Este trabajo fue apoyado por el NIH Blueprint Neurociencia básica de subvención (P30 NS057105, BJS) de la Universidad de Washington, el Programa de Proyecto de Donación NS032636 (BJS) y por el Centro de Esperanza para los Trastornos Neurológicos.

1. Embalaje de vectores lentiviral Lentiviral vectores son producidos por cotransfección de un vector de transferencia lentiviral y otros plásmidos necesarios para el envasado en células 293T por el método de transfección con fosfato de calcio. Usamos 10 de 100 mm placas de cultivo de tejidos en este protocolo. Se puede escalar hacia arriba o hacia abajo dependiendo de las aplicaciones. La línea celular 293T se mantiene en modificado de Dulbecco medio Eagles (DMEM) con glucosa alta (4500 mg / l), suplementado con suero fetal bovino al 10% (FBS), 100 unidades / ml de penicilina, 100 ug / ml de estreptomicina a 37 ° C incubadora con 5% de CO 2. <ol><li> Semillas 293T células de la confluencia del 30-40% a 10 de 100 mm placas de cultivo de tejidos (3 x 10 6 células / placa) en medio de cultivo. Volver a las células a la incubadora. </li><li> Después de 20-24 h de cultivo, comprobar la densidad de las células. Las células debe ser aproximadamente 80% de confluencia en el momento de la transfección. </li><li> Preparar un tubo de 50 ml. Añadir 4,4 ml TE79/10 (1 mM Tris HCl, 0,1 mMEDTA, pH 7,9) menos el volumen total del ADN plásmido siguiente. Añadir 100 g de transferencia lentiviral plásmido (Figura 1), 58 g pMDLg / pRRE, 31 ug pCMV-T, 25 mg pRSV-Ap, 600 l 2M CaCl 2. Mezclar con cuidado. </li><li> Preparar otro tubo de 50 ml. Añadir 5 ml de 2x HBS (0,05 M, HEPES 0,28 M NaCl, 1,5 mM Na 2 HPO 4, pH 7,12). </li><li> Tomar la ADN-CaCl 2 por mezcla de 10 ml pipeta y añadir al tubo que contenía 2 x HBS, gota a gota mientras que el tubo vortex. </li><li> Mantener la reacción de precipitación a temperatura ambiente (TA) durante 30 min. </li><li> Retire las placas de cultivo de la incubadora. Mezclar la reacción de precipitación adecuadamente por agitación. Añadir 1 ml de suspensión a cada uno de las células del plato de 100 mm que contienen. La suspensión se debe añadir lentamente, gota a gota mientras agita suavemente el medio en el plato. Volver estas placas en la estufa y se deja durante 5 horas. </li><li> Retire el medio de la cultura. Añadir 6 ml de medio de cultivo fresco que contiene 6 mM de sodio butyrate a cada plato. Volver a los cultivos a la incubadora. Después de cultivo durante la noche, si hay un reportero fluorescente en el constructo, comprobar la expresión del gen bajo microscopio de fluorescencia. Por lo general, más del 80% de las células expresan el gen reportero si es impulsado por un promotor ubicuo (por ejemplo, promotor CMV). </li><li> Dos días (40-44 h) después de la transfección, recoger el líquido sobrenadante de 10 platos en 2 50-ml tubos (aproximadamente 30 ml cada tubo). Congelar el sobrenadante en el congelador de -80 º C o ir al siguiente paso. </li></ol> 2. La concentración y purificación de los Vectores <ol><li> Centrifugar el recién recogida o descongelado sobrenadante a 900 g (aproximadamente 2000 rpm) durante 10 minutos para eliminar cualquier residuo de células en el sobrenadante. </li><li> Coloque una jeringa de 60 ml a un filtro de 0,2-m SFCA jeringa. Transferir el sobrenadante de 50-ml tubo de la jeringa. Se filtra el sobrenadante en un tubo de centrífuga de polialómero. </li><li> Tomar 5 ml de sacarosa 20% (preparado en PBS) en una pipeta de 5 ml. Insertarla pipeta a la parte inferior del tubo de centrífuga que contiene sobrenadante. Añadir lentamente la solución de sacarosa en el sobrenadante del vector. Repita estos pasos con sobrenadante de otro tubo. </li><li> Centrifugar el sobrenadante a 11000 rpm y 4 ° C durante 4 h con rotor Beckman SW28 oscilación. </li><li> Eliminar el sobrenadante. Añadir 150 l de lactosa 4% (preparado en PBS) a cada tubo de centrífuga. Resuspender las pastillas. </li><li> Transferir el vector de concentrado de todos los tubos de centrífuga a un tubo de 1,5 ml. Dejar el tubo en hielo durante 15 min. </li><li> Mezclar la suspensión de vectores con la pipeta. Gira con la microcentrífuga a máxima velocidad (alrededor de 16.000 g) durante 1 min. </li><li> Transferir el sobrenadante a un nuevo tubo de 1,5 ml. Divida la muestra final en 20 ml de alícuotas y se almacenan en -80 ° C congelador. </li></ol> 3. Valoración de los Vectores <ol><li> Semillas de 5 x 10 4 / pocillo de células HT1080 en 12-pocillos en 1 ml de medio DMEM suplementado con FBS al 10%. </li><li> Después de Overnigla cultura ht, contar las células de un bien y anotar el número de células. </li><li> Hacer 5-pliegue dilución en serie (1:5, 1: 25; 1:125, y 1:625) del vector de concentrado con medio de cultivo. Añadir I l de cada vector diluido a los pocillos separados. Las muestras pueden ser duplicados para aumentar la precisión. </li><li> Añadir 1 l de 4 mg / ml de polibreno (Hexadimetrina Bromuro) en cada vector que contiene bien y en un pozo sin vectorial. Mezcle agitando suavemente la placa. Volver a la incubadora durante 48 h. </li><li> Quitar medio de los pocillos de cultivo de células. Lavar cada pocillo con PBS. Añadir 250 l 1x tripsina-EDTA solución a las células. Cuando las células se separan (3-5 minutos), añadir 1 ml de medio de cultivo. Resuspender las células mediante pipeteo. Transferir la suspensión de células a tubos de 1,5 ml de centrífuga. </li><li> Centrifugar a 900 g durante 6 minutos. Para los vectores con un gen indicador fluorescente (GFP, por ejemplo), vaya al paso 3.7 para el análisis de FACS. Para los vectores sin un periodista, vaya al paso 3.8 para qPCR en tiempo real. </li><li> Para los vectores que contienen un fluogen reportero fluorescentes, separar el sobrenadante y resuspender el precipitado con 300 l de 3,7% de formaldehído en PBS. Determinar el porcentaje de las células informadoras positivos por análisis FACS. El título se representa como unidades de transducción por mililitro concentrado vector (TU / ml). </li></ol><img alt="Ecuación 1" src="/files/ftp_upload/4031/4031eq1.jpg" /> Por ejemplo, si 1 x 10 5 células transducidas con l fue 1/25 (0,04 l) vector y las células 30% son reportero positivo, el título será: <img alt="Ecuación 2" src="/files/ftp_upload/4031/4031eq2.jpg" /> Sólo utilizar las diluciones caer en una relación lineal entre el porcentaje de células positivas y la cantidad de vector añadido para calcular título. El título final debe ser un promedio de los títulos obtenidos a partir de transducciones de al menos 2 diferentes cantidades del vector. <ol start="8"><li> Para los vectores sin ungen reportero fluorescente, extraer el ADN genómico de las células HT1080 con QIAamp DNA Mini Kit (Qiagen), según el protocolo del fabricante. Amplificar la secuencia del vector en el ADN genómico utilizando ABI Prism 7000 secuencia sistema de detección (Applied Biosystems) con los cebadores (VIH-1 en PBS / psi región 17) 5&#39;-CCGTTGTCAGGCAACGTG-3 &#39;y 5&#39;-AGCTGACAGGTGGTGGCAAT-3&#39;, y sonda TaqMan 5 &#39;-FAM-AGCTCTCTCGACGCAGGACTCGGC-TAMRA-3&#39;. Gen de la albúmina que es un gen de copia única en el genoma (2 copias / célula) también fue amplificado con los primers 5&#39;-TGAAACATACGTTCCCAAAGAGTTT-3 &#39;y 5&#39;-CTCTCCTTCTCAGAAAGTGTGCATAT-3&#39;, y la sonda de 5&#39;-FAM-TAMRA TGCTGAAACATTCACCTTCCATGCAGA-3- &quot;como un control interno. Determinar el número de copias del vector y la albúmina por PCR en placa de 96 pocillos de acuerdo con la fabricación de instrucción con el siguiente programa: 50 ° C durante 2 min, 95 ° C durante 10 min, y 35 ciclos de 95 ° C durante 15 segundos y 60 ° C durante 2 min. Diez veces diluciones seriadas de los plásmidos de concentración conocida y representada como una copianúmero) que contiene las secuencias modelo también debe ser amplificada para crear una curva estándar para la cuantificación de muestras desconocidas. El título se representa como unidades de integración por mililitro concentrado vector (UI / ml). </li></ol><img alt="Ecuación 3" src="/files/ftp_upload/4031/4031eq3.jpg" /> 4. Transducción de las Culturas neocorticales Culturas neocorticales que contienen tanto las neuronas y glía se preparó a partir de cortezas de ratón utilizando un procedimiento de chapado de dos pasos como se ha descrito previamente 18. Neocorteza obtenidos de ratones fetales en 14-16 días de gestación se sembraron en una monocapa previamente establecido gliales en MEM suplementado con FBS al 10%, 20 mM de glucosa y glutamina 2 mM en 24-así placa de cultivo de tejido. <ol><li> Después de 5 días in vitro, añadir 10 mM citosina arabinósido (Ara-C) en NEOCcultura ortical para inhibir no neuronal división celular. Continuar cultivar las células durante 2 días. </li><li> Medio de cultivo en caliente 37 ° C baño de agua durante 5-10 minutos. Sustituir Ara-C que contiene el medio con medio de cultivo fresco (500 l / pocillo). </li><li> Añadir vector con deseado MOI (multiplicidad de infección; la relación del número de partículas de vectores para el número de células diana) para el cultivo. Continuar la cultura durante 24 horas. Usamos MOI de 1-10 (generalmente 5) en cultivos primarios corticales. </li><li> Sustituir el medio de cultivo con medio fresco. Continuar la cultura. Si hay un gen reportero en la construcción de vectores, visita células bajo microscopio fluorescente 2 días después de la transducción. La expresión del gen será visible en las neuronas 2-7 después de la transducción, dependiendo del diseño del vector y la dosis utilizada. </li></ol> 5. Los resultados representativos Los títulos de los vectores lentivirales producidos con este rango de protocolo de 10 8 -10 10 UI / ml, which son adecuados para la transducción de una variedad de tipos de células a partir del SNC, tanto in vitro como in vivo. Tabla 1 y Figura 2 muestran un resultado representativo utilizando los vectores producidos por este protocolo. Nos transducidas murinos culturas neocorticales con vectores lentivirales que expresan la proteína verde fluorescente (GFP), controlada por sinapsina (SYN) promotor o promotor de la proteína glial fibrilar ácida (GFAP). Siete días después de la transducción, se realizó inmunotinción a las neuronas y astrocitos de la etiqueta con anticuerpos anti-NeuN y anti-GFAP, respectivamente. Como se muestra en la tabla 1 y fig. 2A, después de la transducción con el vector que lleva el promotor sinapsina, más del 90% de las neuronas (células NeuN +) expresa las buenas prácticas agrarias y los astrocitos no (GFAP + en las células) expresan este gen reportero. Cuando promotor GFAP se utiliza en la construcción de vectores (Fig. 2B), alrededor del 80% de los astrocitos (GFAP + células) expresións las buenas prácticas agrarias; todos GFP + células son los astrocitos según lo confirmado por colocalización con Bosnia y Herzegovina y la ausencia de expresión de GFP en las células NeuN marcadas. Estos resultados demuestran que los vectores lentiviral son muy eficientes para entregar los transgenes de las células de SNC y células específicas de la expresión génica puede lograrse cuando se utilizan promotores apropiados. <img alt="Figura 1" src="/files/ftp_upload/4031/4031fig1.jpg" /> Figura 1. Representación esquemática de VIH-vectores basados ​​en lentivirus y los plásmidos de embalaje. El VIH-1 provirus se muestra en la parte superior. Los elementos para la producción de vectores están separados en cuatro diferentes plásmidos. La transferencia del plásmido lentiviral contiene un híbrido LTR 5 &#39;en la que se sustituye la región U3 con el citomegalovirus (CMV), la señal de empaquetamiento (ψ), la secuencia RRE, el tracto polipurina central (cPPT), un gen de interés (por ejemplo un reportero fluorescente), junto con un promotor de la elección, y LTR 3 &#39;en la que elsecuencias reguladoras cis se elimina completamente de la región U3. pMDLg / pRRE contiene los genes gag y pol y secuencia RRE de VIH-1 bajo el control del promotor CMV. pRSV-Ap contiene la secuencia codificante de Rev impulsado por el promotor RSV. pCMV-G contiene el gen de la proteína VSV-G bajo el control del promotor CMV. PA indica la señal de poliadenilación de β-globina humana gen. <img alt="Figura 2" src="/files/ftp_upload/4031/4031fig2.jpg" /> Figura 2. Expresión de genes reporteros en el ratón cultivo mixto neocortical transducidas con vectores lentivirales que llevan el tipo de células específicas de los promotores. Los cultivos fueron transducidas con el LV-SYN-GFP (A) o LV-GFAP-GFP vectores (B) a una MOI de 5. Siete días después de la transducción, las células se immunostained con anticuerpo anti-NeuN o anti-GFAP. Los paneles superiores muestran la fluorescencia de GFP, los paneles centrales muestran inmunotinción y los paneles inferiores se fusionan imágenes (GFP: verde; NeuN y GFAP: rojo). </p&gt; <table border="1"><tbody><tr><td> Vector </td><td> GFP + células en las neuronas </td><td> GFP + en los astrocitos </td></tr><tr><td> LV-SYN-GFP </td><td> 92,2 ± 7,3 </td><td> 0 </td></tr><tr><td> LV-GFAP-GFP </td><td> 0 </td><td> 78,3 ± 11,5 </td></tr></tbody></table> Tabla 1. Comparación de la expresión de GFP en murinos culturas neocorticales transducidas con vectores lentivirales que llevan a diferentes promotores. unas culturas murinos neocorticales (5 x 10 5 / bien en placa de 24 pocillos) fueron transducidas con el LV-SYN-GFP o LV-GFAP-GFP en una MOI de 5. Siete días después de la transducción, las culturas se fijaron y se immunostained para NeuN y GFAP. El número de células GFP y NeuN / GFAP que expresan fueron contadas en imágenes de 10 campos por condición experimental. Los valores representan el porcentaje de las neuronas (células NeuN +) oastrocitos (GFAP + en las células), que también expresa el gen reportero GFP. Los valores mostrados son medias ± SD de tres experimentos independientes.

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Virol. 73, 1828-1834 (1999).</li><li>Palsson, B., Andreadis, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physico-chemical+factors+that+govern+retrovirus-mediated+gene+transfer.">The physico-chemical factors that govern retrovirus-mediated gene transfer.</a> Exp. Hematol. 25, 94-102 (1997).</li><li>Lizee, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+quantitative+reverse+transcriptase-polymerase+chain+reaction+as+a+method+for+determining+lentiviral+vector+titers+and+measuring+transgene+expression.">Real-time quantitative reverse transcriptase-polymerase chain reaction as a method for determining lentiviral vector titers and measuring transgene expression.</a> Hum. Gene Ther. 14, 497-507 (2003).</li><li>Lee, J. K., Chung, J., McAlpine, F. E., Tansey, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulator+of+G-Protein+Signaling-10+Negatively+Regulates+NF-{kappa}B+in+Microglia+and+Neuroprotects+Dopaminergic+Neurons+in+Hemiparkinsonian+Rats.">Regulator of G-Protein Signaling-10 Negatively Regulates NF-{kappa}B in Microglia and Neuroprotects Dopaminergic Neurons in Hemiparkinsonian Rats.</a> J. Neurosci. 31, 11879-11888 (2011).</li><li>Shevtsova, Z., Malik, J. M., Michel, U., Bahr, M., Kugler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promoters+and+serotypes:+targeting+of+adeno-associated+virus+vectors+for+gene+transfer+in+the+rat+central+nervous+system+in+vitro+and+in+vivo.">Promoters and serotypes: targeting of adeno-associated virus vectors for gene transfer in the rat central nervous system in vitro and in vivo.</a> Exp. Physiol. 90, 53-59 (2005).</li></ol>

No hay conflictos de interés declarado.

En este protocolo, se ha demostrado la producción de vectores lentivirales y la aplicación de estos vectores en cultivos neocorticales. Hemos demostrado eficaz y tipo de células específicas de transducción con los vectores producidos por estos métodos. Cuando el promotor sinapsina se utiliza, la expresión de GFP es estrictamente neurona específica. Cuando el promotor de GFAP se utiliza, la expresión de GFP es exclusivamente en los astrocitos. Si ningún tipo de células específicas de expresión se requiere, u...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> DMEM </td><td> Sigma-Aldrich </td><td> D5796 </td></tr><tr><td> MEM </td><td> Invitrogen </td><td> 11090-081 </td></tr><tr><td> Suero bovino fetal </td><td> Hyclone </td><td> SV3001403 </td></tr><tr><td> PBS </td><td> Mediatech </td><td> 21-040-CM </td></tr><tr><td> Tripsina-EDTA </td><td> Sigma-Aldrich </td><td> T3924 </td></tr><tr><td> Butirato sódico </td><td> Sigma-Aldrich </td><td> B5887 </td></tr><tr><td> Hexadimetrina bromuro (Polybrene) </td><td> Sigma-Aldrich </td><td> H9268 </td></tr><tr><td> Células 293T </td><td> ATCC </td><td> CRL-11268 </td></tr><tr><td> Células HT1080 </td><td> ATCC </td><td> CCL-121 </td></tr><tr><td> Falcon 100 x 20 mm de tejido CULTURAe plato </td><td> BD Biosciences </td><td> 353003 </td></tr><tr><td> 1 x 3 ½ en el tubo de centrífuga polyallomoer </td><td> Beckman-Coulter </td><td> 326823 </td></tr><tr><td> 0.2 micras filtro de jeringa </td><td> Corning </td><td> 431219 </td></tr><tr><td> QIAamp DNA Mini Kit </td><td> Qiagen </td><td> 51304 </td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System at JoVE.com

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

En este protocolo se describe la producción, purificación y valoración de lentiviral vectores. Se presenta un ejemplo de entrega gen vector lentiviral mediada por neuronas cultivadas en la enseñanza primaria y astrocitos. Nuestros métodos pueden aplicarse también a otros tipos de células<em&gt; In vitro</em&gt; Y<em&gt; En vivo</em&gt;.

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 ...

production-lentiviral-vectors-for-transducing-cells-from-central

Research

JoVE Journal

Neuroscience

35.8K vistas.  Washington University School of Medicine. En este protocolo se describe la producción, purificación y valoración de lentiviral vectores. Se presenta un ejemplo de entrega gen vector lentiviral mediada por neuronas cultivadas en la enseñanza primaria y astrocitos. Nuestros métodos pueden aplicarse también a otros tipos de células In vitro Y En vivo.

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

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.

Thorough preclinical testing of drugs that act in the central nervous system often involves assessing and comparing drug biodistribution in association with specific routes of administration. Here, three commonly used methods of systemic delivery (intravenous, intraperitoneal, and oral) as well as a method for local delivery (convection-enhanced delivery) are demonstrated in mice.

Medicamentos sistémicos y locales de entrega para el Tratamiento de Enfermedades del Sistema Nervioso Central en modelos de roedores

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

Investigación

Neurociencias

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

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

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, ...

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 ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

El análisis inmunohistoquímico en el sistema nervioso central y periférico de la rata de nodo linfático secciones de tejido

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

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 ...

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 ...

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central nervous system (CNS). However, its existence also dramatically lowers the accessibility of systemically administered therapeutic agents to the CNS. One method to overcome this, is to inject those agents directly into the cerebrospinal fluid (CSF), thus bypassing the BBB. This can be done via implantation of a catheter for either continuous infusion using an osmotic pump, or for single bolus delivery. In this article, we describe a surgical protocol for delivery of CNS-targeting antisense oligonucleotides (ASOs) via a catheter implanted directly into the cauda equina space of the adult rat spine. As representative results, we show the efficacy of a single bolus ASO intrathecal (IT) injection via this catheterization system in knocking down the target RNA in different regions of the rat CNS. The procedure is safe, effective and does not require expensive equipment or surgical tools. The technique described here can be adapted to deliver drugs in other modalities as well.

Here, we describe a method for delivering drugs to the rat central nervous system by implanting a catheter into the lumbar intrathecal space of the spine. We focus on the delivery of antisense oligonucleotides, though this method is suitable for delivery of other therapeutic modalities as well.

Entrega intratecal de oligonucleótidos antisentido en el sistema nervioso central de Rata

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central ...