Agradecemos Denis Flipo para o conselho de processamento e análise de imagem, e David W. Silversides em cujo laboratório a linha de rato Gata4p [5kb]-GFP foi gerada. Pesquisas em laboratório Pilon é financiado pelo CIHR, NSERC, FRQS e FRQNT.

Declaração de Ética Experimentos envolvendo camundongos foram realizadas seguindo Conselho Canadense de diretrizes de cuidados com animais para o cuidado e manipulação de animais utilizados em pesquisas médicas. Protocolos que envolvem a manipulação de animais foram aprovados pelo comitê de ética institucional da Universidade do Quebec em Montreal (Comité Institutionnel de Proteção des Animaux; número de referência 0512-R3-650-0513). 1. Preparação de Colágeno Gel Trabalhar de forma estéril, sob uma capa de cultura de tecidos. <ol><li> Prepare completa 5x DMEM (meio essencial modificado de Eagle), incluindo antibióticos padrão. Dissolve-se 3,37 g de pó de DMEM e 0,925 g de NaHCO3 em 20 ml de água. Esterilizar por passagem através de um filtro de 0,22 um. Adicionar 2,5 ml de solução estéril de 100x de penicilina / estreptomicina e 25 ml de soro fetal bovino inactivado pelo calor estéril. Armazenar a 4 ° C. </li><li> No gelo, misture 800 mLO colagénio de solução I (3,77 mg / ml em 0,02 N de ácido acético, esterilizada por filtração), 600 ul de DMEM completo 5x e 17 ul de NaOH 1 N. Dilui-se com água estéril para um volume final de 3 ml. Incluir fatores de crescimento relevantes na mistura. Normalmente, utilizar o GDNF a 10 ng / ml para estimular a migração NCC entérico. </li><li> Depositar cerca de 480 mL em cada poço de uma única linha de uma placa de 24 poços. Evite bolhas. As restantes linhas pode ser usado para testar os efeitos de outros factores de crescimento sobre a migração das células. </li><li> Deixe o colagénio polimerizam, pelo menos, 1 hora numa incubadora esterilizada, a 37 ° C. </li></ol> 2. Dissecção de Animais 16 <ol><li> Configure acasalamentos e verificar se há plugs vaginais na manhã seguinte. E0.5 Day sendo meio-dia no dia a ficha vaginal for encontrado, isolar a fêmea e esperar 12 dias até E12.5. </li><li> Anestesiar ratos grávida com isoflurano e eutanásia por inalação de CO 2. </li><li> Pulverizar o mouse com etanol 70%. Levante as s abdômenparentes e abrir a cavidade abdominal com uma tesoura de dissecação. </li><li> Remover o útero para uma placa de Petri de vidro cheio com PBS gelado (soro fisiológico tamponado com fosfato). Corte o útero transversalmente entre inchaços deciduum individuais para isolar cada local de implantação do embrião. </li><li> Trabalhar em cada local de implantação separadamente em outra placa de Petri de vidro cheio de PBS gelado. Sob um microscópio de dissecação, utilizar uma pinça fina para remover a camada muscular do útero. </li><li> Abrir o saco vitelino visceral e âmnio para revelar o embrião. Tome cuidado ao cortar os vasos sanguíneos se juntar o embrião para a placenta / visceral saco vitelino, como eles estão interligados com os intestinos em desenvolvimento. </li><li> Cortar a cabeça do embrião no pescoço. </li><li> Insira um fórceps fechadas na cavidade abdominal de um embrião, logo acima do fígado de cor vermelho escuro e deixar a pinça aberta de si mesmo (deixar de aplicar pressão) para fazer uma abertura transversal na cavidade abdominal. Puxe as pinças abertas para baixo em direçãoa extremidade posterior do embrião para abrir completamente o abdómen. </li><li> Pegue o tecido conjuntivo por trás do fígado e puxar as tripas para fora do abdômen, tomando cuidado para não quebrar o intestino (cólon é ligado ao ânus). </li><li> Cortar o cólon para libertar as entranhas do resto do embrião. O corte pode ser feito em qualquer local ao longo do cólon. Reserve a parte da cauda para mais tarde. </li><li> Tease do tecido conjuntivo a partir do ceco, em seguida, o resto dos intestinos. Tenha cuidado para não ferir os intestinos ao fazê-lo. </li><li> Cortar o fígado e estômago (na extremidade rostral do intestino delgado), bem como os mesonefros e cristas genitais, se algum estiver presente. </li><li> Isolar o intestino delgado. Mais uma vez, tome cuidado para não cortar o intestino. A partir de agora, a orientação rosrrocaudal do tecido intestinal pode ser monitorado usando a curvatura acentuada presente na extremidade rostral. Deixar o intestino delgado em PBS à temperatura ambiente durante um tempo tão curto quanto possível, antes de incama (veja o passo 3.3). </li><li> Por fim, registrar o número de somitos cauda para cada embrião para determinar com precisão o estágio de desenvolvimento embrionário. </li></ol> 3. Seccionamento das embrionárias Intestines <ol><li> Antes de prosseguir com a dissecção de embrião, derreter 1,5 g de agarose em 100 ml de PBS (solução salina tamponada com fosfato) e manter a 50 ° C. </li><li> Despeje agarose fundida num molde de incorporação (por exemplo, um sistema fechado de 2 ml tubo de microcentrífuga que foi cortado longitudinalmente para excisar a cerca de 1/4 da parede do tubo). Deixe a agarose arrefecer para cerca de 42-45 ° C, deve ser apenas ligeiramente quente ao toque. </li><li> Incorporar o intestino embrionário pouco antes das solidifica agarose (esta pode ser avaliada com fórceps dicas e irá ocorrer em torno de 36-38 ° C). Segurando o intestino com uma pinça pela extremidade rostral dobrado, puxá-lo muito lentamente através da agarose ao longo do comprimento do molde. Isso ajuda a manter o intestino reto, enquanto os conjuntos de agarose. Solte o temitir assim que começa a resistir a ser movido. Mantenha o controle da orientação rostral-caudal do intestino. </li><li> Coloque o molde no frigorífico 2-3 minutos, para assegurar que a agarose completamente definido. </li><li> Retire a agarose a partir do molde (deslizando-o para fora do tubo Eppendorf abertas). Com uma lâmina, retirar o excesso de agarose em ambas as extremidades, fazendo cortes perpendiculares para o intestino. </li><li> Cole a extremidade rostral do intestino / agarose a bloquear-se na fase de metal de um micrótomo de lâmina vibrante. Aparar o excesso de agarose sobre os lados do bloco de intestino / agarose. </li><li> Monte a fase de metal na câmara de micrótomo vibrando. Se necessário, ajustar o ângulo da fase de modo que o intestino é mais vertical possível (portanto, perpendicular à lâmina). Cobrir as amostras com PBS gelado. Traga a lâmina de micrótomo até alguns milímetros abaixo da superfície do buffer. </li><li> Fazer 200 mM vibratome transversais cortes do intestino caudal-mais pequenos, garantindo que each fatia agarose contém uma seção intestinal completo. </li></ol> 4. Cultura da Intestinal explantes <ol><li> Gentilmente depositar o fatias intestino / agarose recém-cortadas plana para os géis de colágeno, com uma pinça, colocando uma fatia para o meio de cada poço. </li><li> Incubar 3 dias a 37 ° C, em húmido 5% de CO2, para permitir a migração de NCC fora do explante. </li><li> Leve as fatias de intestino / agarose fora do gel de colágeno muito delicadamente com uma pinça. Tome cuidado para não danificar o gel abaixo. </li><li> Evitar tocar directamente o gel de colagénio, durante o tempo de incubação e de lavagem de passos subsequentes, a fim de garantir que o padrão de migração de células não é perturbado. Fixar com 500 mL de 4% PFA (paraformaldeído) (em PBS) por poço 1 hora à temperatura ambiente. </li><li> Substituir o fixador com 500 mL de DAPI (4 &#39;,6-diamidino-2-fenilindole) de solução (5 ug / ml em PBS) por poço e incubar 10 min a temperatura ambiente. </li><li> Lave cada poço 3xcom 500 ul de PBS durante 5 min. </li><li> Fotografar as células fluorescentes (canais GFP e DAPI) incorporados no gel de colágeno dentro de cada poço. </li></ol> 5. Análise de Imagem Fizemos uso extensivo de ImageJ 17 para processar e quantificar as imagens geradas após cultura explante. <ol><li> Comece imaginando um slide micrômetro com a mesma ampliação como as fotografias de células fluorescentes. Meça o comprimento de um mícron de pixels (utilizando a ferramenta linha reta). </li><li> Defina a escala (Analyze / Set Scale, o número de entrada de pixels / micron). </li><li> Para cada fotografia GFP fluorescência, mude o formato de imagem para 8 bits em tons de cinza (Image/Type/8-bit). </li><li> Ajuste a intensidade do sinal (Imagem / Ajustar / Brilho / Contraste). </li><li> Subtrair o ruído de fundo, se necessário (Processo / Subtrair fundo, ajustar o raio de bola rolando). </li><li> Definir um limite para destacar as células e aglomerados de células (Imagem / Ajustar / Threshold), on aplicar um divisor de águas para dividir as touceiras (Processo / Binário / Bacia Hidrográfica). </li><li> Analisar partículas para especificar regiões de interesse (ROI) e gerar estatísticas de número de células (Analisar / Analisar Partículas; tamanho conjunto mínimo de excluir restantes pixels). </li><li> Definir opções de medida para incluir o diâmetro de Feret (Analyze / Definir Medidas). </li><li> Grupo ROIs e medir como um todo para determinar o diâmetro de Feret, uma indicação de difusão celular (Análise / Ferramentas / ROI Gerente / Mais / OU, então Gerente de ROI / Adicionar e ROI Gerente / Measure). </li></ol>

<ol>
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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophin-3+is+required+for+the+survival-differentiation+of+subsets+of+developing+enteric+neurons.">Neurotrophin-3 is required for the survival-differentiation of subsets of developing enteric neurons.</a> J. Neurosci. 21 (15), 5620-5636 (2001).</li><li>Goldstein, A. M., Brewer, K. C., Doyle, A. M., Nagy, N., Roberts, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+signaling+is+necessary+for+neural+crest+cell+migration+and+ganglion+formation+in+the+enteric+nervous+system.">BMP signaling is necessary for neural crest cell migration and ganglion formation in the enteric nervous system.</a> Mech. Dev. 122 (6), 821-833 (2005).</li><li>Jiang, Y., Liu, M. T., Gershon, M. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signals+regulate+multiple+aspects+of+gastrointestinal+development.">Hedgehog signals regulate multiple aspects of gastrointestinal development.</a> Development. 127 (12), 2763-2772 (2000).</li><li>Natarajan, D., Marcos-Gutierrez, C., Pachnis, V., de Graaf, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+of+signaling+by+receptor+tyrosine+kinase+RET+for+the+directed+migration+of+enteric+nervous+system+progenitor+cells+during+mammalian+embryogenesis.">Requirement of signaling by receptor tyrosine kinase RET for the directed migration of enteric nervous system progenitor cells during mammalian embryogenesis.</a> Development. 129 (22), 5151-5160 (2002).</li><li>Young, H. M., Hearn, C. 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Biol. 293 (1), 203-217 (2006).</li><li>Nagy, A., Gertsenstein, M., Vintersen, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the mouse embryo: a laboratory manual. , 209-250 (2003).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat. Methods. 9, 671-675 (2012).</li><li>Byth, K. F., Thomas, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AZD5438,+a+potent+oral+inhibitor+of+cyclin-dependent+kinases+1,+2,+and+9,+leads+to+pharmacodynamic+changes+and+potent+antitumor+effects+in+human+tumor+xenografts.">AZD5438, a potent oral inhibitor of cyclin-dependent kinases 1, 2, and 9, leads to pharmacodynamic changes and potent antitumor effects in human tumor xenografts.</a> Mol. Cancer Ther. 8 (7), 1856-1866 (2009).</li></ol>

Os autores não têm nada a revelar.

Nós mostramos como nosso ex vivo explante técnica de cultura pode ser usada para quantificar com precisão entérico potencial de migração NCC na presença de GDNF. Essa quantificação precisa é muito facilitada usando 200 seções vibratome intestino m de espessura em vez de grandes pedaços de tamanho aproximado, como descrito anteriormente 11,12,15. Na verdade, isso nos permite trabalhar com um número razoável de células em um ambiente altamente reprodutível. De nota, a distribuição un...

Células da crista neural (NCC) são um tipo de célula transitória única de vertebrados que forma muitos derivados durante o desenvolvimento do embrião. Esta população de células surge na fronteira da placa neural, ao lado ectoderma não-neural 1. Durante neurulação, curvatura da placa neural NCC lugares ao longo do bordo dorsal do tubo neural formando. NCC então sofrer uma transição epitelial-mesenquimal, segregar e migrar para longe do tubo neural. NCC colonizar várias estruturas embrionárias, incluindo o tracto digestivo onde formam todo o sistema nervoso entérico (ENS), uma rede interligada de gânglios neurais embutido na parede do intestino. Como recentemente revisou de 2,3, muitos genes foram envolvidos no desenvolvimento desta estrutura intrincada. A maior parte do ENS é derivado de uma pequena piscina de NCC proveniente do tubo neural vagal (ou seja, em torno do potencial limite rombencéfalo / medula espinal) 4.Estes progenitores neurais chegar ao intestino anterior em torno do dia embrionário (e) 9,0 em ratos e depois migrar caudalmente dentro do mesênquima intestino até aproximadamente e15.0 para colonizar todo o intestino embrionárias. Um subconjunto menor de células progenitoras neurais cólon também é fornecida por sacral NCC, que invadem o intestino posterior na direcção oposta até ao ceco 4. Ambos vagal e sacral NCC requerem múltiplos migração, proliferação, sobrevivência e-sinais de promoção de diferenciação para garantir a formação completa do ENS. A este respeito, os modelos animais - especialmente ratos geneticamente modificados - têm sido fundamentais para a identificação de vários ligandos extracelulares essenciais: GDNF (factor neurotrófico derivado de células gliais),,, (proteínas morfogenéticas do osso) BMPs neurotrofina-3 endotelina-3, Netrin , assim como Sonic e indiano Hedgehog (Shh e Ihh) 5-10. Destes, o GDNF a sinalização através de RET receptor transmembranar da tirosina quinase (Rearranged durante a transfecção), é reconhecido como the caminho mais crítico para a atração e orientação do NCC e para dentro do intestino embrionário. O GDNF é secretada pelo mesênquima intestino e forma um gradiente rosrrocaudal espaço-temporalmente controlado, que é directamente quimioatratante para entérico NCC, que expressam RET 11,12. Entre outras funções, o ENS regula o movimento no interior do tracto digestivo através da sua interacção com o músculo liso na parede intestinal. Ausência de gânglios neural na região terminal dos resultados intestinais na doença de Hirschsprung: a contração tônica do segmento afetado leva ao bloqueio, a acumulação a montante do material digerido e distensão maciça do intestino e abdômen. Doença de Hirschsprung ocorre cerca de um em 5.000 nascidos vivos. O padrão de migração rostro-caudal de entérico NCC é acreditado para ser o principal fator de contribuição à etiologia da doença de Hirschsprung. O cólon, mais afastada da fonte de migrar NCC e última porção de bowel a ser colonizada, é mais suscetível a defeitos na formação ENS. De acordo com o seu papel crucial na migração NCC entérico, a interrupção da sinalização GDNF / RET é a principal causa genética conhecida da doença de Hirschsprung 13. Para melhor estudar NCC e desenvolvimento ENS, geramos uma linha de camundongo transgênico - chamado Gata4p [5kb]-GFP 14 - em que migratório NCC são rotulados com Proteína Fluorescente Verde (GFP). A seguir, aperfeiçoou um ensaio ex vivo, a migração celular, adaptado a partir de trabalhos publicados por outros grupos 11,12,15, que agora permite a quantificação precisa do potencial de migração entérico NCC na presença de vários factores de crescimento, tais como o GDNF.

<table border="1">
	<tbody>
		<tr>
			<td>DMEM powder</td>
			<td>Wisent</td>
			<td>219-010-XK</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Bioshop</td>
			<td>SOB999</td>
			<td>Biotechnology grade</td>
		</tr>
		<tr>
			<td>Steriflip vacuum filtration system (0.22 micron)</td>
			<td>EMD Millipore</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin-Streptomycin solution, 100x</td>
			<td>Wisent</td>
			<td>450-201-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Wisent</td>
			<td>095-150</td>
			<td>High quality grade</td>
		</tr>
		<tr>
			<td>Collagen I</td>
			<td>BD biosciences</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Bioshop</td>
			<td>SHY700</td>
			<td>Diluted from 10 N stock then sterile-filtered</td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>Cedarlane</td>
			<td>CLCYT305</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 24-well Plate</td>
			<td>BD biosciences</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors</td>
			<td>Fisher Scientific</td>
			<td>089515</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Petri dish</td>
			<td>VWR</td>
			<td>89000-306</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma</td>
			<td>P5493</td>
			<td>Cell culture grade</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Leica</td>
			<td>M125</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Bioshop</td>
			<td>AGA001</td>
			<td>Biotechnology grade</td>
		</tr>
		<tr>
			<td>Surgical blade</td>
			<td>Feather</td>
			<td>21</td>
			<td></td>
		</tr>
		<tr>
			<td>All Purpose Instant Krazy Glue Pen</td>
			<td>Krazy Glue</td>
			<td>KG824</td>
			<td></td>
		</tr>
		<tr>
			<td>HM 650V Vibrating-Blade Microtome</td>
			<td>Thermo Scientific</td>
			<td>920110</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9564</td>
			<td></td>
		</tr>
	</tbody>
</table>

a quantitative cell migration assay for murine enteric neural progenitors

Células da crista neural (NCC) é uma população de células multipotentes transitória e que se origina a partir do tubo neural dorsal e migra extensivamente em todo o embrião de vertebrados em desenvolvimento. Além de proporcionar a glia e os neurónios periféricos, NCC gerar melanócitos, bem como a maior parte do esqueleto crânio-faciais. A migração e diferenciação NCC é controlada por uma combinação da sua origem axial ao longo do tubo neural e sua exposição a estímulos extracelulares regionalmente distintas. Tal contribuição de ligandos extracelulares é especialmente evidente durante a formação do sistema nervoso entérico (ENS), uma rede de complexo interligado de gânglios neurais que controla localmente (entre outras coisas) o movimento do músculo do intestino e motilidade intestinal. A maior parte do ENS é derivado a partir de um pequeno conjunto inicial de NCC que empreender um longo percurso, a fim de colonizar - num rostral para caudal moda - todo o comprimento do intestino prospectivo. Entre as várias vias de sinalização conhecidosinfluenciar colonização NCC entérico, GDNF / sinalização RET é reconhecido como o mais importante. Na verdade, a secreção de espaço-temporalmente controlada do RET ligante GDNF pelo mesênquima intestino é o principal responsável para a atração e orientação de expressar-RET entérico NCC para e dentro do intestino embrionário. Aqui, nós descrevemos um ensaio ex vivo, a migração de células, fazendo uso de uma linha de ratinho transgénico possuindo marcado por fluorescência NCC, que permite a quantificação precisa do potencial de migração entérico NCC na presença de vários factores de crescimento, incluindo o GDNF.

Os seguintes resultados são representativos do que pode ser obtida com a técnica descrita aqui (Figura 1). O uso de factores de crescimento (isto é, de GDNF) estimula a migração dos entérico NCC fora do explante intestinal e no gel de colagénio-expressando GFP (Figura 2). Embora algumas células sair do explante, na ausência de factores de crescimento, estes não são mais marcadas com GFP e representam entrada passiva. É necessário remover a fatia intestinal a partir...

Watch this Scientific Journal Video about A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors at JoVE.com

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Apresenta-se um ensaio de migração de células ex vivo que permite a quantificação precisa do potencial de migração de células de crista neural entérico na presença de vários factores de crescimento.

Um ensaio de migração celular quantitativo para murinos progenitores neurais entéricas

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

a-quantitative-cell-migration-assay-for-murine-enteric-neural

Research

JoVE Journal

Neuroscience

9.2K visualizações.  UQAM.  Apresenta-se um ensaio de migração de células ex vivo que permite a quantificação precisa do potencial de migração de células de crista neural entérico na presença de vários factores de crescimento. 

Vídeo: A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

A Técnica de Janela crânio-fino para Crônica de dois fótons In vivo Imaging da Microglia murino em Modelos de Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

Neurociência

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

Estabelecer embrionárias de rato Cultura de Células-tronco neurais utilizando o ensaio Neurosphere

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

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

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

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.