Este trabalho é financiado pelas Ciências Naturais e Pesquisa de Engenharia Council of Canada (Grant # 249669 e # 482986) e Coração and Stroke Foundation of Canada (Grant # 485508). Os autores agradecem Youssef El-Hayek e Dr. Qi Wan por sua assistência no desenvolvimento dos protocolos experimentais.

Todos os procedimentos que envolvem manipulação de animais foram aprovados pela Universidade de Toronto Comitê Animal Care, de acordo com as diretrizes institucionais (protocolo no. 20009387). Os métodos que se seguem devem ser realizados utilizando ferramentas e técnicas estéreis, numa câmara de fluxo laminar, onde aplicável. No texto do protocolo a seguir, a frase &quot;EFH-SFM&quot; refere-se a meio isento de soro suplementado com factor de crescimento epidérmico, factor de crescimento de fibroblastos básico e heparina. EFH-SFM é usado quando a investigar o galvanotaxis de NPCs indiferenciadas, porque esses mitógenos manter NPCs em sua 4 estado indiferenciado. Ao investigar a galvanotaxis de NPCs induzidas a diferenciar-se em tipos de células maduras, &quot;FBS-SFM&quot; refere-se a meio isento de soro suplementado com 1% de soro fetal bovino. FBS promove a diferenciação de maduras NPCs em fenótipos neurais 5. 1. Isolamento e Cultura de precursores neurais (Nãomostrado em video) <ol><li> Anestesiar um rato CD1 (6-8 semanas de idade) com isofluorano e sacrifício através de deslocamento cervical. </li><li> Extinguir a cabeça em etanol a 70% e decapitar o animal com tesouras de dissecação afiadas. </li><li> Enquanto mantém a cabeça com pinça cirúrgica, remover a pele na superfície dorsal para expor o crânio. </li><li> Usando um bisturi e não. Lâmina 11, marcar o crânio no seio frontal ao longo do eixo mediolateral, e também ao longo da sutura sagital na direcção rostrocaudal. </li><li> Descasque os ossos parietais de distância da cabeça com nenhum. 7 pinças curvas, tendo cuidado para não furar o tecido cerebral. </li><li> Insira uma espátula fina embaixo do cérebro a partir de debaixo do cerebelo e avançando para os bulbos olfatórios. Enquanto segurando o crânio no lugar com uma pinça, puxe o cérebro do crânio e imediatamente colocá-lo na gelada líquido cefalorraquidiano artificial (veja receitas abaixo). </li><li> Sob um microscópio de dissecação, utilizando estériltesouras e pinças de dissecção cortar o cérebro de meia ao longo da linha média. Rodar cada hemisfério, de modo que a superfície (cut) medial virado para cima. </li><li> Selecione um hemisfério, e com a superfície medial voltada para cima, localize o esplênio do corpo caloso (região posterior do corpo caloso). </li><li> Fazer uma incisão da superfície do córtex do esplênio do corpo caloso ao longo do eixo dorsoventral. </li><li> Descasque o córtex incisão em direção ao bulbo olfativo para expor as paredes medial e lateral do ventrículo lateral. </li><li> Girar o hemisfério, de modo que a superfície dorsal virada para cima, e usar microtesoura curvadas para cortar e recolher as paredes expostas medial e lateral, que contêm a região periventricular onde residem NPCs 6. </li><li> Repita os passos 1,8-1,11 para o outro hemisfério. </li><li> Pipetar a tecido isolado em 7 ml de solução de tripsina (ver receita abaixo) num tubo de 15 cc, e colocar o tubo sobre um balancim de 37 ° Cincubadora durante 25 min. </li><li> Centrifuga-se o tubo a 1500 rpm durante 5 min, o sobrenadante aspirado, e ressuspender o tecido em 2 ml de solução de inibidor de tripsina (ver receita abaixo). </li><li> Tritura-se o tecido suavemente com um pequeno furo de pipetas de Pasteur 30-50 vezes com cuidado para evitar bolhas de ar. </li><li> Centrifuga-se o tubo a 1500 rpm durante 5 min, o sobrenadante aspirado, e ressuspender em 1-2 ml de SFM (ver receita abaixo) por trituração do pellet 3-5 vezes. </li><li> Centrifuga-se o tubo a 1500 rpm durante 3 minutos, aspirar o sobrenadante e ressuspender em 1 ml de SFM + EFH. </li><li> Conte viver a densidade celular com um hemocitómetro e as células da placa num frasco de cultura T25, a uma densidade de 10 células por uL de SFM + EFH. </li><li> Permitir que a cultura a crescer durante 7 dias não perturbadas para render flutuantes neuroesferas primárias compostas por NPCs. </li></ol> 2. Galvanotaxis Preparação Câmara <ol><li> Coloque 3 de vidro quadrado não. 1 lamelas (22 x 22 x 0,17) ind garrafa de 6N de ácido clorídrico durante a noite. </li><li> No dia seguinte, utilizar uma ponta de diamante, cortador de vidro para cortar seis tiras rectangulares (22 x 5 x 0,17 mm) de vidro a partir de qualquer quadrado. 1 lamínulas. </li><li> Transfira os deslizamentos lavado com ácido quadrados e retangulares deslizamentos em uma capela de fluxo laminar. Lave as tiras rectangulares e quadradas, primeiro com etanol 70%, em seguida com água de cultura de tecidos de grau autoclavado, e deixa-se secar numa Kim Wipe (para esterilidade adicionado, o vidro pode ser permitido para o ar seco). </li><li> Aplique graxa de vácuo para o perímetro de uma superfície das lâminas de vidro quadrados, e vedá-las à base de 60 mm pratos de Petri de plástico. </li><li> Aplique graxa de vácuo ao longo do eixo longo de uma superfície das tiras de vidro rectangulares e selá-los às arestas opostas das lâminas de vidro quadrados (de tal forma que elas fiquem paralelas umas às outras), a fim de criar uma calha central. </li><li> UV-esterilizar as câmaras durante pelo menos 15 min no fluxo laminar. </li><li> Pipetar 250-300 ul de poli-L-Lysine para a calha central das câmaras e incubar à temperatura ambiente durante 2 horas. </li><li> Aproximadamente 15 minutos antes do final do período de incubação, a preparação da solução de Matrigel (ver receita abaixo). </li><li> Aspirar o poli-L-lisina, lave as calhas centrais com 1 ml de água esterilizada, e uL de solução da pipeta 250-300 Matrigel para as calhas centrais. </li><li> Incubar as câmaras, a 37 ° C durante 1 h. </li><li> Aspirar a solução Matrigel e lavar cuidadosamente as calhas centrais com 1-2 ml de SFM. </li><li> Pipetar 100 mL de EFH-SFM ou FBS-SFM para as calhas centrais e transferir as câmaras galvanotaxis para o palco de um microscópio de contagem. </li><li> Pipetar 3-4 ml da cultura neuroesfera contendo em uma placa de Petri de 60 mm e transferir a placa de Petri, para o estágio de microscópio de contagem. </li><li> Se a um objectivo de visualização de 5x, utilizar uma pipeta para transferir P10 5-8 neuroesferas inteiras (até quatro de cada vez) na calha central de cada galvanotaxiscâmara sem dissociar-los, e cuidadosamente espalhado as neuroesferas redor da calha central, sem perturbar o substrato Matrigel. </li><li> Adicionar um adicional de 150-200 ul EFH-SFM ou FBS-SFM para as calhas centrais. </li><li> Transferir as câmaras galvanotaxis em um de 37 ° C, 5% CO 2, 100% incubadora humidificada durante 17-20 h (se analisar NPCs indiferenciadas) para permitir que as neuroesferas a aderir ao substrato de Matrigel e dissociam-se em células individuais, como mostrado na Figura 1 . Se a análise de NPCs diferenciados, o período de incubação deve ser alargado para 69-72 horas para permitir a diferenciação das células. </li></ol> 3. Viver imagens de células Time-Lapse <ol><li> Permitir que o sistema de imagens ao vivo de células para equilibrar a 37 ° C, 5% CO 2 durante um período mínimo de 30 min antes do início da gravação de lapso de tempo. </li><li> Corte dois pedaços de 12 cm de 1 mm de diâmetro prata fio da bobina, eles de um lado, e colocá-los em Clorox blixiviação durante 20 minutos de modo a formar de Ag / AgCl. </li><li> Transferir as câmaras galvanotaxis para a plataforma de um microscópio de contagem e seleccionar que câmara irá ser utilizada para a análise de células live-migração de imagem com base nos seguintes critérios: i) as neuroesferas deve ser quase completamente dissociado em células individuais e, ii) as células devem possuir morfologias redondas com pouco ou nenhum processo que se estende dos corpos celulares. </li><li> Transferir a câmara galvanotaxis seleccionado numa câmara de fluxo laminar, em conjunto com um qualquer quadrado separado. Uma lamínula de vidro e graxa de vácuo. </li><li> Lava-se a tampa desliza em primeiro lugar com 70% de etanol, em seguida, em água esterilizada, e aplicar uma tira de massa lubrificante de vácuo sobre dois bordos paralelos da lâmina de cobertura. </li><li> Aspirar o meio de cultura a partir da calha central da câmara, em seguida, rapidamente colocar a lâmina de cobertura (graxa lado voltado para baixo) para a câmara de tal modo que a gordura restante nas duas tiras paralelas tiras de vidro rectangulares, criando efectivamente um telhadopara a câmara. </li><li> Pipetar 100 mL de fresco EFH-SFM ou FBS-SFM na calha central através de ação capilar. </li><li> Use graxa de vácuo para criar bordas de piscinas de meios de cultura em cada extremidade da calha central, como mostrado na Figura 2. </li><li> Cortar duas peças 15 cm de tubo de PVC, e com uma seringa de 10 cc com uma agulha de calibre 18 para injectar a solução de agarose cuidadosamente no tubo, garantindo não formar bolhas no interior dos tubos, e permitir que o gel solidificar durante 5 min. </li><li> Transferir a câmara galvanotaxis ao sistema vivo de células de imagem, juntamente com o gel de agarose a tubos, Ag / AgCl, e um par de vazios 60 pratos de Petri milímetros que serão utilizados como meios de cultura reservatórios e irá conter o Ag / AgCl. Permitir que a câmara galvanotaxis para descansar dentro do 37 ° C, 5% CO 2 ambiente durante 20-30 min. </li><li> Durante este tempo, preparar as tampas dos dois pratos de Petri vazias e da tampa da caixa de Petri a câmara de galvanotaxis por furos de perfuraçãoneles com uma ferramenta Dremel ou semelhante, como mostrado na Figura 3. </li><li> Pipetar 1-1,5 ml de EFH-SFM ou FBS-SFM para um ou outro lado da calha central, e 7-8 ml de SFM em cada placa de Petri vazia. Coloque uma placa de Petri em cada lado da calha central da câmara e galvanotaxis lugar um eléctrodo de Ag / AgCl em cada prato. Ponte o espaço entre a câmara de galvanotaxis e as placas de Petri para estabelecer a continuidade eléctrica com as pontes de gel de agarose, como se mostra na Figura 4. </li><li> Conectar os eletrodos de Ag / AgCl a uma fonte de alimentação externa, com um amperímetro em série para medir a corrente elétrica e ligue a fonte de alimentação. Use um voltímetro para medir a força do campo eléctrico em frente da calha central, e ajustar a saída da fonte de alimentação até que a intensidade do campo eléctrico desejado é alcançado (os ensaios realizados neste laboratório utilizam uma força dcEF de 250 mV / mm com corrente eléctrica entre 1 e 1,5 mA). </li><li> Iniciativate módulo do lapso de tempo no sistema de célula de imagem ao vivo, e permitir que o experimento de correr para a quantidade de tempo desejada. Após a conclusão do ensaio, as células de fixação em paraformaldeído a 4%, para análise de imunomarcação padrão. </li></ol>

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Não há conflitos de interesse declarados.

Este protocolo foi adaptado a partir de métodos bem estabelecidos de estudos anteriores 7-9. Galvanotactic câmaras podem ser construídas utilizando uma variedade de técnicas diferentes, incluindo a construção de um poço de vidro separada para confinamento de sementeira de células, ou utilizando a ablação por laser de CO 2 para a microfabricação da calha central, 10,11. Algumas técnicas podem ser mais laboriosa ou dispendiosa do que outros. Nós descrevemos um ensaio simples ...

<table border="1"><tbody><tr><td height="44"> Nome do item </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td></td><td></td><td></td><td> Isolamento Celular Neural Precursor </td></tr><tr><td> 2M NaCl </td><td> Sigma </td><td> S5886 </td><td> 11,688 g dissolvido em 100 ml de dH2O </td></tr><tr><td> 1M KCl </td><td> Sigma </td><td> P5405 </td><td> 7,456 g dissolvido em 100 ml de dH2O </td></tr><tr><td> 1M de MgCl2 </td><td> Sigma </td><td> M2393 </td><td> 20,33 g dissolvido em 100 ml de dH2O </td></tr><tr><td> 155 mM de NaHCO3 </td><td> Sigma </td><td> S5761 </td><td> 1,302 g dissolvido em 100 ml de dH2O </td></tr><tr><td> Glicose 0,5 M </td><td> Sigma </td><td> G6152 </td><td> 9,01 g dissolvido em 100ml de dH2O </td></tr><tr><td> 108 mM CaCl 2 </td><td> Sigma </td><td> C7902 </td><td> 1,59 g dissolvido em 100 ml de dH2O </td></tr><tr><td> Penicilina-estreptomicina </td><td> Gibco </td><td> 15070 </td><td></td></tr><tr><td> Tripsina de pâncreas bovino </td><td> Sigma </td><td> T1005 </td><td></td></tr><tr><td> Sheep testículos hialuronidase </td><td> Sigma </td><td> H6254 </td><td></td></tr><tr><td> Ácido quinurênico </td><td> Sigma </td><td> K3375 </td><td></td></tr><tr><td> Ovomucóide inibidor de tripsina </td><td> Worthington </td><td> LS003086 </td><td></td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 12100046 </td><td></td></tr><tr><td> F12 </td><td> Invitrogen </td><td> 21700075 </td><td></td></tr><tr><td> Glucose 30% </td><td> Sigma </td><td> G6152 </td><td></td></tr><tr><td> 7,5% de NaHCO3 </td><td> Sigma </td><td> S5761 </td><td></td></tr><tr><td> HEPES 1M </td><td> Sigma </td><td> H3375 </td><td> 23,83 g dissolvido em 100 ml de dH2O </td></tr><tr><td> L-glutamina </td><td> Gibco </td><td> 25030 </td><td></td></tr><tr><td> EGF </td><td> Invitrogen </td><td> PMG8041 </td><td> Reconstituir em 1 ml de mistura de hormona e alíquota em 20 unidades ul. </td></tr><tr><td> FGF </td><td> Invitrogen </td><td> PHG0226 </td><td> Reconstituir em 0,5 ml da mistura de hormona e alíquota em 20 unidades ul. </td></tr><tr><td> Heparina </td><td> Sigma </td><td> H3149 </td><td></td></tr><tr><td> Apo-transferrina </td><td> R &amp; D Systems </td><td> 3188-AT </td><td> 0,1 g dissolvido em 4 ml de dH 2 0 </td></tr><tr><td> Putrescina </td><td> Sigma </td><td> P7505 </td><td> Dissolver 9,61 mg em Apo-transferrina assimlução </td></tr><tr><td> Insulina </td><td> Sigma </td><td> I5500 </td><td> Dissolver 25 mg em 0,5 ml de HCl 0,1 N e adicionar 3,5 ml de dH 2 0 </td></tr><tr><td> Selênio </td><td> Sigma </td><td> S9133 </td><td></td></tr><tr><td> Progesterona </td><td> Sigma </td><td> P6149 </td><td></td></tr><tr><td> Ferramentas padrão de Dissecção </td><td> Belas Science Tools </td><td></td><td></td></tr><tr><td> Microscópio de dissecção </td><td> Zeiss </td><td> Stemi 2000 </td><td></td></tr><tr><td></td><td></td><td></td><td> Galvanotaxis Preparação Câmara </td></tr><tr><td> Lâminas de vidro tampa quadrada </td><td> VWR </td><td> 16004 </td><td></td></tr><tr><td> Ácido clorídrico 6N </td><td> VWR </td><td> BDH3204-1 </td><td></td></tr><tr><td> Graxa de alto vácuo </td><td> Dow Corning </td><td></td><td></td></tr><tr><td> 60 milímetros placas de Petri </td><td> Fisher Scientific </td><td> 0875713A </td><td></td></tr><tr><td> Poli-L-lisina </td><td> Sigma </td><td> P4707 </td><td></td></tr><tr><td> Matrigel </td><td> BD Biosciences </td><td> 354234 </td><td> Descongelar e alíquota em 150 unidades ul </td></tr><tr><td> FBS </td><td> Invitrogen </td><td> 10082139 </td><td> Só use se induzir a diferenciação NPC, caso contrário use SFM + meios de cultura EFH como indicado acima </td></tr><tr><td> Contando microscópio </td><td> Olimpo </td><td> CKX41 </td><td></td></tr><tr><td></td><td></td><td></td><td> Viver imagens de células Time-Lapse </td></tr><tr><td> Fio de prata </td><td> Alfa Aesar </td><td> 11434 </td><td></td></tr><tr><td> UltraPure Agarose </td><td> Invitrogen </td><td> 15510-027 </td><td></td></tr><tr><td> Calor inatividadeated FBS </td><td> Sigma </td><td> 16140071 </td><td></td></tr><tr><td> Tubos de PVC </td><td> Fisher Scientific </td><td> 80000006 </td><td> 3/32 &quot;ID x 5/32&quot; OD </td></tr><tr><td> Alvejante </td><td> Clorox </td><td></td><td></td></tr><tr><td> Seringa de 10 cc </td><td> BD </td><td> 309604 </td><td></td></tr><tr><td> Agulha de calibre 18 </td><td> BD </td><td> 305195 </td><td></td></tr><tr><td> Broca Dremel </td><td> Dremel </td><td> Modelo 750 </td><td></td></tr><tr><td> Microscópio invertido equipado com câmara, umidificado incubada </td><td> Zeiss </td><td> Axiovert-200M </td><td></td></tr><tr><td></td><td></td><td></td><td> Receitas <table border="1"><tbody><tr><td> Item </td><td> Volume </td></tr><tr><td> 2M NaCl </td><td> 6,2 ml </td></tr><tr><td>1M KCl </td><td> 0,5 ml </td></tr><tr><td> 1M de MgCl2 </td><td> 0,32 ml </td></tr><tr><td> 155mm de NaHCO3 </td><td> 16,9 ml </td></tr><tr><td> A glicose 1M </td><td> 1 ml </td></tr><tr><td> 108 mM CaCl 2 </td><td> 0,09256 ml </td></tr><tr><td> Penicilina-estreptomicina </td><td> 1 ml </td></tr><tr><td> Água esterilizada </td><td> 74 ml </td></tr></tbody></table> Líquido cefalorraquidiano artificial <table border="1"><tbody><tr><td> Item </td><td> Volume ou massa </td></tr><tr><td> Líquido cefalorraquidiano artificial </td><td> 30 ml </td></tr><tr><td> Tripsina de pâncreas bovino </td><td> 40 mg </td></tr><tr><td> Sheep testículos hialuronidase </td><td> 22,8 mg </td></tr><tr><td> Ácido quinurênico </td><td> 5 mg </td></tr></tbody></table> &lt;strong&gt; Solução de tripsina <table border="1"><tbody><tr><td> Item </td><td> Volume ou massa </td></tr><tr><td> SFM </td><td> 15 ml </td></tr><tr><td> Ovomucóide inibidor de tripsina </td><td> 10 mg </td></tr></tbody></table> Solução de tripsina Inhibitor <table border="1"><tbody><tr><td> Item </td><td> Volume </td></tr><tr><td> Água esterilizada </td><td> 37 ml </td></tr><tr><td> DMEM/F12 10X </td><td> 10 ml </td></tr><tr><td> Glucose 30% </td><td> 2 ml </td></tr><tr><td> 7,5% de NaHCO3 </td><td> 1,5 ml </td></tr><tr><td> HEPES 1M </td><td> 0,5 ml </td></tr><tr><td> Transferrina, solução Putrescine </td><td> 4 ml </td></tr><tr><td> Solução a 25 mg de insulina </td><td> 4 ml </td></tr><tr><td> Selênio </td><td> 100 e mu, L </td></tr><tr><td> Progesterona </td><td> 100 ul </td></tr></tbody></table> Mix hormona (100 ml no total loja, a -20 ° C) <table border="1"><tbody><tr><td> Item </td><td> Volume </td></tr><tr><td> Água esterilizada </td><td> 37,5 ml </td></tr><tr><td> DMEM/F12 10X (3:1) </td><td> 5 ml </td></tr><tr><td> Glucose 30% </td><td> 1 ml </td></tr><tr><td> 7,5% de NaHCO3 </td><td> 0,75 ml </td></tr><tr><td> HEPES 1M </td><td> 0,25 ml </td></tr><tr><td> Mix de hormônio </td><td> 5 ml </td></tr><tr><td> L-glutamina </td><td> 0,5 ml </td></tr><tr><td> Penicilina-estreptomicina </td><td> 0,5 ml </td></tr></tbody></table> O soro livre de mídia EFH-SFM: adicionar 10 ul de EGF, 10 | il de FGF, e 3,66 ul de Heparina FBS-SFM: adicionar 0,5 ml de FBS <tafronteira vel = &quot;1&quot;&gt; <tbody><tr><td> Item </td><td> Volume </td></tr><tr><td> Matrigel </td><td> 150 ul </td></tr><tr><td> SFM </td><td> 3,6 ml </td></tr></tbody></table> Matrigel Solução alíquota Matrigel deve ser colocado numa caixa de gelo e deixou-se descongelar lentamente ao longo de 4-5 horas de modo a formar um líquido viscoso antes de misturar com SFM. Isto irá assegurar a formação de uma camada lisa de substrato Matrigel. Se não for descongelado lentamente, o substrato resultante irá conter aglomerados de Matrigel, possivelmente impedindo a migração de células. <table border="1"><tbody><tr><td> Item </td><td> Volume ou massa </td></tr><tr><td> UltraPure Agarose </td><td> 300 mg em 10 ml de DDQ 2 0 </td></tr><tr><td> SFM FBS inactivado pelo calor </td><td> 8 ml 2 ml </td></tr></tbody></table> MatrigelSolução Misture 8 ml de SFM com 2 ml de calor FBS inativado em um tubo falcon 15 cc. Mistura de agarose com 10 ml de DDQ 2 0 em um balão de Erlenmeyer, e de calor num forno de microondas durante 30 segundos em intervalos de 10 seg, assegurando para remover a solução a partir do forno de microondas depois de cada intervalo de 10 segundos e misturar completamente. Após a final microondas período de 10 seg, misture a solução de agarose com a SFM solução FBS / e armazenar em um banho de água 57 ° C. </td></tr></tbody></table>

a galvanotaxis assay for analysis of neural precursor cell migration kinetics in an externally applied direct current electric field

A descoberta de haste neural e células progenitoras (colectivamente denominadas células precursoras neurais) (NPCs) no cérebro de mamíferos adultos tem levado a um corpo da investigação sobre a utilização das propriedades multipotentes e proliferativa destas células para o desenvolvimento de estratégias neuroregenerative. Uma etapa essencial para o sucesso de tais estratégias é a mobilização de NPCs para um local da lesão após transplante exógena ou para aumentar a resposta dos precursores endógenos, que são encontradas na região periventricular do SNC. Assim, é essencial para compreender os mecanismos que promovem, orientar e melhorar a migração NPC. O nosso trabalho centra-se na utilização de corrente contínua de campos elétricos (dcEFs) para promover e migração NPC direta - um fenômeno conhecido como galvanotaxis. Endógenos fisiológicos campos elétricos funcionam como pistas fundamentais para a migração de células durante o desenvolvimento normal e da cicatrização. Perturbação farmacológica dotrans-neural potencial tubo em embriões axolotl provoca graves malformações do desenvolvimento 1. No contexto da cicatrização de feridas, a taxa de reparação de córnea ferida está directamente correlacionada com a magnitude do potencial epitelial que surge após a lesão, como mostrado pela melhoria farmacológica ou ruptura dessa dcEF 2-3. Nós demonstramos que o adulto NPCs subependimários sofrer rápida e dirigido migração catódica in vitro quando exposto a uma dcEF aplicado externamente. Neste protocolo, descrevemos nossas técnicas de laboratório para a criação de um ensaio galvanotaxis simples e eficaz para alta resolução, longo prazo de observação de translocação dirigido célula do corpo (migração) em um nível de uma única célula. Este ensaio seria adequado para investigar os mecanismos que regulam a transdução dcEF em motilidade celular através da utilização de ratinhos transgénicos ou knockout, curtas de ARN de interferência, ou agonistas / antagonistas de receptores específicos.

A análise cinemática revela que, na presença de um 250 dcEF mV / mm, NPCs indiferenciadas exibem galvanotaxis altamente dirigidos e rápida para o cátodo (Figura 5A, Movie 1). Na ausência de um dcEF, o movimento aleatório das células é observada (Figura 5B, Movie 2). Neste intensidade de campo,&gt; 98% de NPCs indiferenciadas migrar para o 6-8 hr inteiro para o qual são gravadas, e uma vez que as células mortas não migram isto sugere que eles permaneçam viáveis ​​durant...

Watch this Scientific Journal Video about A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field at JoVE.com

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

Neste protocolo demonstramos como construir câmaras personalizados que permitem a aplicação de um campo elétrico de corrente contínua para permitir lapso de tempo de imagem do cérebro adulto derivado translocação de células neurais precursor durante galvanotaxis.

Um Ensaio Galvanotaxis para Análise de Precursores Neurais Cinética migração celular em um campo aplicado externamente Corrente Contínua Elétrica

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

a-galvanotaxis-assay-for-analysis-neural-precursor-cell-migration

Research

JoVE Journal

Neuroscience

12.4K visualizações.  University of Toronto. Neste protocolo demonstramos como construir câmaras personalizados que permitem a aplicação de um campo elétrico de corrente contínua para permitir lapso de tempo de imagem do cérebro adulto derivado translocação de células neurais precursor durante galvanotaxis.

Vídeo: A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

Neurais Colony-Forming Assay Cell: um ensaio para Discriminar Bona Fide células-tronco neurais de células progenitoras neurais

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Neurociência

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

Análise de Tronco de Migração da crista neural celular usando uma modificação Zigmond Ensaio Câmara

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Reprodutível mouse esmagamento do nervo ciático e subsequente avaliação de Regeneração por Análise Whole Mount muscular

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

Análise de migração da crista neural e Diferenciação por Cross-espécies de Transplantes

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

O Ensaio neuroblasto: um ensaio para a Geração e Enriquecimento de células progenitoras neuronais de Diferenciando Progeny Neural Stem Cell utilizando citometria de fluxo

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Isolamento e cultivo de células da crista neural do tubo neural embrionário murino

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.

We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

Um ensaio de migração celular quantitativo para murinos progenitores neurais entéricas

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

Gravações simultâneas de potenciais de campo corticais Local, eletrocardiograma, eletromiograma e respirar o ritmo de um rato andando livremente

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural circuits. Due to the fractional change in the light signal, it has been difficult to use this method as a quantitative assay. This article describes special optics and slice handling systems, which render this technique stable and reliable. The present article demonstrates the slice handling, staining, and recording of the VSD-stained hippocampal slices in detail. The system maintains physiological conditions for a long time, with good staining, and prevents mechanical movements of the slice during the recordings. Moreover, it enables staining of slices with a small amount of the dye. The optics achieve high numerical aperture at low magnification, which allows recording of the VSD signal at the maximum frame rate of 10 kHz, with 100 pixel x 100-pixel spatial resolution. Due to the high frame rate and spatial resolution, this technique allows application of the post-recording filters that provide sufficient signal-to-noise ratio to assess the changes in neural circuits.

We introduce a reproducible and stable optical recording method for brain slices using voltage-sensitive dye. The article describes voltage-sensitive dye staining and recording of optical signals using conventional hippocampal slice preparations.

Gravação ótica do único-fóton do largo-campo em fatias do cérebro usando a tintura tensão-sensível

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural ...

High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance between in vitro simplification and recreating the complex in vivo environment, along with the main aim, shared by all screening strategies, of obtaining robust and reliable data, highly predictive for in vivo translation.
In the field of demyelinating diseases, the majority of drug screening strategies are based on immortalized cell lines or pure cultures of isolated primary oligodendrocyte precursor cells (OPCs) from newborn animals, leading to strong biases due to the lack of age-related differences and of any real pathological condition or complexity.
Here we show the setup of an in vitro system aimed at modeling the physiological differentiation/maturation of neural stem cell (NSC)-derived OPCs, easily manipulated to mimic pathological conditions typical of demyelinating diseases. Moreover, the method includes isolation from fetal and adult brains, giving a system which dynamically differentiates from OPCs to mature oligodendrocytes (OLs) in a spontaneous co-culture which also includes astrocytes. This model physiologically resembles the thyroid hormone-mediated myelination and myelin repair process, allowing the addition of pathological interferents which model disease mechanisms. We show how to mimic the two main components of demyelinating diseases (i.e., hypoxia/ischemia and inflammation), recreating their effect on developmental myelination and adult myelin repair and taking all the cell components of the system into account throughout, while focusing on differentiating OPCs.
This spontaneous mixed model, coupled with cell-based high-content screening technologies, allows the development of a robust and reliable drug screening system for therapeutic strategies aimed at combating the pathological processes involved in demyelination and at inducing remyelination.

We describe the production of mixed cultures of astrocytes and oligodendrocyte precursor cells derived from fetal or adult neural stem cells differentiating into mature oligodendrocytes, and in vitro modeling of noxious stimuli. The coupling with a cell-based high-content screening technique builds a reliable and robust drug screening system.

Análise de diferenciação e maturação de rastreamento de alto conteúdo das culturas celulares precursoras do Oligodendrocyte, derivadas de células-tronco-tronco-tronco-de-alto teor

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance ...