>&nbsp;</td>
<td>A-M Systems</td>
<td>573000</td>
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<td>Adhesive</td>
<td>&nbsp;</td>
<td>JB Weld</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Adhesive: Silicone caulk</td>
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<td>Xylene</td>
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<td>Insect pins: Austerlitz 0.1mm</td>
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<td>Fine Science Tools</td>
<td>26002-10</td>
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<td>Magnetic Stand</td>
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<td>Narishige</td>
<td>GJ-8</td>
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<td>Micromanipulator</td>
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<td>Polyview Adaptor Unit</td>
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<td>Bipolar Portable Physiodata Amplifier System</td>
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<td>Grass Industries</td>
<td>15LT</td>
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<td>ANALOG TO DIGITAL CARD</td>
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<td>National Instruments</td>
<td>6035E</td>
<td>&nbsp;</td>
<tr>
<td>Air Table; Vibraplane</td>
<td>&nbsp;</td>
<td>Kinetic Systems</td>
<td>&nbsp;</td>
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spinal cord electrophysiology ii: extracellular suction electrode fabrication

Desenvolvimento de circuitos neurais e locomoção podem ser estudadas usando neonatal da medula espinhal de roedores centro gerador de padrão de comportamento (CPG). Demonstramos um método para fabricar eletrodos de sucção que são usados ​​para examinar a atividade CPG, ou locomoção fictícia, em cabos de roedores dissecados espinhal. Os cabos de roedor da coluna vertebral são colocados no líquido cefalorraquidiano artificial e as raízes ventral são atraídos para o eletrodo de sucção. O eletrodo é construído através da modificação de um eletrodo de sucção disponível comercialmente. Um fio mais pesado de prata é usado em vez do fio padrão dado pelo eletrodo disponíveis comercialmente. A ponta do eletrodo de vidro em comercial é substituído com uma ponta de plástico para maior durabilidade. Nós preparamos mão eletrodos desenhado e eletrodos feitos de tamanhos específicos de tubos, permitindo a consistência e reprodutibilidade. Os dados são coletados por meio de um amplificador e software de aquisição neurograma. As gravações são realizadas em uma mesa de ar dentro de uma gaiola de Faraday para evitar interferências mecânicas e elétricas, respectivamente.

Watch this Scientific Journal Video about Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication at JoVE.com

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

A demonstração da fabricação e uso de um eletrodo de sucção extracelular usado para medir gravações eletrofisiológicas de cordas de roedores neonatal espinhal<em&gt; In vitro</em

Spinal Cord Eletrofisiologia II: Fabricação Eletrodo de Sucção Extracelular

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

spinal-cord-electrophysiology-ii-extracellular-suction-electrode

Research

JoVE Journal

Neuroscience

15.0K visualizações.  The Salk Institute for Biological Studies. A demonstração da fabricação e uso de um eletrodo de sucção extracelular usado para medir gravações eletrofisiológicas de cordas de roedores neonatal espinhal In vitro

Vídeo: Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

Embrião de galinha Spinal Cord Protocolo Cultura Slice

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Neurociência

Spinal Cord Transection in the Larval Zebrafish

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.

Transecção medular nos larval Zebrafish

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.

Um<em&gt; Ex Vivo</em&gt; Induzida por laser Lesão Medular modelo para avaliar mecanismos de axonal Degeneration em tempo real

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 ...

Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to stimuli remain to be fully understood. To overcome the difficulty of accessing the brainstem in the whole animal, isolation of the brainstem and part of the spinal cord is performed. This preparation is maintained in artificial cerebro-spinal fluid where gases, concentrations, and temperature are controlled and monitored. The output signal from the respiratory network is recorded by a suction electrode placed on the fourth ventral root. In this manner, stimuli can be directly applied onto the brainstem, and the effect can be recorded directly. The signal recorded is linked to the inspiratory signal sent to the diaphragm via the phrenic nerve, and can be described as bursts (around 8 bursts per minute). Analysis of these bursts (frequency, amplitude, length, and area under the curve) allows precise characterization of the stimulus effect on the respiratory network. The main limitation of this method is the viability of the preparation beyond the early post-natal stages. Thus, this method greatly focuses on the study of the whole network without the peripheral inputs in the newborn rat.

The central respiratory drive is located in the brainstem. Spontaneous respiratory motor output from an isolated brainstem-spinal cord is recorded by placing an electrode on the fourth ventral root. This experimental approach is valuable for pharmacological investigations or the assessment of respiratory challenges and genetic manipulations on rhythmic motor behavior.

Eletrofisiologia no isolado do tronco encefálico-espinal Preparações do cordão umbilical de recém-nascidos Roedores permite a gravação de saída Neural Rede Respiratory

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

Investigando regeneração funcional em organotï¿½icas Medula Espinhal Co-culturas cultivadas em Arrays Multi-eletrodo

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in the brain stem. Loss of hair cells is a major cause of sensory hearing loss. Prosthetic devices such as cochlear implants function by bypassing lost hair cells and directly stimulating SGNs electrically, allowing for restoration of hearing in deaf patients. The performance of these devices depends on the functionality of SGNs, the implantation procedure and on the distance between the electrodes and the auditory neurons. 
We hypothesized, that reducing the distance between the SGNs and the electrode array of the implant would allow for improved stimulation and frequency resolution, with the best results in a gapless position. Currently we lack in vitro culture systems to study, modify and optimize the interaction between auditory neurons and electrode arrays and characterize their electrophysiological response. To address these issues, we developed an in vitro bioassay using SGN cultures on a planar multi electrode array (MEA). With this method we were able to perform extracellular recording of the basal and electrically induced activity of a population of spiral ganglion neurons. We were also able to optimize stimulation protocols and analyze the response to electrical stimuli as a function of the electrode distance. This platform could also be used to optimize electrode features such as surface coatings.

We present a protocol to culture primary murine spiral ganglion neuron explants on multi electrode arrays to study neuronal response profiles and optimize stimulation parameters. Such studies aim to improve the neuron-electrode interface of cochlear implants to benefit hearing in patients as well as the energy consumption of the device.

Do gânglio espiral Neuron Explant Cultura e Eletrofisiologia em multi-eléctrodos Arrays

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in ...

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.

Imagem de atividade Neural no córtex somatossensorial primário usando Thy1-GCaMP6s de ratos transgênicos

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

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.

Treinamento baseado em atividade em uma esteira com medula espinhal ferido de ratos Wistar

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

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.

Entrega intra-arterial de células-tronco neurais ao cérebro de ratos e ratos: aplicação à isquemia cerebral

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

Uptake of Fluorescent Labeled Small Extracellular Vesicles In Vitro and in Spinal Cord

Small extracellular vesicles (sEVs) are 50-150 nm vesicles secreted by all cells and present in bodily fluids. sEVs transfer biomolecules such as RNA, proteins, and lipids from donor to acceptor cells, making them key signaling mediators between cells. In the central nervous system (CNS), sEVs can mediate intercellular signaling, including neuroimmune interactions. sEV functions can be studied by tracking the uptake of labeled sEVs in recipient cells both in vitro and in vivo. This paper describes the labeling of sEVs from the conditioned media of RAW 264.7 macrophage cells using a PKH membrane dye. It shows the uptake of different concentrations of labeled sEVs at multiple time points by Neuro-2a cells and primary astrocytes in vitro. Also shown is the uptake of sEVs delivered intrathecally in mouse spinal cord neurons, astrocytes, and microglia visualized by confocal microscopy. The representative results demonstrate time-dependent variation in the uptake of sEVs by different cells, which can help confirm successful sEVs delivery into the spinal cord.

Uptake of Fluorescent Labeled Small Extracellular Vesicles In Vitro and in Spinal Cord

We describe a protocol to label macrophage-derived small extracellular vesicles with PKH dyes and observe their uptake in vitro and in the spinal cord after intrathecal delivery.

Absorção de Vesículos Extracelulares Fluorescentes Em Vitro e na Medula Espinhal

Small extracellular vesicles (sEVs) are 50-150 nm vesicles secreted by all cells and present in bodily fluids. sEVs transfer biomolecules such as RNA, ...