Agradecemos Bakhos A. Tannous, Ph.D., Diretor de Desenvolvimento de Vector e Produção no Centro de Neurociências de Massachusetts General Hospital, em Charlestown, Massachusetts, para fornecer-nos com o vetor rAAV-EGFP e João Whang para assistência técnica. Este trabalho foi financiado por subvenções R01 NS050408 (a JS) do Instituto Nacional de Distúrbios Neurológicos e Derrame.

O procedimento cirúrgico descrito foi aprovado pelo Animal Care Institucional e Comitê de Uso (IACUC) da Universidade de Columbia. 1. Preparação de Equipamentos e suspensão de partículas de vírus <ol><li> Limpar e desinfectar o equipamento, esterilizar os instrumentos cirúrgicos e os picos de entalhe em V que serão utilizados para fixar vértebra L1. </li><li> Puxe e pipetas de vidro bisel. Usamos pipetas que têm um diâmetro da ponta de 40 um e são chanfrados num ângulo de 20 °. Esterilizar as pipetas de vidro. </li><li> Configure o quadro estereotáxico, montar o injetor micro para o manipulador e conectar o injetor para o controlador. </li><li> Prenda uma das pipetas de vidro para o micro usando o kit de conexão de compressão. </li><li> Remover o êmbolo da microsseringa e encher a seringa com óleo mineral. Oil Red O (1 - (2,5-dimetil-4-(2,5-dimethylphenylazo) fenilazo)-2-naftol), podem ser adicionados ao óleo mineral para aumentar a sua visibility. Reinserir o êmbolo e empurre-o para todo o caminho até a ponta. Cuidadosamente evitar a criação de uma bolha de ar. </li><li> Preparar a suspensão de partículas de vírus em um armário de biossegurança. Descongelar o virus congelada em gelo e, imediatamente antes da utilização, diluir com estéril salina tamponada com fosfato com a concentração de partículas desejado. </li><li> Insira o micro para o suporte no injector. </li><li> Pipetar 5 ul da suspensão de vírus para uma folha de plástico, por exemplo Parafilm. Diminuir a ponta da pipeta de vidro para o soltar e puxar o êmbolo para encher a microsseringa. Criar uma pequena bolha de ar na ponta da pipeta para evitar que o entupimento. </li></ol> 2. Laminectomia <ol><li> Prepare a área de cirurgia limpando banco e almofada de aquecimento com desinfetante. </li><li> Anestesiar o mouse. Nós usamos anestesia inalatória com isoflurano (3% durante a indução, 2% a 3% durante a manutenção). </li><li> Coloque lubrificante em cada olho, para proteger os olhos de secagem durante o períodooperação. </li><li> Raspar a pele a partir da parte inferior das costas do pescoço do rato e desinfectar a pele com a alternância de toalhetes de um anti-séptico tópico tal como clorhexidina ou povidona-iodo e etanol a 70%. Isolar o local assepticamente preparada com tecido cirúrgico e infiltrar o local da incisão com bupivacaína (0,25%, 1:10 diluídas com solução salina fisiológica). </li><li> Incisão na pele na extremidade caudal da caixa torácica ao longo da linha média (2-3 cm) e separar a fáscia que cobre a lombada. </li><li> Porque a medula espinhal pára de crescer mais cedo durante o desenvolvimento pós-natal do que a coluna vertebral, o segmento vertebral L4 está por baixo da primeira vértebra lombar (L1). Vértebra L1 está localizado à vértebra caudal que mantém o último par de costelas. Identificar e expor vértebra L1, removendo os pequenos músculos e ligamentos da coluna vertebral presos a sua superfície dorsal. </li><li> Ligeiramente levantar e segurar vértebra L1 com uma pinça de Adson. Use uma pinça laminectomia dedicados para remover o portio dorsaln da vértebra (coluna e lâmina) e expor a medula espinhal. Evitar danos na medula espinhal. </li><li> Transferir o rato sobre a placa de aquecimento na armação estereotáxica. Monitorar a temperatura do mouse durante a operação subseqüente. </li></ol> 3. Injeção <ol><li> Fix vértebra L1 com picos de entalhe em V. Os picos devem estabilizar a coluna vertebral, de modo que a vértebra não se mover durante a respiração. </li><li> Traga a micro mais perto de modo que a ponta da pipeta é acima do local da laminectomia. Abaixe o êmbolo até que a suspensão de vírus sair da pipeta. Remover a gota, com uma ponta de algodão estéril. </li><li> Posicionar a ponta da pipeta na porção rostral do mais medula espinal exposta. Centro da pipeta acima do sulco posterior mediana, em seguida, passar a ponta 500 mm lateralmente. Diminuir a ponta para a superfície do cabo e punção da dura-máter ou, se estiver a trabalhar com uma pipeta de vidro unbeveled, utilizar uma cânula de aço biselado para perfurar o dura. Baixar a ponta da pipeta de vidro 300 um para a medula espinhal. </li><li> Injectar 1 ul de suspensão de vírus, a uma taxa de 200 nl / min. </li><li> No final da injecção, esperar, pelo menos, 2 minutos antes lentamente retraindo a pipeta. </li><li> Repita os passos 3,3-3,5 na porção mais caudal da medula espinal exposta para obter uma distribuição completa do vector viral em L4 segmento da coluna vertebral. Os dois locais de injecção situam-se rostral e caudal do segmento de L4 para evitar danos no tecido na região-alvo. </li></ol> 4. O fechamento da ferida <ol><li> Lançamento vértebra L1 dos grampos de entalhe em V e remover o mouse do quadro estereotáxico. </li><li> Suturar a fascia com 5,0 vicryl. A fáscia fechado oferece cobertura para o site laminectomia. </li><li> Feche a pele com fio de náilon ou grampos cirúrgicos. </li></ol> 5. Cuidados no pós-operatório <ol><li> Transferir o mouse para uma gaiola de recuperação, com cama, macio não-particular. Coloque-o em tele laterais para respiração confortável. Monitorar o animal até que ele é totalmente alerta, ambulatório e começa a beber. </li><li> Fornecemos analgesia pós-operatória, durante 72 h, com injecções subcutâneas diárias de carprofeno (5,0 mg / kg). </li><li> Monitorar a recuperação pós-cirúrgica por inspeção diária para os primeiros 3 dias, depois a cada dois dias ou três dias por semana, até que a experiência é completa. </li><li> Remover as suturas ou agrafos 7 a 10 dias após a operação, quando a cicatrização de feridas é completa. </li><li> Eutanásia do animal após a conclusão da experiência. </li></ol>

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Os autores declaram que não têm interesses conflitantes financeiros.

Injecção estereotáxica vector permite a segmentação neurónios da espinal medula de aplicações, tais como mapeamento de rede neuronal com base em vírus transsináptica espalhando 6,7 ou optogenética dissecção 8, durante a regeneração axonal de orientação a partir de 9,10 lesão, ou terapia de gene para a prevenção ou tratamento da neurodegeneração 11, 12. Os vectores virais têm sido utilizados para a manipulação de genes na medula espinhal de estudar vias...

As tecnologias avançadas de manipulação genética condicional no rato permitir abordagens multifacetadas para a exploração de vias sinápticas e ligações funcionais no sistema nervoso central. Os transgenes podem ser regulados por moléculas pequenas efectoras, tais como a doxiciclina que actuam sobre um transactivador controlado por tetraciclina, que pode ser concebido para funcionar como um repressor ou um activador da transcrição de genes, ou tamoxifeno reconhecendo um domínio mutado de ligação ao ligando do receptor de estrogénio 1 . Modificação irreversível transgene geralmente é conseguido por o ácido desoxirribonucléico (DNA) recombinases. Cre (recombinação causas) e FLP (flippase enzima de recombinação) catalisam a excisão, inversão ou translocação de fragmentos de DNA que são ladeadas por loxP (locus de cruzamento x mais, P1) ou Frt (flippase alvo de reconhecimento) sites, respectivamente 1. As aplicações incluem a ativação do gene ou silenciamento e ácido induzível ribonucléico (RNA) interferência <sup&gt; 2. Expressão condicional de repórteres fluorescentes ou enzimáticos, tais como β-galactosidase ou fosfatase alcalina pode ser utilizada para rotular os neurónios e examinar a sua organização tópica e conectividade 3. Grandes projectos de mutagênese na América do Norte ( <a href="http://www.norcomm.org/index.htm" target="_blank">http://www.norcomm.org/index.htm</a> ) e Europa ( <a href="http://www.knockoutmouse.org/about/eucomm" target="_blank">http://www.knockoutmouse.org/about/eucomm</a> ) estão produzindo bibliotecas de clones de rato com células-tronco embrionárias condicionais genes alvos e armadilhas que eventualmente cobrem o genoma do rato inteiro. Ratos gerados a partir destes clones podem ser cruzadas com um número crescente de linhas de ratinhos que expressam ADN sob recombinases promotores ou loci específicos para uma determinada população de neurónios para a manipulação de genes selectiva ( <a href="http://nagy.mshri.on.ca/cre_new/index.php" target="_blank">http://nagy.mshri.on.ca/cre_new/index . php</a> ). <p class= &quot;Jove_content&quot;&gt; No entanto, restringir a manipulação de genes a diferentes populações de neurónios ou de regiões de interesse particular não pode ser obtida por segmentação genética sozinho, se um promotor específico para a população de neurónios de interesse não é conhecido ou não expresso por todos os neurónios na região de interesse. Na medula espinal, os desenhos experimentais podem exigir restrição espacial da manipulação genética de um ou dois segmentos crânio-caudal. Injecção estereotáxica de um vector viral que expressa Cre ou Flp permite limitar a recombinação de genes para as regiões na medula espinal de ratos em que os fragmentos de DNA são flanqueados por sítios loxP ou FRT, os chamados alelos floxed ou flrted. Ao contrário de rearranjo do DNA constituinte, que resultaria da hibridização, os animais com ratos que expressam recombinase, esta estratégia também proporciona um controlo temporal sobre a activação do gene ou silenciamento. Os vectores virais que codificam floxed ou flertou transgenes oferecem uma opção inversa de manipulação de genes em ratos expressando a corresponding recombinase jusante de um promotor específico para neurónios. Vários vetores recombinantes com afinidade para os neurônios estão disponíveis 4. De elevada capacidade de adenovírus (gutless), vírus adeno-associados, vírus do herpes simplex e lentivírus são comumente utilizados vectores neurotrópicas. Selecionando o vírus apropriado para uma questão de pesquisa é uma parte crucial do projeto experimental. Tamanho do transgene, via de administração, a especificidade da infecção para os neurónios, em oposição às células da glia, a eficácia da infecção, os efeitos secundários inflamatórios e tóxicos precisam ser considerados 4. Aqui descrevemos a injecção estereotáxica de um vector viral no corno dorsal da medula espinhal, uma técnica que empregam para a regulação do gene condicional na nossa pesquisa sobre a neurobiologia da dor. O corno dorsal recebe entrada aferente primário neurônios somatossensoriais incluindo neurónios nociceptivos. Interneurônios locais processar as informações antes de neurônios de projeção transmiti-lo a partir decorno dorsal para o cérebro 5. Nós demonstramos que a infecção de neurónios do corno dorsal da coluna vertebral ao nível segmental L4 com um neurotrópica de vírus adeno-associado recombinante (rAAV) que expressa a proteína verde fluorescente melhorada (EGFP) no âmbito de um promotor de citomegalovírus constitucionalmente activo.

<table border="1"><tbody><tr><td> Nome de material </td><td> Companhia </td><td> Número de Catálogo </td><td></td></tr><tr><td> Placa de base da coluna vertebral </td><td> David Kopf Instruments </td><td> 912 </td><td></td></tr><tr><td> Instrumento estereotáxico de pequenos animais </td><td> David Kopf Instruments </td><td> 900 </td><td></td></tr><tr><td> Rato gás suporte da cabeça anestesia </td><td> David Kopf Instruments </td><td> 923-B </td><td></td></tr><tr><td> Ajustáveis ​​montagens de base </td><td> David Kopf Instruments </td><td> 982 </td><td></td></tr><tr><td> Picos de entalhe em V </td><td> David Kopf Instruments </td><td> 987 </td><td></td></tr><tr><td> Pequeno animal, sistema de controlo de temperatura </td><td> David Kopf Instruments </td><td> TCAT-2LV </td><td></td></tr><tr><td> Adson fórceps </td><td> Belas Science Tools </td><td> 11006-12 </td><td></td></tr><tr><td> Fórceps laminectomia </td><td> Belas Science Tools </td><td> 11223-20 </td><td></td></tr><tr><td> UltraMicroPump (um) com SYS-Micro4 Controlador </td><td> Instrumentos de Precisão mundo </td><td> UMP3-1 </td><td></td></tr><tr><td> Micro-65RN </td><td> Hamilton </td><td> 7633-01 </td><td></td></tr><tr><td> RN de compressão apropriado, 1 mm </td><td> Hamilton </td><td> 55750-01 </td><td></td></tr><tr><td> Borosilicato capilares de vidro </td><td> Instrumentos de Precisão mundo </td><td> 1B100F-4 </td><td></td></tr><tr><td> Microgrinder </td><td> Narishige </td><td> EG-44 </td><td></td></tr></tbody></table>

stereotaxic injection of a viral vector for conditional gene manipulation in the mouse spinal cord

Intraparenquimatosa injecção de um vector viral permite a manipulação genética condicional em populações distintas de neurônios ou regiões específicas do sistema nervoso central. Nós demonstramos uma técnica de injecção estereotáxica que permite a expressão do gene alvo ou silenciamento no corno dorsal da medula espinal do rato. O procedimento cirúrgico é breve. Ele requer laminectomia de uma única vértebra, fornecendo para a recuperação rápida do animal e da motilidade perfeita da coluna vertebral. Injecção controlada de um pequeno volume vector suspensão a baixa velocidade e à utilização de uma micro-seringa com cânula de vidro biselado minimizar a lesão dos tecidos. A resposta imunitária local do vector depende das propriedades intrínsecas do vírus empregue, na nossa experiência, é menor e de curta duração quando um vírus adeno-associado recombinante é utilizado. Um gene repórter, tais como a proteína fluorescente verde melhorada facilita a distribuição espacial de controlo do vector, e a eficácia e celular specipecificidade da transfecção.

Sucesso rendimentos de transfecção expressão gênica robusto em neurônios do corno dorsal injetado (Figura 1), poupando o corno dorsal do lado contralateral, o corno anterior e gânglios da raiz dorsal. <img src="/files/ftp_upload/50313/50313fig1.jpg" alt="Figura 1" /> Figura 1. Transfecção dos neurónios do corno dorsal. (A) Expressão do EGFP repórter fluorescente (verde) no corno dorsal esquerda da medula espinhal L4, duas semanas após a inje...

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Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Vetores virais permitem a manipulação de genes alvo. Nós demonstramos um método para a expressão de genes condicionais ou ablação da medula espinhal de rato, utilizando a injecção estereotáxica de um vector viral no corno dorsal, um local proeminente de contacto sináptico entre os aferentes primários e os neurónios de somatossensorial no sistema nervoso central.

Injeção estereotáxica de um vetor viral para manipulação genética condicional no Cabo de Rato Spinal

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

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30.1K visualizações.  Centre National de la Recherche Scientifique (CNRS). Vetores virais permitem a manipulação de genes alvo. Nós demonstramos um método para a expressão de genes condicionais ou ablação da medula espinhal de rato, utilizando a injecção estereotáxica de um vector viral no corno dorsal, um local proeminente de contacto sináptico entre os aferentes primários e os neurónios de somatossensorial no sistema nervoso central.

Vídeo: Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

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

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 ...

Neurociência

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.

Em Imaging vivo do Cordão do rato Spinal Usando Two-photon Microscopia

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 ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Expansão da embrionárias e adultas células-tronco neurais por<em&gt; In Utero</em&gt; Eletroporação ou injeção estereotáxica Viral

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

Injection Viral intracerebroventricular do Neonatal do rato do cérebro para a transdução neuronal persistente e generalizada

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

Um Processo de Implantação de uma Câmara Spinal para Longitudinal<em&gt; In Vivo</em&gt; Criação de Imagens do Cordão do rato Spinal

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

Dois fótons de imagem da Cellular Dynamics na Medula Espinhal Rato

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

Cirurgia estereotáxica para manipulação genética em células estriadas de cérebro de rato Neonatal

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

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.

Implante de janela de laminectomia e medula espinhal no 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 ...

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

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.

Injeção direta de um vetor de Lentivirus destaca várias vias motoras na medula espinhal de ratos

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.

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 ...