<p class="jove_content"&gt; O trabalho do autor foi apoiado por bolsa de investigação-ID BOF 2006 da Universidade de Antuérpia (concedido a PPO e AVdL), pela bolsa de investigação G.0136.11 e G.0130.11 (concedido a AVdL, ZB e PPO) e 1.5.021.09. N.00 (concedido a PPO) do Fundo de Pesquisa Científica-Flanders (FWO-Vlaanderen, Bélgica), pela SBO bolsa de investigação IWT-60838: BRAINSTIM do Instituto Flamengo para a Ciência e Tecnologia (concedida a ZB e AVDL), em parte de uma pesquisa Methusalem concessão do governo flamengo (concedido a ZB), em parte, pela EC-FP6-NoE DIMI (LSHB-CT-2005-512146), EC-FP6-NoE EMIL (LSHC-CT-2004-503569) , e pelo Inter Universidade atração poloneses IUAP-NIMI-P6/38 (concedido a AVDL). Nathalie De Vocht tem um PhD bolsa de estudo a partir do FWO-Vlaanderen. Peter Ponsaerts é um pós-doutorado da FWO-Vlaanderen.</p

1. Preparação de células <ol><li> Experiências deve ser iniciada usando ex vivo populações de células-tronco cultivadas geneticamente modificadas para expressar as proteínas repórter da luciferase e EGFP. Aqui usamos luciferase / eGFP-expressam murinos derivadas da medula óssea (células estromais BMSC-Luc/eGFP) como anteriormente descrito por Bergwerf et al. 2, 5. </li><li> Dois dias antes de marcação celular, as células BMSC-Luc/eGFP em placas a uma densidade de 8 x 10 células por 5 T75 frasco de cultura em meio de expansão 15 ml completo (CEM) suplementado com 1 ug / ml Puromycine. </li><li> Permitir que as células a crescer durante 48 horas a 37 ° C e 5% de CO 2. </li><li> Adicionar 5 x 10 6 Glacial azul micron de tamanho de partículas de óxido de ferro (GB MPIO) por ml de meio de cultura para a cultura de células em crescimento (total: 75 x 10 6 partículas por meio de cultura 15 ml em um frasco de cultura T75). </li><li> Incubar durante 16 horas para permitir a captação de partículas através de endocitose. </li><li> Lavar as partículas restantese permitir que as células crescer durante uma hora adicional 24 para obter uma distribuição de células de confluência e homogénea da MPIO GB. </li><li> Validate captação de partículas visualmente usando microscopia de fluorescência através da contagem do rácio de EGFP + e MPIO GB + de células para a quantidade total de EGFP + de células. </li><li> Lavar as células GB MPIO-rotulados BMSC-Luc/eGFP duas vezes com 10 ml de PBS e as células de colheita sequência de tripsina-EDTA tratamento (5 ml por frasco de cultura T75, durante 5 minutos). </li><li> Lave as células colhidas e ressuspender em concentração final de 133 x 10 6 células / ml em PBS. </li><li> Manter as células em gelo até ao transplante. </li></ol> 2. Implantação de células no SNC de Ratos <ol><li> Anestesiar ratinhos por uma injecção intraperitoneal de uma cetamina (80 mg / kg) + xilazina (16 mg / kg) mistura. </li><li> Aguarde 5 minutos para permitir que os ratos para adormecer. </li><li> Raspar a cabeça de rato para permitir manipulações estéreis. </li><li> Posicione o mouse em um quadro estereotáxico. </li><li> Desinfectares aa pele e os olhos molhado dos ratinhos para evitar a desidratação. </li><li> Fazer uma incisão na linha média do couro cabeludo para expor o crânio, e perfurar um orifício no crânio utilizando uma broca dental rebarba nas coordenadas dada: 2 mm posterior e 2 mm lateral à bregma. </li><li> Vórtice da suspensão de células de forma breve e aspirar a suspensão de células na seringa. </li><li> Coloque a agulha da seringa, a uma profundidade de 2,5 mm sob a dura-máter e permitir que o equilíbrio de pressão durante 1 minuto. </li><li> Injectar uma suspensão de células 3 ul (4 x 10 5 células) a uma profundidade de 2 mm sob a dura-máter e com uma velocidade de 0,70 ul / min utilizando uma bomba de injecção de micro-automatizado. </li><li> Esperar por um período adicional de 5 minutos, para evitar o refluxo da suspensão de células injectadas. </li><li> Lentamente, retirar a agulha e desinfectar as fronteiras da pele antes da sutura da pele. </li><li> Injectar 300 solução de NaCl a 0,9% uL por via subcutânea, a fim de evitar a desidratação, e colocar o rato sob uma lâmpada de aquecimento para recuperar da anestesia. Administrar post-operatório analgésico em conformidade com as normas institucionais. </li></ol> 3. Na bioluminescência in vivo <ol><li> Anestesiar os ratos por uma mistura de 3% de isoflurano e oxigénio. </li><li> Coloque os ratos no Imager Photon e reduzir o nível de anestesia para 1,5%, isoflurano e oxigênio. </li><li> Injectar 150 mg de D-luciferina por kg de peso corporal por via intravenosa. </li><li> Adquirir imagem por 5 minutos, utilizando o software Vision Photo. </li><li> Executar o processamento de imagem usando o software M3vision. Quantificar o sinal observado utilizando regiões de juro fixa. </li></ol> 4. Em Ressonância Magnética vivo <ol><li> Anestesiar os ratos com 3% de isoflurano em uma mistura de O 2: N 2 O (3:7). </li><li> Colocar os ratinhos no limitador de um sistema horizontal MR 9.4T e reduzir a anestesia nível de 1% de isoflurano em uma mistura de O 2: N 2 O (3:7). </li><li> Molhe os olhos dos camundongos para evitar desidrataçãoção, anexar uma sonda rectal de controlar a temperatura do corpo e monitorar a taxa de respiração pela colocação de um sensor de baixo da barriga do rato. </li><li> Manter a taxa de respiração em 110 ± 10 respirações por minuto e manter a temperatura corporal constante dentro de uma estreita faixa de 37 ± 0,5 ° C. </li><li> Coloque a bobina RF superfície sobre a parte superior da cabeça do rato e da posição do rato no meio do magneto. </li><li> Adquirir um conjunto de 10 coronais ponderadas em T2 spin-eco (SE) imagens para obter informações anatômica específica e T2 * ponderadas gradiente eco (GE) imagens, a fim de estudar a migração de células-tronco com uma resolução no plano de 70 mM 2. Definir parâmetros de seqüência da seguinte forma: tempo de repetição (TR): 500 ms, tempo de eco (TE): 8 ms (seqüência GE) e tempo de repetição (TR): 4200 ms, tempo de eco (TE): 12,16 ms (SE seqüência); campo de visão (FOV): 18x18 mm 2, matriz: 256x256, 1 fatia de espessura mm e 1 fatia de separação mm (Paravision software 5.1). </li><li> Realizar o processamento de dadosusando software Amira 4,0. </li></ol> 5. Post Mortem Histologia <ol><li> Anestesiar os ratos muito profundamente por inalação de um oxigénio isofluorano (4%), (0,5 L / min) e azoto (1 l / min) a mistura durante 2 minutos. Sacrifício dos camundongos por deslocamento cervical. </li><li> Remover o cérebro do rato a partir do crânio e fixar o tecido cerebral em paraformaldeído a 4% em PBS durante 2 horas. </li><li> Desidratar o tecido do cérebro, colocando o cérebro, posteriormente, em diferentes gradientes de sacarose: 2 h em sacarose a 5% em PBS, 2 horas em 10% de sacarose em PBS, durante a noite em sacarose a 20% em PBS. </li><li> Congelar o tecido do cérebro utilizando azoto líquido e armazenar o tecido à temperatura de -80 ° C até seccionamento. </li><li> Seção tecido do cérebro em 10 mM de espessura, utilizando um criostato. </li><li> Tela unstained criossecções para a fluorescência azul a partir das partículas GB MPIO e fluorescência verde a partir das células que expressam EGFP usando um microscópio de fluorescência. </li><li> Tela imaculada criosecções paraverde / vermelho fluorescência de fundo a partir de células inflamatórias. </li><li> Realizar colorações mais imuno-histoquímica e imunofluorescência para identificar populações de células endógenas (por exemplo, a microglia, astrócitos, células T, ...) que interagem com enxertos celulares. </li></ol> 6. Os resultados representativos Nós aqui apresentadas visualmente uma sequência otimizada de eventos para imagiologia multimodal sucesso da luciferase / eGFP expressar-populações (caule) de células no sistema nervoso central de camundongos. Na primeira, os descritos GB MPIO resultados de rotulagem procedimento em rotulagem altamente eficiente de células BMSC-Luc/eGFP, que podem facilmente ser validados utilizando microscopia de fluorescência (Figura 2A). Em seguida, mediante enxerto de GB MPIO rotulado BMSC-Luc/eGFP no SNC de ratinhos, a sobrevivência do enxerto celular pode ser monitorizada por in vivo BLI com base na actividade da luciferase (Figura 2B). Além disso, a localização exacta das células enxertadas pode ser monitorizada por in vivo </em&gt; RM com base no teor de ferro do MPIO GB (Figura 2C). Finalmente, a análise histológica permite a validação dos resultados obtidos por BLI e RM. Sobrevivência estável foi confirmada por uma expressão eGFP estável no tempo (Figura 2D). Para isso, colocalização de EGFP-expressando células com azul fluorescente MPIO permite a determinação exacta da localização e sobrevivência das células transplantadas. Além disso, este rotulagem dupla fluorescente das células-tronco reduz a possibilidade de observar resultados falsos positivos baseados em fluorescência de fundo criado por células inflamatórias 5. <img alt="Figura 1." src="/files/ftp_upload/3906/3906fig1.jpg" /> Figura 1. Visão geral da seqüência de manipulação <ol class="lroman"><li> Marcação celular: BMSC-Luc/eGFP são rotulados com 5 x 10 6 partículas GB MPIO por ml de meio de cultura. Após 16 hr de incubação e um período de repouso seguinte de 24 horas, as células são colhidas paraimplante intracerebral. </li><li> Implante de células: GB MPIO rotulado BMSC-Luc/eGFP são enxertados no cérebro do rato nas coordenadas seguinte: bregma -2 mm, 2 mm direita da linha média e 2 mm sob dura. </li><li> In vivo BLI: administração intravenosa de 150 mg D-luciferin/kg peso corporal, a luz gerada foi adquirida durante 5 minutos usando o Imager Photon. </li><li> In vivo RM: Um conjunto de 10 coronais ponderadas em T2 spin-eco (SE) imagens e T2 * ponderadas gradiente eco (GE) imagens com uma resolução no plano de 70 mM 2 foram adquiridos. Parâmetros de sequência foram as seguintes: tempo de repetição (TR): 500 ms, tempo de eco (TE): 8 ms (sequência GE) e tempo de repetição (TR): 4200 ms, tempo de eco (TE): 12,16 ms (sequência SE); campo de visão (FOV): 18x18 mm 2, matriz: 256x256, 1 fatia de espessura mm e 1 fatia de separação mm. </li><li> Post-mortem histologia: cryosection espessura 10μm foram selecionados para eGFP expressar e azul fluorescente etiquetado BMSC ucantar um microscópio de fluorescência. </li></ol><img alt="Figura 2." src="/files/ftp_upload/3906/3906fig2.jpg" /> Figura 2. Imagem Multimodal de enxertos de células-tronco <ol class="ualpha"><li> Microscopia de imunofluorescência direta da Glacial azul (GB) MPIO rotulado BMSC-Luc/eGFP. </li><li> In vivo BLI de camundongos injetados com 4x10 5 GB MPIO rotulado células BMSC-Luc/eGFP na semana 1 e semana 2 após a implantação. A análise estatística dos sinais detectados BLI na semana 1 e semana 2 pós-implantação. Os dados são expressos como o número médio de fotões por segundo por esterradiano por centímetro quadrado (pH / s / SR / cm 2) a partir de um período de tempo 5 minutos + / - erro padrão calculados a partir de uma região de interesse fixa em ratinhos apresentando um sinal claro BLI no local da injecção. </li><li> Coronal bidimensional de ressonância magnética GB MPIO rotulado BMSC-Luc/eGFP (painel da esquerda: (i) ponderação T2 spin echo e (ii) T2 *-weighted imagem eco gradiente) e sem rótulo BMSC-LUC / eGFP (painel da direita: (iii) ponderação T2 spin echo e (vi) T2 *-weighted imagem eco gradiente) enxertos em cérebro de camundongo. </li><li> A análise histológica do MPIO GB rotulado implantes BMSC-Luc/eGFP na semana 2 pós-implantação. (I) a análise de imunofluorescência directa para a localização de EGFP-expressar e MPIO GB enxertos BMSC-Luc/eGFP rotulados. (Ii) detalhe ampliação elevada de (i). (Iii) coloração imunofluorescente para o antigénio GFAP, indicando o desenvolvimento de tecido cicatricial no astrocítica circundante de GB MPIO rotulado BMSC-Luc/eGFP. (Vi) coloração de imunofluorescência para Iba-1 antígeno, indicando a invasão e ao redor da GB MPIO rotulado BMSC-Luc/eGFP por microglia. </li></ol>

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P., Neal, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ABCG2/BCRP+expression+modulates+D-Luciferin+based+bioluminescence+imaging.">ABCG2/BCRP expression modulates D-Luciferin based bioluminescence imaging.</a> Cancer Res. 67, 9389-9397 (2007).</li><li>Brightwell, G., Poirier, V., Cole, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-dependent+and+cell+cycle-dependent+expression+from+a+cytomegalovirus-based+mammalian+expression+vector.">Serum-dependent and cell cycle-dependent expression from a cytomegalovirus-based mammalian expression vector.</a> Gene. 194, 115-123 (1997).</li><li>Grassi, G., Maccaroni, P., Meyer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+of+DNA+methylation+and+histone+deacetylation+activate+cytomegalovirus+promoter-controlled+reporter+gene+expression+in+human+glioblastoma+cell+line+U87.">Inhibitors of DNA methylation and histone deacetylation activate cytomegalovirus promoter-controlled reporter gene expression in human glioblastoma cell line U87.</a> Carcinogenesis. 24, 1625-1635 (2003).</li><li>Krishnan, M., Park, J. M., Cao, F. <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+epigenetic+modulation+on+reporter+gene+expression:+implications+for+stem+cell+imaging.">Effects of epigenetic modulation on reporter gene expression: implications for stem cell imaging.</a> FASEB J. 20, 106-108 (2006).</li><li>Svensson, R. U., Barnes, J. M., Rokhlin, O. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapeutic+agents+up-regulate+the+cytomegalovirus+promoter:+implications+for+bioluminescence+imaging+of+tumor+response+to+therapy.">Chemotherapeutic agents up-regulate the cytomegalovirus promoter: implications for bioluminescence imaging of tumor response to therapy.</a> Cancer Res. 67, 10445-10454 (2007).</li></ol>

Os autores declaram nenhum conflito de interesse.

Neste relatório, nós descrevemos um protocolo otimizado para a combinação de três modalidades de imagem complementares (BLI, ressonância magnética e histologia) para a caracterização detalhada dos implantes celulares no sistema nervoso central de camundongos imunes competentes. Uma combinação de rotulagem de gene repórter de células, com base na modificação genética com os genes repórter luciferase do pirilampo e EGFP, e uma célula de rotulagem directa com a GB MPIO, conduz a uma avaliação precisa de...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> IMDM </td><td> Lonza </td><td> BE12-722F </td><td> Componente do CEM meio de crescimento celular </td></tr><tr><td> Soro fetal de bovino </td><td> Gibco </td><td> 10270-106 </td><td> Componente do CEM meio de crescimento celular </td></tr><tr><td> De soro de cavalo </td><td> Gibco </td><td> 1605-122 </td><td> Componente do CEM meio de crescimento celular </td></tr><tr><td> Penicilina-estreptomicina </td><td> Gibco </td><td> 15140 </td><td> Componente do CEM meio de crescimento celular </td></tr><tr><td> Fungizone </td><td> Gibco </td><td> 15290-018 </td><td> Componente do CEM meio de crescimento celular </td></tr><tr><td> PBS </td><td> Gibco </td><td> 14190 </td><td></td></tr><tr><td> Puromycine </td><td> Invivogen </td><td> formiga-pr-1 </td><td></td></tr><tr><td> tripsina </td&gt; <td> Gibco </td><td> 25300 </td><td></td></tr><tr><td> GB MPIO </td><td> Bangs Laboratories </td><td> ME04F/7833 </td><td></td></tr><tr><td> D-luciferina </td><td> Promega </td><td> E1601 </td><td></td></tr><tr><td> De cetamina (Ketalar) </td><td> Pfizer </td><td></td><td></td></tr><tr><td> Xilazina (Rompun) </td><td> Bayer Os cuidados de saúde </td><td></td><td></td></tr><tr><td> Isoflurano </td><td> Isoflo </td><td> 05260-05 </td><td></td></tr><tr><td> 0,9% de solução de NaCl </td><td> Baxter </td><td></td><td></td></tr><tr><td> paraformaldeído </td><td> Merck </td><td> 1.04005.1000 </td><td></td></tr><tr><td> sacarose </td><td> Applichem </td><td> A1125 </td><td></td></tr><tr><td> Micro-injecção de bomba </td><td> KD científica </td><td> KDS100 </td><td></td></tr><tr><td> Photon imager </td><td> Biospace Lab </td><td></td><td></td></tr><tr&gt; <td> 9.4T scanner de MR </td><td> Bruker BioSpin </td><td> Biospec 94/20 USR </td><td></td></tr><tr><td> BX51 microscópio </td><td> Olimpo </td><td> BX51 </td><td></td></tr><tr><td> Mycrom HM criostato </td><td> Prosan </td><td> HM525 </td><td></td></tr><tr><td> seringa </td><td> Hamilton </td><td> 7635-01 </td><td></td></tr><tr><td> Agulha de calibre 30 </td><td> Hamilton </td><td> 7762-03 </td><td></td></tr><tr><td> Foto Visão software </td><td> Biospace Lab </td><td></td><td></td></tr><tr><td> M3vision software </td><td> Biospace Lab </td><td></td><td></td></tr><tr><td> Paravision software 5,1 </td><td> Bruker BioSpin </td><td></td><td></td></tr><tr><td> Amira 4,0 software </td><td> Imagem Visage </td><td></td><td></td></tr></tbody></table>

multimodal imaging of stem cell implantation in the central nervous system of mice

Durante a última década, o transplante de células estaminais ganhou interesse crescente como primário ou secundário modalidade terapêutica para uma variedade de doenças, tanto em estudos pré-clínicos e clínicos. No entanto, até à data resultados referentes resultado funcional e / ou tecido regeneração transplante de células estaminais seguinte são bastante diversos. Geralmente, um benefício clínico é observado sem uma profunda compreensão do mecanismo subjacente (s) 1. Portanto, os esforços têm múltiplas levou ao desenvolvimento de diferentes modalidades de imagens moleculares para monitorar enxerto de células-tronco com o objectivo último para avaliar com precisão o destino, sobrevivência e fisiologia de células estaminais enxertados e / ou do seu ambiente de micro-. Alterações observadas em um ou mais parâmetros determinados por imagem molecular pode estar relacionado com o efeito observado clínica. Neste contexto, os nossos estudos focalizam o uso combinado de imagem de bioluminescência (BLI), ressonância magnética (MRI) e Analysi histológicas para avaliar células estaminais do miocárdio. BLI é normalmente usado para executar de forma não invasiva de rastreamento de células e monitorar a sobrevivência das células no tempo após o transplante de 2-7, com base numa reacção bioquímica onde as células expressando o gene repórter da luciferase-são capazes de emitir luz interacção seguinte com o seu substrato (por exemplo, D- luciferina) 8, 9. RM, por outro lado é uma técnica não-invasivo que é clinicamente aplicável 10 e pode ser usado para localizar precisamente os enxertos celulares com resolução muito elevada 11-15, embora a sua sensibilidade altamente depende do contraste gerado após marcação das células com um agente de contraste para IRM . Finalmente, post-mortem análise histológica é o método de escolha para validar os resultados de pesquisa obtidos com técnicas não-invasivas com maior resolução e sensibilidade. Além disso end-point análise histológica nos permite realizar uma análise detalhada fenotípica das células enxertadas e / ou tecido circundante, based sobre a utilização de proteínas repórter fluorescentes e / ou rotulagem célula directa com anticorpos específicos. Em resumo, estamos aqui visualmente demonstrar a complementaridade de BLI, ressonância magnética e histologia para desvendar células-tronco e diferente / ou meio ambiente associada seguintes características de células-tronco de enxerto no sistema nervoso central de camundongos. Como um exemplo, derivadas da medula óssea células estromais, geneticamente modificados para expressar o reforço de Proteína Fluorescente Verde (eGFP) e luciferase do pirilampo (FLUC), e marcadas com azul fluorescentes micron de tamanho de partículas de óxido de ferro (MPIOs), vai ser enxertados na SNC de camundongos imuno-competentes e os resultados serão monitorados por BLI, ressonância magnética e histologia (Figura 1).

Watch this Scientific Journal Video about Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice at JoVE.com

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

Este artigo descreve uma sequência otimizada de eventos para imagiologia multimodal de enxertos celulares no cérebro de roedores por meio de: (i) na bioluminescência vivo e ressonância magnética, e (ii) a análise post mortem histológica. A combinação destas modalidades de imagem em um único animal permite uma avaliação do enxerto celular com alta resolução, sensibilidade e especificidade.

Imagem Multimodal de implante de células-tronco no sistema nervoso central de camundongos

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

multimodal-imaging-stem-cell-implantation-central-nervous-system

Research

JoVE Journal

Neuroscience

11.0K visualizações.  University of Antwerp. Este artigo descreve uma sequência otimizada de eventos para imagiologia multimodal de enxertos celulares no cérebro de roedores por meio de: (i) na bioluminescência vivo e ressonância magnética, e (ii) a análise post mortem histológica. A combinação destas modalidades de imagem em um único animal permite uma avaliação do enxerto celular com alta resolução, sensibilidade e especificidade.

Vídeo: Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

Transecção do nervo óptico: Um Modelo de Apoptose Neuron Adultos no Sistema Nervoso Central

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

Neurociência

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

Produção de lentivirais para Células Transdução do Sistema Nervoso Central

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

Marcação de células individuais no Sistema Nervoso Central de<em&gt; Drosophila melanogaster</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Entrega intraventricular direta de medicamentos para o Sistema Nervoso Central Rodent

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

In vivo Interrogatório de Sistema Nervoso Central Translatome por Polyribosome Fracionamento

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

In vivo Optogenetic estimulação do sistema nervoso central de roedores

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and &#947;-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

A custom protocol is described to differentiate mouse ES cells into defined populations of highly pure neurons exhibiting functioning synapses and emergent network behavior. Electrophysiological analysis demonstrates the loss of synaptic transmission following exposure to botulinum neurotoxin serotypes /A-/G and tetanus neurotoxin.

Avaliação Funcional de neurotoxinas Biológica em Culturas em Rede de Células-Tronco derivadas do Sistema Nervoso Central Neurônios

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, ...

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

In Situ Ca 2+ Imagem do Sistema Nervoso Entérico

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

Clearing óptica do Sistema Nervoso Central Rato Usando a claridade passiva

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster

This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways.

Eletrofisiológica gravação da atividade do sistema nervoso Central de terceiro ínstar Drosophila Melanogaster

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield ...