Este trabajo es financiado por las Ciencias Naturales e Ingeniería de Investigación de Canadá (subvención # 249669, 482986 y #) y Heart and Stroke Foundation de Canadá (subvención # 485508). Los autores agradecen a Youssef El-Hayek y el Dr. Qi Wan por su ayuda en la elaboración de los protocolos experimentales.

Todos los procedimientos que impliquen la manipulación de animales fueron aprobados por la Universidad de Toronto Comité de Cuidado de Animales de acuerdo con las directrices institucionales (Protocolo n º. 20009387). Los siguientes métodos se debe realizar utilizando instrumentos estériles y técnicas, en una campana de flujo laminar en su caso. En el texto siguiente protocolo, la frase &quot;EFH-SFM&quot; se refiere a medio libre de suero suplementado con factor de crecimiento epidérmico, factor de crecimiento de fibroblastos básico y la heparina. EFH-SFM se utiliza en la investigación de la galvanotaxia de NPCs no diferenciados debido a que estos mitógenos mantener NPCs en su estado indiferenciado 4. Al investigar la galvanotaxia de NPCs inducidas a diferenciarse en tipos de células maduras, &quot;FBS-SFM&quot; se refiere a medio libre de suero suplementado con suero fetal bovino al 1%. FBS promueve la diferenciación de NPCs en maduros fenotipos neurales 5. 1. Aislamiento y cultivo de precursores neurales (Nose muestra en vídeo) <ol><li> Anestesiar un ratón CD1 (6-8 semanas) con isofluorano y el sacrificio por dislocación cervical. </li><li> Apaga la cabeza en 70% de etanol y decapitar al animal con tijeras de disección cortante. </li><li> Mientras sostiene la cabeza con fórceps quirúrgico, quitar la piel en la superficie dorsal para exponer el cráneo. </li><li> Utilizando un escalpelo y no. 11 blade, anotar el cráneo en el seno frontal a lo largo del eje mediolateral, y también a lo largo de la sutura sagital en la dirección rostrocaudal. </li><li> Pelar los huesos parietales lejos de la cabeza con no. 7 pinzas curvas, teniendo cuidado de no perforar el tejido cerebral. </li><li> Insertar una espátula fina debajo del cerebro a partir de debajo del cerebelo y avanzando hacia los bulbos olfatorios. Mientras sostiene el cráneo en su lugar con pinzas, tire suavemente del cerebro del cráneo y de inmediato se coloca en el hielo frío artificial líquido cefalorraquídeo (ver recetas a continuación). </li><li> Bajo un microscopio de disección, usando estériltijeras y pinzas de disección del cerebro cortar por la mitad a lo largo de la línea media. Gire cada hemisferio a fin de que la media (corte) de la superficie hacia arriba. </li><li> Seleccione un hemisferio, y con la superficie medial hacia arriba, busque el esplenio del cuerpo calloso (región posterior del cuerpo calloso). </li><li> Hacer una incisión en la superficie de la corteza de la rodilla del cuerpo calloso a lo largo del eje dorsoventral. </li><li> Pelar la corteza incisa hacia el bulbo olfatorio para exponer las paredes medial y lateral del ventrículo lateral. </li><li> Girar el hemisferio para que la superficie dorsal esté orientada hacia arriba, y el uso de micro tijeras curvadas para cortar y recoger las paredes expuestas medial y lateral, que contienen la región periventricular NPCs donde residen 6. </li><li> Repita los pasos 1.8 a 1.11 para el otro hemisferio. </li><li> Pipetear el tejido aislado en 7 ml de solución de tripsina (ver receta a continuación) en un tubo de 15 cc, y coloque el tubo en un rockero en un 37 ° Cincubadora durante 25 min. </li><li> Centrifugar el tubo a 1.500 rpm durante 5 min, aspirar el sobrenadante, y resuspender el tejido en 2 ml de solución de inhibidor de tripsina (véase la receta a continuación). </li><li> Suavemente se tritura el tejido con una pipeta Pasteur de pequeñas perforaciones 30-50 veces con cuidado para evitar burbujas de aire. </li><li> Centrifugar el tubo a 1.500 rpm durante 5 min, aspirar el sobrenadante, y resuspender en 1-2 ml de SFM (ver receta más abajo) triturando el pellet 3-5 veces. </li><li> Centrifugar el tubo a 1.500 rpm durante 3 min, aspirar el sobrenadante y se resuspende en 1 ml de SFM + EFH. </li><li> Contar vivir densidad de células con un hemocitómetro y la placa de las células en un matraz de cultivo T25 a una densidad de 10 células por microlitro en SFM + EFH. </li><li> Deje que la cultura a crecer durante 7 días no perturbados para producir flotan libremente neuroesferas primarias compuestas de NPCs. </li></ol> 2. Galvanotaxis cámara de preparación <ol><li> Coloque 3 cristal cuadrado no. Un cubreobjetos (22 x 22 x 0,17 mm) ina botella de ácido clorhídrico 6 N durante la noche. </li><li> El día siguiente, utilizar una punta de diamante de cristal del cortador para cortar 6 tiras rectangulares (22 x 5 x 0,17 mm) de vidrio de no cuadrado. Un cubreobjetos. </li><li> Transfiera los resbalones cuadrados lavados con ácido e injertos rectangulares en una campana de flujo laminar. Lavar las tiras rectangulares y cuadradas primero con 70% de etanol, luego con el cultivo de tejidos de grado agua tratada en autoclave, y permitir que se seque en un Kim Wipe (para esterilidad añadido, el cristal se puede permitir que el aire seco). </li><li> Aplicar grasa de vacío para el perímetro de una superficie de las placas de vidrio cuadrados, y los sella a la base de 60 mm de placas Petri de plástico. </li><li> Aplicar grasa de vacío a lo largo del eje largo de una superficie de las tiras rectangulares de vidrio, y sellar los bordes opuestos de las placas de vidrio cuadrados (de forma que son paralelas entre sí) con el fin de crear un canal central. </li><li> UV-esterilizar las cámaras durante al menos 15 min en la campana de flujo laminar. </li><li> Pipetear 250-300 l de poli-L-Lysine en el canal central de las cámaras y se incuban a temperatura ambiente durante 2 h. </li><li> Aproximadamente 15 min antes de la finalización del periodo de incubación, preparar la solución de Matrigel (ver receta más abajo). </li><li> Aspirar el poli-L-lisina, se lavan los canales centrales con 1 ml de agua esterilizada, y la pipeta 250-300 l de solución de Matrigel en las depresiones centrales. </li><li> Incubar las cámaras a 37 º C durante 1 hr. </li><li> Aspirar la solución Matrigel y lavar suavemente los valles centrales con 1-2 ml de SFM. </li><li> Añadir 100 l de EFH-SFM SFM-FBS o en los valles centrales y la transferencia de las cámaras Galvanotaxis en el escenario de un microscopio contando. </li><li> Ml pipeta 3-4 de la cultura neurosphere que contiene en una placa de Petri de 60 mm y transferir la placa de Petri para la etapa del recuento con microscopio. </li><li> En un objetivo de visión de 5x, utilizar una pipeta para transferir P10 5-8 neuroesferas enteros (hasta cuatro a la vez) en el canal central de cada galvanotaxiacámara sin disociar, y extiende con cuidado las neuroesferas alrededor de la depresión central sin interrumpir el sustrato Matrigel. </li><li> Añadir un adicional de 150-200 l de EFH-SFM SFM-FBS o en los valles centrales. </li><li> Transferir las cámaras Galvanotaxis en un 37 ° C, 5% de CO 2, 100% incubador humidificado durante 17-20 horas (si el análisis de NPCs indiferenciadas) para permitir que las neuroesferas para adherirse al sustrato Matrigel y se disocian en células individuales como se muestra en la Figura 1 . Si el análisis de NPCs diferenciados, el período de incubación debe ser extendido a 69-72 horas para permitir la diferenciación de las células. </li></ol> 3. Vivo Celular Time-lapse de imágenes <ol><li> Permitir que el sistema de células de imágenes en vivo se equilibre a 37 ° C, 5% de CO 2 durante un mínimo de 30 min antes de la iniciación de la grabación de lapso de tiempo. </li><li> Corte dos piezas de 12 cm de diámetro a 1 mm Plata alambre, la bobina de un lado, y colocarlos en Clorox blixiviación durante 20 minutos para formar Ag / AgCl electrodos. </li><li> Transferir las cámaras Galvanotaxis sobre la platina de un microscopio contar y seleccionar qué cámara se utilizan para análisis en vivo de células migración de imagen basado en los siguientes criterios: i) las neuroesferas debe ser casi completamente disocia en células individuales y ii) las células deben poseer morfologías circulares con poco o ningún proceso que se extiende desde los cuerpos celulares. </li><li> Transferir la cámara galvanotaxia seleccionado en una campana de flujo laminar, junto con un no cuadrado separado. 1 vaso cubreobjetos y grasa de vacío. </li><li> Lavar la tapa de deslizarse primero con 70% de etanol, luego con agua tratada en autoclave, y aplicar una tira de grasa de vacío en dos bordes paralelos de la hoja de cubierta. </li><li> Aspirar los medios de cultivo de la cubeta central de la cámara, a continuación, colocar rápidamente la hoja de la cubierta (grasa-lado que mira hacia abajo) en la cámara de tal manera que el resto grasa tiras de las dos tiras paralelas rectangulares de vidrio, creando efectivamente un techoa la cámara. </li><li> Añadir 100 l de nuevo EFH-SFM SFM-FBS o en la pila central a través de la acción capilar. </li><li> Use grasa de vacío para crear bordes de piscinas de medios de cultivo en cada extremo de la cubeta central, como se muestra en la Figura 2. </li><li> Cortar dos piezas de 15 cm de tubo de PVC, y utilizar una jeringa de 10 cc con una aguja de calibre 18 para inyectar cuidadosamente una solución de agarosa en el tubo, asegurándose de que no se forman burbujas en los tubos, y permitir que el gel solidifique durante 5 min. </li><li> Transferir la cámara galvanotaxia al sistema de células de imágenes en vivo, junto con los tubos de gel de agarosa, Ag / AgCl, y un par de vacío 60 mm placas de Petri que serán utilizados como medios de cultivo embalses y contendrá el Ag / AgCl. Permitir la cámara galvanotaxia a descansar dentro de la 37 ° C, 5% de CO 2 ambiente durante 20-30 min. </li><li> Durante este tiempo, preparar las tapas de las cajas de Petri vacías 2 y la tapa de la cámara de cápsula de Petri galvanotaxia por los agujeros de perforaciónen ellos con una herramienta Dremel o similar como se muestra en la Figura 3. </li><li> Pipetear 1-1.5 ml de EFH-SFM o FBS SFM-en ambos lados de la depresión central, y 7-8 ml de SFM en cada placa de Petri vacía. Colocar una placa de Petri en cada lado del canal central de la cámara de galvanotaxia y coloque un electrodo Ag / AgCl en cada plato. Salvar la distancia entre la cámara de galvanotaxia y las placas Petri a establecer la continuidad eléctrica con los puentes en gel de agarosa, tal como se muestra en la Figura 4. </li><li> Conecte los electrodos de Ag / AgCl a una fuente de alimentación externa, con un amperímetro en serie para medir la corriente eléctrica y encienda la fuente de alimentación. Use un voltímetro para medir la fuerza del campo eléctrico directamente a través de la depresión central, y ajustar la salida de la fuente de alimentación hasta que la fuerza deseada de campo eléctrico se consigue (los ensayos realizados en este laboratorio utilizan una fuerza dcEF de 250 mV / mm con corriente eléctrica entre 1 y 1,5 mA). </li><li> IniciativaTE el módulo de lapso de tiempo en el sistema de células de imágenes en directo, y permitir el experimento para funcionar para la cantidad de tiempo deseada. Después de completar el ensayo, fijar las células en 4% de paraformaldehído para el análisis de inmunotinción estándar. </li></ol>

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No hay conflictos de interés declarado.

Este protocolo ha sido adaptado de los métodos bien establecidos de estudios previos 7-9. Galvanotactic cámaras pueden construirse utilizando una variedad de técnicas diferentes, incluyendo la construcción de un vaso separado bien para el confinamiento de la siembra de células, o el uso de CO 2 ablación con láser para la microfabricación de la depresión central 10,11. Algunas técnicas pueden ser más laborioso o costosos que otros. Se ha descrito un ensayo simple y rentable par...

<table border="1"><tbody><tr><td height="44"> Nombre del elemento </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td></td><td></td><td></td><td> Precursoras neurales de aislamiento de células </td></tr><tr><td> 2 M NaCl </td><td> Sigma </td><td> S5886 </td><td> 11,688 g disueltos en 100 ml de dH 2 O </td></tr><tr><td> 1 M KCl </td><td> Sigma </td><td> P5405 </td><td> 7,456 g disueltos en 100 ml de dH 2 O </td></tr><tr><td> 1 M MgCl 2 </td><td> Sigma </td><td> M2393 </td><td> 20,33 g disueltos en 100 ml de dH 2 O </td></tr><tr><td> 155 mM NaHCO 3 </td><td> Sigma </td><td> S5761 </td><td> 1,302 g disueltos en 100 ml de dH 2 O </td></tr><tr><td> La glucosa 0,5 M </td><td> Sigma </td><td> G6152 </td><td> 9,01 g disueltos en 100ml dH 2 O </td></tr><tr><td> 108 mM CaCl 2 </td><td> Sigma </td><td> C7902 </td><td> 1,59 g disueltos en 100 ml de dH 2 O </td></tr><tr><td> Penicilina-estreptomicina </td><td> Gibco </td><td> 15070 </td><td></td></tr><tr><td> Tripsina de páncreas bovino </td><td> Sigma </td><td> T1005 </td><td></td></tr><tr><td> Ovejas testículos hialuronidasa </td><td> Sigma </td><td> H6254 </td><td></td></tr><tr><td> Ácido quinurénico </td><td> Sigma </td><td> K3375 </td><td></td></tr><tr><td> Inhibidor de tripsina de ovomucoide </td><td> Worthington </td><td> LS003086 </td><td></td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 12100046 </td><td></td></tr><tr><td> F12 </td><td> Invitrogen </td><td> 21700075 </td><td></td></tr><tr><td> 30% de glucosa </td><td> Sigma </td><td> G6152 </td><td></td></tr><tr><td> 7,5% NaHCO 3 </td><td> Sigma </td><td> S5761 </td><td></td></tr><tr><td> HEPES 1 M </td><td> Sigma </td><td> H3375 </td><td> 23,83 g disueltos en 100 ml de dH 2 O </td></tr><tr><td> L-glutamina </td><td> Gibco </td><td> 25030 </td><td></td></tr><tr><td> FEAG </td><td> Invitrogen </td><td> PMG8041 </td><td> Reconstituir en 1 ml de mezcla de la hormona y la alícuota en 20 unidades mu l. </td></tr><tr><td> FGF </td><td> Invitrogen </td><td> PHG0226 </td><td> Reconstituir en 0,5 ml de mezcla de la hormona y la alícuota en 20 unidades mu l. </td></tr><tr><td> La heparina </td><td> Sigma </td><td> H3149 </td><td></td></tr><tr><td> Apo-transferrina </td><td> R &amp; D Systems </td><td> 3188-AT </td><td> 0,1 g disueltos en 4 ml de dH 2 0 </td></tr><tr><td> Putrescina </td><td> Sigma </td><td> P7505 </td><td> Disolver 9,61 mg en Apo-transferrina asíción </td></tr><tr><td> Insulina </td><td> Sigma </td><td> I5500 </td><td> Disolver 25 mg en 0,5 ml de HCl 0,1 N y añade a 3,5 ml de dH 2 0 </td></tr><tr><td> Selenio </td><td> Sigma </td><td> S9133 </td><td></td></tr><tr><td> Progesterona </td><td> Sigma </td><td> P6149 </td><td></td></tr><tr><td> Herramientas generales Dissection </td><td> Herramientas Artes Ciencias </td><td></td><td></td></tr><tr><td> Disección microscopio </td><td> Zeiss </td><td> Stemi 2000 </td><td></td></tr><tr><td></td><td></td><td></td><td> Galvanotaxis cámara de preparación </td></tr><tr><td> Square portaobjetos de vidrio cubierta </td><td> VWR </td><td> 16004 </td><td></td></tr><tr><td> Ácido clorhídrico 6N </td><td> VWR </td><td> BDH3204-1 </td><td></td></tr><tr><td> Grasa de alto vacío </td><td> Dow Corning </td><td></td><td></td></tr><tr><td> 60 mm Placas de Petri </td><td> Fisher Scientific </td><td> 0875713A </td><td></td></tr><tr><td> Poli-L-lisina </td><td> Sigma </td><td> P4707 </td><td></td></tr><tr><td> Matrigel </td><td> BD Biosciences </td><td> 354234 </td><td> Descongelar y alícuota en 150 unidades mu l </td></tr><tr><td> FBS </td><td> Invitrogen </td><td> 10082139 </td><td> Utilice únicamente si la inducción de diferenciación NPC, de lo contrario usar SFM + EFH medios de cultivo como se ha indicado anteriormente </td></tr><tr><td> Contando microscopio </td><td> Olimpo </td><td> CKX41 </td><td></td></tr><tr><td></td><td></td><td></td><td> Vivo Celular Time-lapse de imágenes </td></tr><tr><td> Alambre de plata </td><td> Alfa Aesar </td><td> 11434 </td><td></td></tr><tr><td> UltraPure agarosa </td><td> Invitrogen </td><td> 15510-027 </td><td></td></tr><tr><td> Calentar la inactividadATED FBS </td><td> Sigma </td><td> 16140071 </td><td></td></tr><tr><td> Tubería de PVC </td><td> Fisher Scientific </td><td> 80000006 </td><td> 3/32 &quot;ID x 5/32&quot; OD </td></tr><tr><td> Blanqueador </td><td> Clorox </td><td></td><td></td></tr><tr><td> 10 cc jeringa </td><td> BD </td><td> 309604 </td><td></td></tr><tr><td> Calibre 18 aguja </td><td> BD </td><td> 305195 </td><td></td></tr><tr><td> Dremel taladro </td><td> Dremel </td><td> Modelo 750 </td><td></td></tr><tr><td> Microscopio invertido equipado con cámara de humidificado, incubados </td><td> Zeiss </td><td> Axiovert-200M </td><td></td></tr><tr><td></td><td></td><td></td><td> Recetas <table border="1"><tbody><tr><td> Artículo </td><td> Volumen </td></tr><tr><td> 2 M NaCl </td><td> 6,2 ml </td></tr><tr><td>1 M KCl </td><td> 0,5 ml </td></tr><tr><td> 1 M MgCl 2 </td><td> 0,32 ml </td></tr><tr><td> 155mm NaHCO 3 </td><td> 16,9 ml </td></tr><tr><td> La glucosa 1M </td><td> 1 ml </td></tr><tr><td> 108 mM CaCl 2 </td><td> 0,09256 ml </td></tr><tr><td> Penicilina-estreptomicina </td><td> 1 ml </td></tr><tr><td> Agua autoclave </td><td> 74 ml </td></tr></tbody></table> Líquido cefalorraquídeo artificial <table border="1"><tbody><tr><td> Artículo </td><td> Volumen o Masa </td></tr><tr><td> Líquido cefalorraquídeo artificial </td><td> 30 ml </td></tr><tr><td> Tripsina de páncreas bovino </td><td> 40 mg </td></tr><tr><td> Ovejas testículos hialuronidasa </td><td> 22,8 mg </td></tr><tr><td> Ácido quinurénico </td><td> 5 mg </td></tr></tbody></table> &lt;strong&gt; solución de tripsina <table border="1"><tbody><tr><td> Artículo </td><td> Volumen o Masa </td></tr><tr><td> SFM </td><td> 15 ml </td></tr><tr><td> Inhibidor de tripsina de ovomucoide </td><td> 10 mg </td></tr></tbody></table> Solución de inhibidor de tripsina <table border="1"><tbody><tr><td> Artículo </td><td> Volumen </td></tr><tr><td> Agua autoclave </td><td> 37 ml </td></tr><tr><td> 10 veces DMEM/F12 </td><td> 10 ml </td></tr><tr><td> 30% de glucosa </td><td> 2 ml </td></tr><tr><td> 7,5% NaHCO 3 </td><td> 1,5 ml </td></tr><tr><td> HEPES 1 M </td><td> 0,5 ml </td></tr><tr><td> Transferrina, putrescina solución </td><td> 4 ml </td></tr><tr><td> 25 mg solución de insulina </td><td> 4 ml </td></tr><tr><td> Selenio </td><td> 100 y mu; L </td></tr><tr><td> Progesterona </td><td> 100 l </td></tr></tbody></table> Hormona Mix (100 ml en total, almacenan a -20 ° C) <table border="1"><tbody><tr><td> Artículo </td><td> Volumen </td></tr><tr><td> Agua autoclave </td><td> 37,5 ml </td></tr><tr><td> 10X DMEM/F12 (3:1) </td><td> 5 ml </td></tr><tr><td> 30% de glucosa </td><td> 1 ml </td></tr><tr><td> 7,5% NaHCO 3 </td><td> 0,75 ml </td></tr><tr><td> HEPES 1 M </td><td> 0,25 ml </td></tr><tr><td> Hormona de mezcla </td><td> 5 ml </td></tr><tr><td> L-glutamina </td><td> 0,5 ml </td></tr><tr><td> Penicilina-estreptomicina </td><td> 0,5 ml </td></tr></tbody></table> Serum Free Media EFH-SFM: añadir 10 l de EGF, 10 l de FGF, y 3,66 l de heparina FBS-SFM: añadir 0,5 ml de FBS <table frontera = &quot;1&quot;&gt; <tbody><tr><td> Artículo </td><td> Volumen </td></tr><tr><td> Matrigel </td><td> 150 l </td></tr><tr><td> SFM </td><td> 3,6 ml </td></tr></tbody></table> Matrigel Solución alícuota Matrigel debe ser colocado en una caja de hielo y se dejó descongelar lentamente durante 4-5 horas para formar un líquido viscoso antes de mezclar con SFM. Esto asegurará la formación de una capa lisa de sustrato Matrigel. Si no descongelaron lentamente, el sustrato resultante contendrá grupos de Matrigel, posiblemente obstaculizar la migración celular. <table border="1"><tbody><tr><td> Artículo </td><td> Volumen o Masa </td></tr><tr><td> UltraPure agarosa </td><td> 300 mg en 10 ml ddH 2 0 </td></tr><tr><td> SFM FBS inactivado por calor </td><td> 8 ml 2 ml </td></tr></tbody></table> MatrigelSolución Mezcle 8 ml de SFM con 2 ml de FBS inactivado por calor en un tubo de 15 cc halcón. Mezcla de agarosa con 10 ml ddH 2 0 en un matraz Erlenmeyer, y el calor en un horno de microondas durante 30 segundos en intervalos de 10-sec, asegurando para eliminar la solución del microondas después de cada intervalo de 10-sec y mezclar bien. Tras el final de 10 segundos período de microondas, mezclar la solución de agarosa con el SFM / solución de SFB y guárdelo en un baño de agua 57 ° C. </td></tr></tbody></table>

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

El descubrimiento de células madre neurales y células progenitoras (colectivamente denominadas células precursoras neurales) (CPN) en el cerebro adulto de mamíferos ha conducido a un cuerpo de la investigación dirigida a la utilización de las propiedades multipotentes y proliferativa de estas células para el desarrollo de estrategias neuroregenerativas. Un paso crítico para el éxito de tales estrategias es la movilización de NPCs hacia un sitio de la lesión después de un trasplante exógeno o para aumentar la respuesta de los precursores endógenos que se encuentran en la región periventricular del SNC. Por consiguiente, es esencial para comprender los mecanismos que promueven, orientar y mejorar la migración NPC. Nuestro trabajo se centra en la utilización de los campos eléctricos de corriente directa (dcEFs) promover y migración NPC directa - un fenómeno conocido como galvanotaxia. Endógenos campos eléctricos funcionan como señales fisiológicas esenciales para la migración celular durante el desarrollo normal y la reparación de heridas. Interrupción farmacológica de latrans-neural en embriones de potencial tubo axolotl provoca graves malformaciones en el desarrollo 1. En el contexto de la cicatrización de heridas, la tasa de reparación de córnea heridos se correlaciona directamente con la magnitud del potencial de heridas epiteliales que surge después de la lesión, como se muestra por la mejora farmacológica o la interrupción de este dcEF 2-3. Hemos demostrado que los adultos NPCs subependimales someterse a migración catódica rápida y dirigida in vitro cuando se exponen a un dcEF aplicada externamente. En este protocolo se describen técnicas de nuestro laboratorio para la creación de un ensayo de galvanotaxia simple y eficaz para la alta resolución, observación a largo plazo de la translocación dirigida cuerpo celular (migración) en un nivel de una sola célula. Este ensayo sería adecuado para la investigación de los mecanismos que regulan la transducción de dcEF en la motilidad celular mediante el uso de ratones transgénicos o knockout, ARN corto de interferencia, o agonistas / antagonistas de receptores específicos.

Análisis cinemático revela que en presencia de un 250 mV / mm dcEF, NPCs indiferenciadas exponer galvanotaxia altamente dirigidos y rápido hacia el cátodo (Figura 5A, Película 1). En ausencia de un dcEF, movimiento al azar de las células se observó (Figura 5B, Película 2). En esta intensidad de campo,&gt; 98% de NPCs indiferenciadas migrar para toda 6-8 hr para los que se explora, y ya que las células muertas no migran esto sugiere que permanecen viables durante este período e...

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

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

En este protocolo, se muestran cómo construir cámaras personalizados que permitan la aplicación de un campo eléctrico de corriente directa para activar el tiempo de imágenes a intervalos de cerebro adulto derivado translocación célula precursora neural durante galvanotaxia.

Un ensayo Galvanotaxis por análisis de la cinética precursoras neurales migración celular en un campo aplicado externamente directo Corriente Eléctrica

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

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

Research

JoVE Journal

Neuroscience

12.4K vistas.  University of Toronto. En este protocolo, se muestran cómo construir cámaras personalizados que permitan la aplicación de un campo eléctrico de corriente directa para activar el tiempo de imágenes a intervalos de cerebro adulto derivado translocación célula precursora neural durante galvanotaxia.

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

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

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

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

Neural formadoras de colonias ensayo de células: un ensayo para discriminar Bona Fide células troncales nerviosas de células progenitoras neurales

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). ...

Investigación

Neurociencias

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.

El análisis de la migración de células troncales neurales Crest utilizando una versión modificada del ensayo Zigmond Cámara

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.

Ratón reproducible aplastamiento del nervio ciático y posterior evaluación de la regeneración mediante el análisis de músculo entero Monte

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.

Análisis de la migración de la cresta neural y la diferenciación de las especies cruzadas Trasplante

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.

El ensayo neuroblasto: un ensayo para la generación y enriquecimiento de las células progenitoras neuronales de la diferenciación de la progenie de células madre neurales Usando citometría de flujo

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.

Aislamiento y cultivo de células de la cresta neural de embriones murinos del Tubo Neural

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

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

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

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

Un ensayo de migración celular cuantitativa para murinos entéricas Neuronales Progenitores

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

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

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

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

Grabaciones simultáneas de potenciales de campo Local Cortical, Electrocardiograma, electromiograma y respirar a ritmo de una rata libremente móvil

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

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

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

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

Grabación óptica de campo ancho de un solo fotón en cortes cerebrales con tinte sensible al voltaje

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

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

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

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

Diferenciación de cribado de alto contenido y análisis de maduración de cultivos de células precursoras de oligodendrocitos derivados de células madre neurales fetales y adultas

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