Este trabalho foi financiado pelo National Institutes of Health Grant RO1DC002756 ea Purdue Research Foundation. Agradecemos a Doris Chang Wu e Wiese para o conselho com experimentos e McPhail Rodney para ajudar com números.

Receitas Pinto Ringers <table border="1"><tbody><tr><td> 7,2 g </td><td> NaCl </td></tr><tr><td> 0,23 g </td><td> CaCl 2 + 2H 2 O </td></tr><tr><td> 0,37 g </td><td> KCl </td></tr><tr><td> 0,115 g </td><td> Na 2 HPO 4 </td></tr><tr><td> 900 ml </td><td> Água (grau de cultura de tecidos) </td></tr></tbody></table> Nota: Ajustar o pH para 7,4. Trazer um volume final de 1000 ml com água. PBS 10X <table border="1"><tbody><tr><td> 40 g </td><td> NaCl </td></tr><tr><td> 1 g </td><td> KCl </td></tr><tr><td> 7 g </td><td> Na 2 HPO 4 </td></tr><tr><td> 1,2 g </td><td> KH 2 PO 4 </td></tr><tr><td> 450 ml </td><td> Água DEPC </td></tr></tbody></table> Nota: Ajustar o pH para 7,4. Traga finaisvolume para 500 ml com água. Concentração de trabalho (1X) é feita por diluição de 1 parte de ações 10x com 9 partes de cultura de tecidos de água de grau, como o obtido com um sistema de irradiação UV Millipore para gerar água com 18 cm de condutividade mohms. SAG médio explante segurando <ul><li> DMEM/F12 com L-glutamina, 10 mM Hepes </li><li> Insulina 10%, transferindo, Selenium (ITS +1) </li><li> 1% pen-strep </li></ul> Explante meio de cultura <ul><li> SAG médio explante segurando </li><li> 10 ng / ml neurotrofina-3 (NT-3) </li><li> 10 ng / ml fator neurotrófico ciliar (CNTF) </li></ul> Dissecção da medula espinhal e médias empresas holding <ul><li> L-15 </li><li> 10% de soro fetal bovino </li><li> 1% pen-strep </li></ul> Ovos incubar a 37-38 ° C. Esterilizar ferramentas e dissecar prato Sylgard dissecar em etanol 70%. Dissecando pratos e ferramentas também podem ser esterilizados UV durante a noite. <ol><li> Coloque as contas em um microtubo com 1 ml estéril PBS, misturar, e esperar que os grânulos se depositem no fundo do tubo. </li><li> Lave as contas várias vezes através da remoção do sobrenadante após as contas se instalaram e ressuspender em PBS. </li><li> Incubar as contas em proteína purificada (diluído em PBS) ou PBS sozinho (Control) por 1 hora em temperatura ambiente. </li><li> Lave as contas em PBS e armazenar em uma placa de 24 poços com 1ml de PBS até à sua colocação em colágeno. </li></ol> 2. Dissecção ganglionar Statoacoustic <ol><li> E4 em remover o embrião do ovo e coloque-o em um prato com solução de Ringer pinto para remover membranas embrionárias. </li><li> Coloque o embrião numa placa de Petri forrada Sylgard © preenchido com HBSS frio e posição do embrião em seu lado com a vesícula ótica voltada para cima. Pin o embrião para o prato utilizando os pinos de dissecação. </li><li> Usando dois pinos de dissecação, faça um corte horizontal através da pele imediatamente ventral aobolsas canal, aproximadamente onde se encontra com o ducto coclear. Faça um corte vertical anterior e posterior ao SAG. Use uma pinça ou um pino de dissecação para levantar e remover o retalho de pele fronteira com os três cortes. Nota: O SAG está localizado na borda do anteroventral otocyst, a meio caminho entre a bolsa dorsal que é facilmente visível com iluminação brightfield base, eo ducto coclear ventral quais projetos ventromedially e é obscurecido pelos arcos da faringe. O ducto coclear se alonga para sua dimensão dorsoventral aumentará progressivamente em E4 entre Hamburger-Hamilton estágios 23-25 ​​setembro. </li><li> Puxe a otocyst em uma direção posterior para separá-lo do SAG. Deslocar o tecido ao redor da SAG com pinos de dissecção e remova cuidadosamente o SAG do embrião usando # 55 fórceps. Remover grandes pedaços de tecido mesenquimal e feixes de nervos que se projeta da SAG. </li><li> Usando a ponta da pipeta wide-boca, a transferência do explante a um24 poços da placa com 0,5 ml explante SAG segurando médio. Mantenha explantes no gelo ou em temperatura ambiente por até 4 horas. </li></ol> 3. Dissecção da medula espinhal <ol><li> Remover o embrião E6 do ovo e coloque-o em um prato contendo solução de Ringer com pinto. Retire a cabeça e as membranas embrionárias. </li><li> Colocar o corpo em um prato Sylgard © dissecar contendo meio de dissecção fria da medula espinhal. Posição e pin o embrião &quot;para baixo ventral&quot; e caudal de frente para o experimentador. Coloque os pinos de dissecação através de cada membro e até o final anterior. </li><li> Utilizando uma pinça e / ou dissecar pinos, remova cuidadosamente a pele e tecido, se movendo em uma direção ventral, até que a superfície dorsal da medula espinhal é visível. </li><li> Cortar a linha média dorsal da medula espinhal com um pino de dissecação, em uma posterior à direção anterior, em todo o comprimento da medula espinhal. Isso cria um &quot;livro aberto&quot;, como os lados direito e esquerdo da medula espinhal em separado. </li><li&gt; Remover o tecido circundante, lateralmente, para isolar a medula espinhal e expor o gânglio da raiz dorsal (DRG). Remover o DRG e meninges, esfregando um pino de dissecação ou fórceps entre a medula espinhal e DRG. </li><li> Remover a medula espinhal do corpo, segurando a extremidade caudal-a maior parte da medula espinhal com uma pinça e levantando-o e longe do experimentador. Remover DRG restantes e meninges. </li><li> Segure uma das extremidades do cabo da coluna vertebral com uma pinça (ou fixá-lo ao prato) e usar a tesoura para cortar Vannas ao longo da linha média ventral, a bissetriz da medula espinhal. </li><li> Segure uma das extremidades do explante com uma pinça para imobilizar o tecido e usar a tesoura para cortar Vannas explantes pequenos (100-500 hm de comprimento) ao longo do comprimento do tecido. A placa de piso pode ser reconhecido como um espessamento do tecido claro ao longo de uma borda explante. </li><li> Use a ponta da pipeta de boca larga para transferir explantes a uma placa de 24 poços com 0,5 ml de médio dissecção fria. Mantenha explantes no gelo para manter a viabilidade do tecido, fou até 4 horas. </li></ol> 4. Explante cultura com proteínas purificadas para testar capacidade de resposta neurite <ol><li> Prepare uma solução de colágeno 1,5 mg / ml em um tubo de 15 ml cônico, no gelo, de acordo com as instruções do fabricante. </li><li> Verificar a solução de colágeno tem um pH de 7-7,4 papel usando indicador de pH. Ajustar o pH pela adição de NaOH em volumes de 1 mL. </li><li> Transferência de explantes a uma placa de 24 poços usando uma pipeta de boca larga e excesso de líquido aspirado. Explantes múltiplas podem ser cultivadas em um bem, mas deve ser colocada, pelo menos, 500 mm afastados uns dos outros. </li><li> Adicionar 0,5 ml de colágeno para o bem que contém o explante. Posição do explante sobre a superfície inferior do poço. Certifique-se de definir cada cultura completamente antes de prosseguir com o bem próxima, pois o colágeno começa a polimerizar em temperatura ambiente. </li><li> Coloque a placa de cultura sobre a 37 ° C mais quente deslizar por 30-45 min para polimerizar o colágeno. Quando o colágeno tem Polymeterizada ele vai se assemelhar a um gel. </li><li> Adicionar 0,5 ml de meio de cultura morno explante, suplementada com proteínas purificadas (diluído em PBS) ou PBS (controle) e incubar a 37 ° C CO, 5% 2. Efeitos sobre o crescimento de neuritos deve ser visível por 24 horas. NOTA: Nós temos utilizado o mesmo protocolo para explantes SAG cultura até 42 horas. </li></ol> 5. Explante co-cultura com proteína revestido contas para testar crescimento de neuritos direcional <ol><li> Comece com as etapas da cultura explante 4,1-4,4. </li><li> Usando uma pipeta, transferir 1-5 grânulos de PBS para o bem que contém a solução de colágeno e explante. </li><li> Use uma pinça para posicionar contas 5-50 mM a partir da borda do explante. </li><li> Coloque o prato sobre a 37 ° C mais quente deslizar por 30-45 min para polimerizar o colágeno. O tecido ou talão pode tornar-se deslocado à medida que o colágeno sofre polimerização. Verifique as culturas e re-posição tecido e miçangas durante primeiros 5 min. </li><li> Adicionar 0,5 ml SAG mídia para cada poço e incubar por 24 horas. </li></ol> 6. Visualização do crescimento de neuritos por imuno-histoquímica <ol><li> Enxágüe em PBS culturas e correção para uma hora em temperatura ambiente em paraformaldeído 4% em PBS. </li><li> Solte o gel das paredes dos poços, traçando em torno da borda dos géis com a ponta arredondada de uma espátula micro teflon. Executar a immunostain com o gel nos poços. Lavar diversas vezes em PBS. </li><li> Incubar em 0,5 ml de solução de bloqueio (10% soro bovino, 0,1% Triton X-100, azida de sódio a 0,1% em PBS) durante a noite a 4 ° C. </li><li> Incubar em 0,5 ml do anticorpo primário (anti-β-tubulina anticorpo diluído 1:500 em solução de bloqueio) overnight a 4 ° C. </li><li> Lavar diversas vezes em PBS. Incubar em 0,5 ml de anticorpo secundário (Alexa flúor 488 cabra anti-camundongo IgG 2b diluído 1:500) durante a noite a 4 ° C. A partir deste passo em frente, manter as placas no escuro ou embrulhe em papel alumínio, devido à sensibilidade à luz deanticorpos secundários. </li><li> Lavar diversas vezes em PBS e armazenar a 4 ° C em PBS por até uma semana. </li></ol> 7. Resultados representante <img alt="Figura 1" src="/files/ftp_upload/3600/3600fig1.jpg" /> Figura 1. Resultados representativos dos efeitos de promoção de neurite purificada NT-3 e NT-3-revestido contas em explantes SAG E4 pinto. SAG corpos de neurônios e células neurites foram imunocoloração com β-tubulina e fotografada com a objetiva de 10x em um microscópio confocal. Após 24 horas in vitro, explantes SAG exibido conseqüência maior neurite na presença de 100 ng / ml humano purificado NT-3 adicionado ao meio de cultura celular (1B), comparados aos controles (1A). Neurônios SAG foram co-cultivados com contas embebido em 100 ng / ml NT-3 para demonstrar que uma fonte pontual de NT-3 pode promover localmente crescimento de neuritos. Explantes exibidos mais longos e mais densos conseqüência neurite do lado daexplante de frente para o talão em comparação com o lado oposto (1D) e comparados com controle de culturas com contas embebido em PBS (1C). Barra de escala = 200 mM. <img alt="Figura 2" src="/files/ftp_upload/3600/3600fig2.jpg" /> Figura 2. Resultados representativos dos efeitos de promoção de neurite purificada Wnt5a e Wnt5a revestido contas em E6 cabo pinto espinhal. Explantes medula espinhal foram imunocoloração com β-tubulina e fotografada com um microscópio confocal. Após 24 horas de crescimento na presença de purificada do mouse Wnt5a (400ng/ml), crescimento de neuritos a partir de explantes medula espinhal pintinho foi aumentada (2B) comparados aos controles cultivados sem Wnt5a (2A). Contas embebido em 500 ng / ml Wnt5a, colocado 300-500 mM a partir da borda de explantes da medula espinhal, também promoveu crescimento de neuritos (2D) em relação ao controle de culturas (2C). Similar co-cultura resultados foram publicados anteriormente com Wnt5a células que expressam 8. Barra de escala = 200mM.

<ol>
	<li>Fekete, D. M., Campero, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+guidance+in+the+inner+ear.">Axon guidance in the inner ear.</a> Int. J. Dev. Biol. 51 (6-7), 549-549 (2007).</li><li>Fritzsch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+inner+ear+afferent+connections:+forming+primary+neurons+and+connecting+them+to+the+developing+sensory+epithelia.">Development of inner ear afferent connections: forming primary neurons and connecting them to the developing sensory epithelia.</a> Brain Res. Bull. 60 (5-6), 423-423 (2003).</li><li>Gu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropilin-1+conveys+semaphorin+and+VEGF+signaling+during+neural+and+cardiovascular+development.">Neuropilin-1 conveys semaphorin and VEGF signaling during neural and cardiovascular development.</a> Dev. Cell. 5 (1), 45-45 (2003).</li><li>Tessarollo, L., Coppola, V., Fritzsch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NT-3+replacement+with+brain-derived+neurotrophic+factor+redirects+vestibular+nerve+fibers+to+the+cochlea.">NT-3 replacement with brain-derived neurotrophic factor redirects vestibular nerve fibers to the cochlea.</a> J. Neurosci. 24 (10), 2575-2575 (2004).</li><li>Avila, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-derived+neurotrophic+factor+and+neurotrophin-3+support+the+survival+and+neuritogenesis+response+of+developing+cochleovestibular+ganglion+neurons.">Brain-derived neurotrophic factor and neurotrophin-3 support the survival and neuritogenesis response of developing cochleovestibular ganglion neurons.</a> Dev. Biol. 159 (1), 266-266 (1993).</li><li>Bianchi, L. M., Cohan, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+neurotrophins+and+CNTF+on+developing+statoacoustic+neurons:+comparison+with+an+otocyst-derived+factor.">Effects of the neurotrophins and CNTF on developing statoacoustic neurons: comparison with an otocyst-derived factor.</a> Dev. Biol. 159 (1), 353-353 (1993).</li><li>Juurlink, B. e. r. n. h. a. r. d. H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+Spinal+Somatic+Motoneurons+in+Culture.">Chick Spinal Somatic Motoneurons in Culture.</a> Protocols for Neural Cell Culture.. 59, (2001).</li><li>Fantetti, K. N., Zou, Y., Fekete, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnts+and+Wnt+inhibitors+do+not+influence+axon+outgrowth+from+chicken+statoacoustic+ganglion+neurons.">Wnts and Wnt inhibitors do not influence axon outgrowth from chicken statoacoustic ganglion neurons.</a> Hear. Res. , (2011).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> Journal of Morphology. 88, 49-49 (1951).</li><li>Bourikas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+guides+commissural+axons+along+the+longitudinal+axis+of+the+spinal+cord.">Sonic hedgehog guides commissural axons along the longitudinal axis of the spinal cord.</a> Nat. Neurosci. 8 (3), 297-297 (2005).</li><li>Charron, F., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+brain+wiring+functions+for+classical+morphogens:+a+role+as+graded+positional+cues+in+axon+guidance.">Novel brain wiring functions for classical morphogens: a role as graded positional cues in axon guidance.</a> Development. 132 (10), 2251-2251 (2005).</li><li>Augsburger, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMPs+as+mediators+of+roof+plate+repulsion+of+commissural+neurons.">BMPs as mediators of roof plate repulsion of commissural neurons.</a> Neuron. 24 (1), 127-127 (1999).</li><li>Lyuksyutova, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anterior-posterior+guidance+of+commissural+axons+by+Wnt-frizzled+signaling.">Anterior-posterior guidance of commissural axons by Wnt-frizzled signaling.</a> Science. 302 (5652), 1984-1984 (2003).</li></ol>

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Nós apresentamos um método para dissecar e cultura SAG E4 e E6 explantes medula espinhal, de bico, sob soro livre de condições. Este procedimento é usado atualmente em nosso laboratório para estudar os efeitos de vários ligantes secretada na sobrevivência SAG neurônio e crescimento de neuritos. Aspectos inovadores deste protocolo incluem o uso de um sistema isento de soro, para explantes cultura eo uso de contas embebido em fatores de crescimento para estudar os efeitos sobre crescimento de neuritos SAG. Um mé...

<table border="1"><tbody><tr><td> Reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentário </td></tr><tr><td> Equipamento </td><td></td><td></td><td></td></tr><tr><td> Microscópio de dissecção </td><td></td><td></td><td> Nós usamos uma Leica M80 estereomicroscópio com brightfield base de iluminação </td></tr><tr><td> Deslize mais quentes </td><td> CS &amp; E. </td><td></td><td> Colágeno polimerização </td></tr><tr><td> Frango ovo incubadora </td><td></td><td></td><td></td></tr><tr><td> 37 ° C / 5% CO 2 umidificado de cultura de células incubadora </td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Materiais dissecção </td><td></td><td></td><td></td></tr><tr><td> Sylgard &amp;cópia; placa de Petri revestidas </td><td></td><td></td><td></td></tr><tr><td> 24 poços da placa de cultura de células </td><td> Corning </td><td> 3526 </td><td></td></tr><tr><td> # 5 Dumont forceps </td><td> Multa Ferramentas Ciência </td><td> 11252-30 </td><td></td></tr><tr><td> # 55 Dumont forceps </td><td> Multa Ferramentas Ciência </td><td> 11295-51 </td><td></td></tr><tr><td> Pinos de aço inoxidável de dissecação </td><td> Multa Ferramentas Ciência </td><td> 26002-10 </td><td> Embrião pinagem, dissecção fina </td></tr><tr><td> Moria colher perfurada </td><td> Multa Ferramentas Ciência </td><td> 10370-17 </td><td> Colheita de embriões / transferência </td></tr><tr><td> 200 mL pontas de pipeta de boca larga </td><td> Dot Scientific Inc </td><td> 118-96R </td><td> Transferência de explante </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> E4 e E6 ovos de galinha </td><td></td><td></td><td> &amp; Nbsp; </td></tr><tr><td> Pinto solução de Ringer </td><td></td><td></td><td> Colheita de embriões </td></tr><tr><td> HBSS </td><td> Sigma </td><td> H8264 </td><td> SAG dissecção </td></tr><tr><td> L15 médio </td><td> Sigma </td><td> L5520 </td><td> Médio dissecção da medula espinhal </td></tr><tr><td> Soro fetal bovino </td><td> Atlanta Biologicals </td><td> S11150 </td><td> Médio dissecção da medula espinhal </td></tr><tr><td> Vannas Tesoura </td><td> Precision Instruments mundo </td><td> 501778 </td><td> Dissecção da medula espinhal </td></tr><tr><td>&nbsp;</td><td></td><td></td><td></td></tr><tr><td> Preparação de colágeno </td><td></td><td></td><td></td></tr><tr><td> Rat-tail colágeno tipo I </td><td> BD Biosciences </td><td> 354249 </td><td> Cultura de explantes </td></tr><tr><td> PBS 10X (estéril) </td><td></td><td></td><td> Para neutralize colágeno </td></tr><tr><td> NaOH 1N (estéril) </td><td></td><td></td><td> Para neutralizar colágeno </td></tr><tr><td> Água destilada (estéril) </td><td></td><td></td><td> Para neutralizar colágeno </td></tr><tr><td> pH papéis indicador (6,0-8,1) </td><td> Whatman </td><td> 2629-990 </td><td> Para verificar o pH colágeno </td></tr><tr><td> 15 ml tubos cônicos </td><td> Dot Scientific Inc </td><td> 818-PG </td><td></td></tr><tr><td> 500 mL pontas de pipeta de boca larga </td><td> Dot Scientific Inc </td><td> 119-R100 </td><td> Ao colágeno pipeta </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Cultura de explantes </td><td></td><td></td><td></td></tr><tr><td> DMEM/F12 médio </td><td> Sigma </td><td> D8437 </td><td> Explante meio de cultura </td></tr><tr><td> ITS 1 </td><td> Sigma </td><td> I2521 </td><td> Suplemento médio </td&gt; </tr><tr><td> Penicilina-estreptomicina </td><td> Invitrogen </td><td> 15140-122 </td><td> Suplemento médio </td></tr><tr><td> CNTF (rato) </td><td> Sigma </td><td> C3835 </td><td> Suplemento médio </td></tr><tr><td> NT-3 (humano) </td><td> Sigma </td><td> N1905 </td><td> Suplemento médio </td></tr><tr><td> Purificada do mouse Wnt5a </td><td> R &amp; D Systems </td><td> WN-645-010 </td><td></td></tr><tr><td> Affi-gel gel contas azuis </td><td> Bio-Rad </td><td> 153-7302 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> Imuno-histoquímica </td><td></td><td></td><td></td></tr><tr><td> Paraformaldeído </td><td> Sigma </td><td> P6148 </td><td> Fixação </td></tr><tr><td> PBS </td><td></td><td></td><td> Lavagens </td></tr><tr><td> Triton X-100 </td><td> Sigma </td><td> T9284 </td><td> Solução de bloqueio </td></tr><tr><td> Sódioazida </td><td> Sigma </td><td> S8032 </td><td> Solução de bloqueio </td></tr><tr><td> Soro bovino </td><td> Invitrogen </td><td> 16170-078 </td><td> Solução de bloqueio </td></tr><tr><td> Monoclonal anti-tubulina IgG2b β 1 + anticorpo II </td><td> Sigma </td><td> T8535 </td><td> Etiquetas corpos celulares e neurites </td></tr><tr><td> Alexa flúor 488 cabra anti-rato IgG2 de anticorpos b </td><td> Invitrogen </td><td> A21141 </td><td> Detecta anticorpos β tubulina </td></tr><tr><td> Teflon micro espátula </td><td> VWR </td><td> 57949-033 </td><td> Géis de colágeno liberação do bem </td></tr></tbody></table>

dissection and culture of chick statoacoustic ganglion and spinal cord explants in collagen gels for neurite outgrowth assays

Os órgãos sensoriais do ouvido interno de frango são inervados pelos processos periféricos do gânglio statoacoustic (SAG) neurônios. Inervação órgão sensorial depende de uma combinação de pistas de orientação axônio 1 e fatores de sobrevivência duas localizadas ao longo da trajetória de axônios em crescimento e / ou dentro de seus alvos órgão sensorial. Por exemplo, interferência funcional com uma via clássica de sinalização de orientação do axônio, Semaforina-neuropilin, gerado misrouting de axônios ótica 3. Além disso, vários fatores de crescimento expressos nas metas sensoriais do ouvido interno, incluindo Neurotrofina-3 (NT-3) e Fator neurotrófico derivado do cérebro (BDNF), têm sido manipulados em animais transgênicos, mais uma vez levando a misrouting de axônios SAG 4. Essas mesmas moléculas promover a sobrevivência e crescimento de neuritos de neurônios SAG pinto in vitro 5,6. Aqui, descrevemos e demonstrar o método in vitro estamos atualmente ucantar para testar a capacidade de resposta do neurites SAG pinto de proteínas solúveis, incluindo morfogenes conhecido como o Wnts, bem como fatores de crescimento que são importantes para promover o crescimento SAG neurite e sobrevivência dos neurônios. Usando este sistema modelo, esperamos tirar conclusões sobre os efeitos que ligantes secretados podem exercer sobre a sobrevivência SAG neurônio e crescimento de neuritos. Explantes SAG são dissecados no dia embrionárias 4 (E4) e cultivadas em géis tridimensionais de colágeno sob condições sem soro por 24 horas. Primeiro, a capacidade de resposta neurite é testada por explantes de cultura suplementado com proteína-médio. Então, perguntar se fontes pontuais de ligantes secretada pode ter efeitos direcionais no crescimento de neuritos, são explantes co-cultivados com proteína revestido contas e ensaiadas para a capacidade do talão até localmente promovem ou inibem a conseqüência. Nós também incluímos uma demonstração da dissecção (protocolo modificado 7) e cultura de explantes E6 medula espinhal. Nósrotineiramente usam explantes medula espinhal para confirmar bioatividade das proteínas e proteína-embebido contas, e verificar as espécies reatividade cruzada com tecido de bico, nas condições mesma cultura como explantes SAG. Estes ensaios in vitro são convenientes para a rápida triagem para as moléculas que exercem tróficos (sobrevivência) ou trópico (direcional) efeitos sobre os neurônios SAG, especialmente antes de realizar estudos in vivo. Além disso, este método permite que o teste de moléculas individuais sob condições sem soro, com alta sobrevivência dos neurônios 8.

Watch this Scientific Journal Video about Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays at JoVE.com

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

Demonstramos como dissecar e cultura pinto gânglio statoacoustic E4 e E6 explantes medula espinhal. Explantes são cultivados sob condições sem soro em gel de colágeno 3D por 24 horas. Neuritos resposta é testado com o meio do fator de crescimento suplementado com proteína e revestido contas.

Dissecção e Cultura do Gânglio pintainho Statoacoustic e Explantes da Medula Espinhal em géis de colágeno para Ensaios crescimento de neuritos

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

dissection-culture-chick-statoacoustic-ganglion-spinal-cord-explants

Research

JoVE Journal

Neuroscience

12.0K visualizações.  Purdue University. Demonstramos como dissecar e cultura pinto gânglio statoacoustic E4 e E6 explantes medula espinhal. Explantes são cultivados sob condições sem soro em gel de colágeno 3D por 24 horas. Neuritos resposta é testado com o meio do fator de crescimento suplementado com proteína e revestido contas.

Vídeo: Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

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

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

Dissecção e Cultura de Neurônios da Medula Espinhal comissural embrionárias

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

Neurociência

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

A Unidade Funcional Motor na placa de cultura: Co-cultivo de explantes da Medula Espinhal e as células musculares

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra2. The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or &beta;III tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.

Dissecção e Cultura do mouse e dopaminérgico do estriado explantes em três dimensões Ensaios matriz de colágeno

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

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, ...

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

Dissecção e montagem lateral de embriões Zebrafish: Análise de Desenvolvimento da Medula Espinhal

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

Spinal Cord Injury completo e Cérebro Dissection Protocolo para Wholemount Subsequente<em&gt; In Situ</em&gt; Hibridização em larval da lampreia de mar

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

RNA-Seq Análise de Gene Expression Diferencial em electroporados Pintainho Embryonic Medular

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

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

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

Isolamento e cultura de neurônios da medula espinhal de ratos neonatais

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

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 ...

Induction of Complete Transection-Type Spinal Cord Injury in Mice

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, such as cell transplantation methods, are being researched to overcome the debilitating disabilities arising from SCI. Most pre-clinical animal trials are conducted in rodent models of SCI. While rat models of SCI have been widely used, mouse models have received less attention, even though mouse models can have significant advantages over rat models. The small size of mice equates to lower animal maintenance costs than for rats, and the availability of numerous transgenic mouse models is advantageous for many types of studies. Inducing repeatable and precise injury in the animals is the primary challenge for SCI research, which in small rodents requires high-precision surgery. The transection-type injury model has been a commonly used injury model over the last decade for transplantation-based therapeutic research, however a standardized method for inducing a complete transection-type injury in mice does not exist. We have developed a surgical protocol for inducing a complete transection type injury in C57BL/6 mice at thoracic vertebral level 10 (T10). The procedure uses a small tip drill instead of rongeurs to precisely remove the lamina, after which a thin blade with rounded cutting edge is used to induce the spinal cord transection. This method leads to reproducible transection-type injury in small rodents with minimal collateral muscle and bone damage and therefore minimizes confounding factors, specifically where behavioral functional outcomes are analyzed.

This protocol describes how to create a precise laminectomy for induction of stable transection-type spinal cord injury in the mouse model, with minimal collateral damage for spinal cord injury research.

Indução da lesão da medula espinhal tipo transssecção completa em camundongos

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, ...