<p class="jove_content"&gt; El trabajo de los autores fue apoyado por una beca de investigación ID-BOF 2006, de la Universidad de Amberes (concedida a PPO y AVDL), por la beca de investigación y G.0136.11 G.0130.11 (concedida a AVDL, ZB y PPO) y 1.5.021.09. N.00 (concedida a PPO) del Fondo para la Investigación Científica de Flandes (FWO-Vlaanderen, Bélgica), por la obstrucción del intestino delgado beca de investigación de TVN-60838: BRAINSTIM del Instituto Flamenco de Ciencia y Tecnología (otorgado a ZB y AVDL), en parte por una beca de investigación de Matusalén, el gobierno flamenco (concedida a ZB), en parte por la CE-FP6-NoE Dimi (LSHB-CT-2005 a 512.146), EC-FP6-NoE EMIL (LSHC-CT-2004-503.569) , y por el Banco Interamericano polacos de la Universidad de atracción IUAP-NIMI-P6/38 (concedida a AVDL). Nathalie De Vocht, tiene un doctorado-beca del FWO-Vlaanderen. Pedro Ponsaerts es un compañero post-doctoral de la FWO-Vlaanderen.</p

1. Preparación de la célula <ol><li> Los experimentos se debe iniciar con ex vivo de células madre cultivadas de poblaciones genéticamente modificadas para expresar las proteínas reportero luciferasa y eGFP. Aquí se utiliza luciferasa / eGFP expresando murinos derivados de médula ósea células del estroma (BMSC-Luc/eGFP) como se ha descrito por Bergwerf et al. 2, 5. </li><li> Dos días antes de marcado de la célula, las células de la placa BMSC-Luc/eGFP a una densidad de 8 x 10 5 células por matraz de cultivo T75 en 15 ml de medio de expansión completa (CEM), suplementado con 1 mg / ml Puromycine. </li><li> Permiten a las células a crecer durante 48 horas a 37 ° C y 5% de CO 2. </li><li> Añadir 5 x 10 6 azul glacial micras de tamaño de partículas de óxido de hierro (GB MPIO) por medio de cultivo ml para el cultivo celular cada vez más (total: 75 x 10 6 partículas por 15 ml de medio de cultivo en un frasco de cultivo T75). </li><li> Incubar durante 16 horas para permitir la captación de partículas a través de la endocitosis. </li><li> Eliminar las partículas que quedany permitir que las células crecen para un período adicional de 24 h para obtener la distribución de células confluencia y homogénea de la MPIO GB. </li><li> Validar la captación de partículas visualmente utilizando microscopía de fluorescencia, contando la relación de eGFP + y células MPIO GB + para la cantidad total de eGFP + en las células. </li><li> Lavar GB MPIO células marcadas BMSC-Luc/eGFP dos veces con 10 ml de PBS y las células después de la cosecha de tripsina-EDTA tratamiento (5 ml por matraz de cultivo T75 durante 5 minutos). </li><li> Lavar las células cosechadas y resuspender en concentración final de 133 x 10 6 células / ml en PBS. </li><li> Mantener las células en hielo hasta el trasplante. </li></ol> 2. Implante celular en el SNC de ratones <ol><li> Anestesiar ratones con una inyección intraperitoneal de una ketamina (80 mg / kg) + xilazina (16 mg / kg) mezcla. </li><li> Espere 5 minutos para permitir que los ratones para conciliar el sueño. </li><li> Afeitarse la cabeza del ratón para permitir manipulaciones estériles. </li><li> Coloque el ratón en un marco estereotáctico. </li><li> Desinfectar ella piel y la humedad de los ojos de los ratones para prevenir la deshidratación. </li><li> Haga una incisión del cuero cabelludo línea media para exponer el cráneo, y perfore un agujero en el cráneo con un taladro dental de rebabas en las coordenadas dadas: 2 mm por detrás y 2 mm lateral a Bregma. </li><li> La suspensión de células vórtice brevemente y aspirar la suspensión de células en la jeringa. </li><li> Colocar la aguja de la jeringa a una profundidad de 2,5 mm bajo la duramadre y permitir el equilibrio de presión durante 1 minuto. </li><li> Inyectar una suspensión de células 3 l (4 x 10 5 células) a una profundidad de 2 mm por debajo de la duramadre y a una velocidad de 0,70 l / min utilizando un sistema automatizado de micro-inyección de la bomba. </li><li> Esperar 5 minutos adicionales para evitar el reflujo de la suspensión celular inyectada. </li><li> Poco a poco retraer la aguja y desinfectar las fronteras de la piel antes de suturar la piel. </li><li> Inyectar 300 l solución NaCl al 0,9% por vía subcutánea con el fin de evitar la deshidratación, y colocar el ratón bajo una lámpara de calentamiento para recuperarse de la anestesia. Administrar post-operatorio analgésico en conformidad con las normas institucionales. </li></ol> 3. En bioluminiscencia in vivo <ol><li> Anestesiar los ratones por una mezcla de 3% de isoflurano y oxígeno. </li><li> Coloque los ratones en el reproductor de imágenes de fotones y reducir el nivel de la anestesia con isoflurano al 1,5% y el oxígeno. </li><li> Inyectar 150 mg de D-luciferina por kg de peso corporal por vía intravenosa. </li><li> Adquirir la imagen durante 5 minutos utilizando el software de visión de fotos. </li><li> Lleve a cabo el procesamiento de imágenes utilizando el software M3vision. Cuantificar la señal observada con las regiones de interés fijas. </li></ol> 4. En vivo de imágenes de resonancia magnética <ol><li> Anestesiar los ratones con 3% de isoflurano en una mezcla de O 2: N 2 O (3:7). </li><li> Coloque los ratones en la inmovilización de un sistema horizontal 9.4T MR y reducir el nivel de anestesia a 1% de isoflurano en una mezcla de O 2: N 2 O (3:7). </li><li> Humedezca los ojos de los ratones para evitar la deshidrataciónción, conecte una sonda rectal para controlar la temperatura corporal y controlar la tasa de respiración mediante la colocación de un sensor debajo del vientre del ratón. </li><li> Mantener la tasa de respiración a 110 ± 10 respiraciones por minuto y mantener la temperatura corporal constante dentro de un estrecho rango de 37 ± 0,5 ° C. </li><li> Colocar la bobina de RF en la superficie superior de la cabeza del ratón y la posición del ratón en el centro del imán. </li><li> Adquirir un conjunto de 10 coronales potenciadas en T2 eco del espín (SE) en las imágenes para obtener información específica anatómica y T2 * imágenes de eco de gradiente (GE) con el fin de estudiar la migración de células madre con una resolución en el plano de 70 m 2. Establezca los parámetros de la secuencia de la siguiente manera: tiempo de repetición (TR): 500 ms, tiempo de eco (TE): 8 ms (secuencia GE) y el tiempo de repetición (TR): 4200 ms, tiempo de eco (TE): 12,16 m (SE secuencia); campo de visión (FOV): 18x18 mm 2, la matriz: 256x256, un grosor de corte mm y una separación de corte mm (5,1 Paravision de software). </li><li> Lleve a cabo el procesamiento de datosutilizando el software Amira 4.0. </li></ol> 5. Post Mortem Histología <ol><li> Anestesiar los ratones muy profundamente por la inhalación de un oxígeno isofluorano (4%), (0,5 l / min) y nitrógeno (1 l / min) mezcla durante 2 minutos. El sacrificio de los ratones por dislocación cervical. </li><li> Retire el cerebro del ratón en el cráneo y fijar el tejido cerebral en paraformaldehído al 4% en PBS durante 2 horas. </li><li> Deshidratar el tejido cerebral mediante la colocación del cerebro posteriormente en diferentes gradientes de sacarosa: 2 horas en 5% de sacarosa en PBS, 2 h en 10% de sacarosa en PBS, durante toda la noche en 20% de sacarosa en PBS. </li><li> Congelar el tejido cerebral utilizando nitrógeno líquido y almacenar el tejido a -80 ° C hasta que el corte. </li><li> Sección del tejido cerebral en 10 secciones m de espesor con un criostato. </li><li> Pantalla sin mancha cryosections de fluorescencia azul de las partículas GB MPIO y la fluorescencia verde de las células que expresan eGFP utilizando un microscopio de fluorescencia. </li><li> Pantalla sin mancha de cryosectionsverde / rojo de fondo de fluorescencia de las células inflamatorias. </li><li> Realizar tinciones inmunohistoquímicas y de inmunofluorescencia más para identificar las poblaciones endógenas de células (por ejemplo, la microglia, astrocitos, células T, ...) que interactúan con los injertos celulares. </li></ol> 6. Los resultados representativos Estamos aquí presentada visualmente una secuencia optimizada de eventos para el éxito de imagen multimodal de las poblaciones de luciferasa / eGFP célula que expresa (STEM) en el sistema nervioso central de ratones. Al principio, los que se describen GB MPIO los resultados del procedimiento de etiquetado en los alimentos de alta eficiencia de las células BMSC-Luc/eGFP, que fácilmente pueden ser validados mediante microscopía de fluorescencia (Figura 2A). A continuación, sobre el injerto de GB MPIO etiquetados BMSC-Luc/eGFP en el SNC de ratones, la supervivencia del injerto celular puede ser controlado por in vivo BLI basado en la actividad de luciferasa (Figura 2B). Además, la localización exacta de las células injertadas pueden ser controlados por in vivo </em&gt; RM basado en el contenido de hierro de la MPIO GB (Figura 2C). Finalmente, el análisis histológico permite la validación de los resultados obtenidos por BLI y RMN. Estable la supervivencia fue confirmada por un eGFP expresión estable en el tiempo (Figura 2). Para esto, colocalización de eGFP-expresión de las células con azul fluorescente MPIO permite la determinación exacta de la localización y la supervivencia de las células injertadas. Además, este doble etiquetado fluorescente de las células madre reduce la posibilidad de observar resultados positivos falsos basados ​​en fluorescencia de fondo creado por las células inflamatorias 5. <img alt="Figura 1." src="/files/ftp_upload/3906/3906fig1.jpg" /> Figura 1. Visión general de la secuencia de manejo <ol class="lroman"><li> Etiquetado de la célula: BMSC-Luc/eGFP están etiquetados con 5 x 10 6 partículas GB MPIO por medio de cultivo ml. Después de 16 horas de incubación y un período de descanso de la siguiente 24 horas, las células se cosechan parala implantación intracerebral. </li><li> Implantación de las células: GB MPIO etiquetados BMSC-Luc/eGFP se injertan en el cerebro de ratones en las siguientes coordenadas: bregma -2 mm, 2 mm de la derecha de la línea media y 2 mm en la duramadre. </li><li> En vivo BLI: Después de la administración intravenosa de 150 mg D-luciferin/kg peso corporal, la luz generada fue adquirida durante 5 minutos usando el reproductor de imágenes de fotones. </li><li> En vivo RM: Un conjunto de 10 coronales potenciadas en T2 eco del espín (SE), las imágenes y T2 * imágenes eco de gradiente (GE) con una resolución en el plano de 70 m 2 fueron adquiridas. Parámetros de la secuencia fueron los siguientes: tiempo de repetición (TR): 500 ms, tiempo de eco (TE): 8 ms (secuencia GE) y el tiempo de repetición (TR): 4200 ms, tiempo de eco (TE): 12,16 m (secuencia SE); campo de visión (FOV): 18x18 mm 2, la matriz: 256x256, un grosor de corte mm y 1 mm de separación corte. </li><li> La histología post-mortem: criosección 10μm de espesor fueron seleccionados para expresar eGFP y azul marcado con fluorescencia u BMSCcantar un microscopio de fluorescencia. </li></ol><img alt="Figura 2." src="/files/ftp_upload/3906/3906fig2.jpg" /> Figura 2. Multimodal de imágenes de los injertos de células madre <ol class="ualpha"><li> Microscopio de inmunofluorescencia directa de azul glacial (GB) MPIO etiquetados BMSC-Luc/eGFP. </li><li> En vivo BLI de los ratones inyectados con 4x10 5 GB MPIO células marcadas BMSC-Luc/eGFP en la semana 1 y 2 semanas después de la implantación. El análisis estadístico de las señales detectadas de BLI en la semana 1 y 2 semanas después de la implantación. Los datos se expresan como el número promedio de fotones por segundo por estereorradián por centímetro cuadrado (ph / s / sr / cm 2) a partir de un período de tiempo de 5 minutos + / - error estándar calculado a partir de una región de interés fijo en ratones que muestran una clara señal de BLI en el sitio de inyección. </li><li> Coronal de dos dimensiones de la resonancia magnética GB MPIO etiquetados BMSC-Luc/eGFP (a la izquierda del panel: (i) en T2 la imagen eco de espín y (ii) T2 * imagen de eco de gradiente) y sin etiqueta BMSC L-UC / eGFP (panel de la derecha: (iii) la imagen ponderada en T2 eco de espín y (vi) T2 * imagen de eco de gradiente) injertos en el cerebro del ratón. </li><li> El análisis histológico de los implantes GB MPIO etiquetados BMSC-Luc/eGFP en la semana 2 post-implantación. (I) el análisis de inmunofluorescencia directa para la localización de eGFP de expresar y GB MPIO etiquetados injertos BMSC-Luc/eGFP. (Ii) los detalles a gran aumento de (i). (Iii) la tinción de inmunofluorescencia para el antígeno de GFAP, lo que indica el desarrollo de tejido cicatrizal los astrocitos en los alrededores de la etiqueta BMSC-Luc/eGFP GB MPIO. (Vi) la tinción de inmunofluorescencia para el antígeno de Iba-1, lo que indica la invasión y los alrededores de GB MPIO BMSC-Luc/eGFP etiquetados por la microglia. </li></ol>

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

Los autores declaran no tener conflicto de intereses.

En este reporte se describe un protocolo optimizado para la combinación de tres técnicas de imagen complementarias (BLI, la resonancia magnética y la histología) para la caracterización detallada de los implantes celulares en el sistema nervioso central de ratones inmunes competentes. Una combinación de etiquetado del gen de las células, sobre la base de la modificación genética con los genes reporteros de luciferasa de luciérnaga y eGFP, y un etiquetado directo de células con GB MPIO, conduce a una evaluaci?...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> IMDM </td><td> Lonza </td><td> BE12-722F </td><td> Componente de la CEM, el medio de cultivo celular </td></tr><tr><td> Suero bovino fetal </td><td> Gibco </td><td> 10270-106 </td><td> Componente de la CEM, el medio de cultivo celular </td></tr><tr><td> El suero de caballo </td><td> Gibco </td><td> 1605-122 </td><td> Componente de la CEM, el medio de cultivo celular </td></tr><tr><td> La penicilina-estreptomicina </td><td> Gibco </td><td> 15140 </td><td> Componente de la CEM, el medio de cultivo celular </td></tr><tr><td> Fungizone </td><td> Gibco </td><td> 15290-018 </td><td> Componente de la CEM, el medio de cultivo celular </td></tr><tr><td> PBS </td><td> Gibco </td><td> 14190 </td><td></td></tr><tr><td> Puromycine </td><td> Invivogen </td><td> ANT-PR-1 </td><td></td></tr><tr><td> tripsina </td&gt; <td> Gibco </td><td> 25300 </td><td></td></tr><tr><td> GB MPIO </td><td> Bangs Laboratorios </td><td> ME04F/7833 </td><td></td></tr><tr><td> D-luciferina </td><td> Promega </td><td> E1601 </td><td></td></tr><tr><td> La ketamina (Ketalar) </td><td> Pfizer </td><td></td><td></td></tr><tr><td> Xilazina (Rompun) </td><td> Bayer Health Care </td><td></td><td></td></tr><tr><td> El isoflurano </td><td> Isoflo </td><td> 05260-05 </td><td></td></tr><tr><td> 0,9% de NaCl solución </td><td> Baxter </td><td></td><td></td></tr><tr><td> paraformaldehído </td><td> Merck </td><td> 1.04005.1000 </td><td></td></tr><tr><td> sacarosa </td><td> Applichem </td><td> A1125 </td><td></td></tr><tr><td> Micro-bomba de inyección </td><td> KD científica </td><td> KDS100 </td><td></td></tr><tr><td> Photon imágenes </td><td> Biospace Lab </td><td></td><td></td></tr><tr&gt; <td> 9.4T MR escáner </td><td> Bruker BioSpin </td><td> Biospec 94/20 USR </td><td></td></tr><tr><td> BX51 </td><td> Olimpo </td><td> BX51 </td><td></td></tr><tr><td> Mycrom HM criostato </td><td> Prosan </td><td> HM525 </td><td></td></tr><tr><td> jeringa </td><td> Hamilton </td><td> 7635-01 </td><td></td></tr><tr><td> Aguja de calibre 30 </td><td> Hamilton </td><td> 7762-03 </td><td></td></tr><tr><td> Foto Visión de software </td><td> Biospace Lab </td><td></td><td></td></tr><tr><td> M3vision de software </td><td> Biospace Lab </td><td></td><td></td></tr><tr><td> Paravision 5.1 del software </td><td> Bruker BioSpin </td><td></td><td></td></tr><tr><td> Amira software 4.0 </td><td> Visage imágenes </td><td></td><td></td></tr></tbody></table>

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

Durante la década pasada, el trasplante de células madre ha ganado un creciente interés como modalidad terapéutica primaria o secundaria para una variedad de enfermedades, tanto en estudios preclínicos y clínicos. Sin embargo, hasta la fecha los resultados con respecto a los resultados funcionales y / o regeneración de los tejidos después de un trasplante de células madre son muy diversas. Por lo general, un beneficio clínico que se observa, sin conocimiento profundo de los mecanismo (s) 1. Por lo tanto, los múltiples esfuerzos han llevado al desarrollo de las diferentes modalidades de imágenes moleculares para controlar el injerto de células madre con el fin último de evaluar con precisión la supervivencia, el destino y la fisiología de las células madre injertadas y / o su entorno micro. Los cambios observados en uno o más parámetros determinados por imagen molecular podría estar relacionado con el efecto clínico observado. En este contexto, nuestros estudios se centran en el uso combinado de imágenes de bioluminiscencia (BLI), imágenes por resonancia magnética (RM) e histológicos analysis para evaluar el injerto de células madre. BLI se utiliza comúnmente para no invasiva realizar el seguimiento de celda y vigilar la supervivencia celular en el tiempo después del trasplante 2-7, basado en una reacción bioquímica en donde las células que expresan el gen de luciferasa-reportero son capaces de emitir luz siguiente interacción con su sustrato (por ejemplo, D- luciferina) 8, 9. RM por otro lado es una técnica no invasiva que es aplicable clínicamente 10 y se puede utilizar para localizar con precisión los injertos celulares con una resolución muy alta 11-15, aunque su sensibilidad depende en gran medida el contraste generado después de marcado de la célula con un agente de contraste para MRI . Por último, post mortem análisis histológico es el método de elección para validar los resultados de investigación obtenidos con técnicas no invasivas con mayor resolución y sensibilidad. Por otra parte punto final el análisis histológico nos permite realizar un análisis detallado fenotípica de las células injertadas y / o el tejido circundante, baSED en el uso de proteínas fluorescentes reportero y / o etiquetado directo de células con anticuerpos específicos. En resumen, aquí visualmente demostrar la complementariedad de BLI, la resonancia magnética y la histología de desentrañar las células madre y diferente / o asociados con el medio ambiente las siguientes características injerto de células madre en el SNC de ratones. A modo de ejemplo, la médula ósea derivados de las células del estroma, la ingeniería genética para expresar la mayor proteína verde fluorescente luciferasa (eGFP) y Firefly (fluctuaciones), y marcado con color azul fluorescente micras de tamaño de partículas de óxido de hierro (MPIOs), serán injertados en el sistema nervioso central de ratones inmunocompetentes y los resultados serán monitoreados por BLI, la resonancia magnética y la histología (Figura 1).

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

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

En este artículo se describe una secuencia optimizada de eventos para obtener imágenes multimodal de los injertos celulares en el cerebro de los roedores por medio de: (i) en la bioluminiscencia in vivo y la resonancia magnética, y (ii) el análisis post mortem histológico. La combinación de estas modalidades de imágenes en un solo animal permite la evaluación del injerto celular con alta resolución, sensibilidad y especificidad.

Imagen multimodal de implante de células madre en el sistema nervioso central de ratones

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

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

Research

JoVE Journal

Neuroscience

11.0K vistas.  University of Antwerp. En este artículo se describe una secuencia optimizada de eventos para obtener imágenes multimodal de los injertos celulares en el cerebro de los roedores por medio de: (i) en la bioluminiscencia in vivo y la resonancia magnética, y (ii) el análisis post mortem histológico. La combinación de estas modalidades de imágenes en un solo animal permite la evaluación del injerto celular con alta resolución, sensibilidad y especificidad.

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

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

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

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

La transección del nervio óptico: un modelo de apoptosis neuronal de adultos en el sistema nervioso central

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

Investigación

Neurociencias

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

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

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

La producción de vectores lentivirales de células transductoras del Sistema Nervioso Central

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

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

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

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

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

El marcaje de células individuales en el Sistema Nervioso Central de<em&gt; Drosophila melanogaster</em

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

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

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

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

Entrega intraventricular directa de medicamentos al roedor Sistema Nervioso Central

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

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

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

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

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

En Interrogatorio vivo del Sistema Nervioso Central Translatome por Polyribosome Fraccionamiento

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

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

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

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

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

In vivo optogenético la estimulación del sistema nervioso central de roedores

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

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

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

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

Evaluación Funcional de neurotoxinas Biológica en Culturas en Red de Células Madre deriva-Sistema Nervioso Central Neuronas

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

In Situ Ca2+ Imaging of the Enteric Nervous System

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

In Situ Ca2+ Imaging of the Enteric Nervous System

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

In Situ Ca 2+ Imaging del sistema nervioso entérico

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

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

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

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

Clearing óptico del sistema nervioso central del ratón Utilizando CLARITY pasiva

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

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

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

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

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

Registro electrofisiológico de la actividad del sistema nervioso Central de tercer estadio de Drosophila Melanogaster

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