Damos las gracias a A. Bakhos Tannous, Ph.D., Director de Desarrollo y Producción del vector en el Centro de Neurociencia del Hospital General de Massachusetts en Charlestown, Massachusetts, para que nos proporciona el vector rAAV-EGFP, Whang y Juan de asistencia técnica. Este trabajo recibió el apoyo de subvención R01 NS050408 (a JS) del Instituto Nacional de Trastornos Neurológicos y Accidentes Cerebrovasculares.

El procedimiento quirúrgico descrito ha sido aprobado por el Cuidado de Animales institucional y el empleo Comisión (IACUC) de la Universidad de Columbia. 1. Preparación de equipos y suspensión de partículas de virus <ol><li> Limpiar y desinfectar el equipo, esterilizar los instrumentos quirúrgicos y los picos de primera categoría V que se utilizan para fijar L1 vértebra. </li><li> Tire y pipetas de cristal biselado. Nosotros usamos pipetas que tienen un diámetro de punta de 40 micras y están biselados en un ángulo de 20 °. Esterilice las pipetas de vidrio. </li><li> Establecer el marco estereotáxico, montar el inyector microjeringa sobre el manipulador y conectar el inyector al controlador. </li><li> Conecte una de las pipetas de vidrio a la microjeringa utilizando el kit de conexión de compresión. </li><li> Retire el émbolo de la microjeringa y llene la jeringa con aceite mineral. Oil Red O (1 - (2,5-dimetil-4-(2,5-dimethylphenylazo) fenilazo)-2-naftol) se puede añadir al aceite mineral para aumentar su visibility. Vuelva a insertar el émbolo y empujar a todo el camino hasta la punta. Con cuidado, evitar la creación de una burbuja de aire. </li><li> Preparar la suspensión de partículas de virus en una cabina de bioseguridad. Descongelar el virus congelado en hielo y, justo antes de su uso, diluir con agua estéril salina tamponada con fosfato a la concentración de partículas deseado. </li><li> Inserte la microjeringa en el soporte del inyector. </li><li> Verter de 5 l de la suspensión de virus en una hoja de plástico, por ejemplo Parafilm. Baje la punta de la pipeta de vidrio en la gota y tire del émbolo para llenar la microjeringa. Crear una pequeña burbuja de aire en la punta de la pipeta para evitar que se obstruya. </li></ol> 2. Laminectomía <ol><li> Prepare el área de la cirugía, limpiando banco y almohada eléctrica con desinfectante. </li><li> Se anestesia el ratón. Usamos anestesia por inhalación con isoflurano (3% durante inducción, 2% -3% durante el mantenimiento). </li><li> Coloque lubricante en cada ojo para proteger los ojos se sequen durante laoperación. </li><li> Afeitar el pelo de la espalda baja para el cuello del ratón y desinfectar la piel con alternancia de toallitas de un antiséptico tópico como la clorhexidina o povidona yodada y 70% de etanol. Aislar el sitio preparado asépticamente con paño quirúrgico y se infiltran en el sitio de la incisión con bupivacaína (0,25%, diluido 1:10 con solución salina fisiológica). </li><li> Incisión en la piel en el extremo caudal de la caja torácica a lo largo de la línea media (2-3 cm) y separar la fascia que cubre la columna vertebral. </li><li> Debido a que la médula espinal deja de crecer antes durante el desarrollo postnatal de la columna vertebral, segmento vertebral L4 se encuentra debajo de la primera vértebra lumbar (L1). Vértebra L1 se encuentra caudal a la vértebra que sostiene el último par de costillas. Identificar y exponer vértebra L1 mediante la eliminación de los pequeños músculos de la columna vertebral y los ligamentos adheridos a la superficie dorsal. </li><li> Levante ligeramente y mantén presionado L1 vértebra con una pinza Adson. Use unas pinzas laminectomía dedicados a eliminar el dorsal Portion de la vértebra (columna vertebral y de la lámina) y exponer la médula espinal. Evitar daños en la médula espinal. </li><li> Transferir el ratón sobre la placa de calentamiento en el marco estereotáxico. Controlar la temperatura del ratón durante la operación posterior. </li></ol> 3. Inyección <ol><li> Fijar vértebra L1 con puntas de primera clase V. Las espigas deben estabilizar la columna vertebral de modo que la vértebra no se mueve durante la respiración. </li><li> Llevar la microjeringa más cerca de modo que la punta de la pipeta está por encima del sitio de laminectomía. Baje el émbolo hasta que vea la suspensión de virus que sale de la pipeta. Retire la gota con una punta de algodón estéril. </li><li> Coloque la punta de la pipeta en la parte rostral más de la médula espinal expuesta. Centro de la pipeta por encima del surco mediano posterior, a continuación, mover la punta 500 m lateralmente. Baje la punta a la superficie de la médula y punción de la duramadre o, si usted está trabajando con una pipeta de vidrio unbeveled, use una cánula de acero biselado para perforar el dura. Baje la punta de la pipeta de vidrio 300 micras en la médula espinal. </li><li> Inyectar 1 l de suspensión de virus a una tasa de 200 NL / min. </li><li> Al final de la inyección, espere al menos 2 min antes de retraer lentamente la pipeta. </li><li> Repetir los pasos desde 3,3 hasta 3,5 en la porción caudal más-de la médula espinal expuesta para conseguir una distribución completa del vector viral en L4 segmento espinal. Los dos sitios de inyección se encuentran rostral y caudal del segmento L4 para evitar el daño tisular en la región diana. </li></ol> 4. Cierre de la herida <ol><li> Release vértebra L1 de las abrazaderas de primera clase V y quitar el ratón del marco estereotáxico. </li><li> Sutura de la fascia con vicryl 5.0. La fascia cerrado proporciona cobertura para el sitio de laminectomía. </li><li> Cierre la piel con suturas de nylon o grapas quirúrgicas. </li></ol> 5. Cuidado Postoperatorio <ol><li> Transferir el ratón en una jaula de recuperación con ropa de cama blanda, nonparticular. Colócalo en tél laterales para respirar cómodo. Supervisar el animal hasta que esté completamente alerta, ambulante y empieza a beber. </li><li> Nos proporcionar analgesia posquirúrgica durante 72 h con inyecciones subcutáneas diarias de carprofeno (5,0 mg / kg). </li><li> Monitorear la recuperación postquirúrgica mediante la inspección diaria durante los primeros 3 días, luego cada dos días o 3 días a la semana hasta que el experimento está completo. </li><li> Eliminar las suturas de la piel o las grapas 7 a 10 días después de la operación, cuando la curación de heridas es completa. </li><li> Sacrificar al animal después de la finalización del experimento. </li></ol>

<ol>
	<li>Lewandoski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+control+of+gene+expression+in+the+mouse.">Conditional control of gene expression in the mouse.</a> Nature Reviews Genetics. 2, 743-755 (2001).</li><li>Couto, L. B., High, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+vector-mediated+RNA+interference.">Viral vector-mediated RNA interference.</a> Curr. Opin. Pharmacol. 10, 534-542 (2010).</li><li>Luo, L., Callaway, E. M., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+neural+circuits.">Genetic dissection of neural circuits.</a> Neuron. 57, 634-660 (2008).</li><li>Davidson, B. L., Breakefield, X. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+vectors+for+gene+delivery+to+the+nervous+system.">Viral vectors for gene delivery to the nervous system.</a> Nature Reviews Neuroscience. 4, 353-364 (2003).</li><li>Todd, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+circuitry+for+pain+processing+in+the+dorsal+horn.">Neuronal circuitry for pain processing in the dorsal horn.</a> Nature Reviews Neuroscience. 11, 823-836 (2010).</li><li>Wall, N. R., Wickersham, I. R., Cetin, A., De La Parra, M., Callaway, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monosynaptic+circuit+tracing+in+vivo+through+Cre-dependent+targeting+and+complementation+of+modified+rabies+virus.">Monosynaptic circuit tracing in vivo through Cre-dependent targeting and complementation of modified rabies virus.</a> Proceedings of the National Academy of Sciences of the United States of America. 107, 21848-21853 (2010).</li><li>Lo, L., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Cre-dependent,+anterograde+transsynaptic+viral+tracer+for+mapping+output+pathways+of+genetically+marked+neurons.">A Cre-dependent, anterograde transsynaptic viral tracer for mapping output pathways of genetically marked neurons.</a> Neuron. 72, 938-950 (2011).</li><li>Zhao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-specific+channelrhodopsin-2+transgenic+mice+for+optogenetic+dissection+of+neural+circuitry+function.">Cell type-specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function.</a> Nature Methods. 8, 745-752 (2011).</li><li>Tang, X. Q., Heron, P., Mashburn, C., Smith, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+sensory+axon+regeneration+in+adult+spinal+cord.">Targeting sensory axon regeneration in adult spinal cord.</a> J. Neurosci. 27, 6068-6078 (2007).</li><li>Cameron, A. A., Smith, G. M., Randall, D. C., Brown, D. R., Rabchevsky, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+of+intraspinal+plasticity+after+spinal+cord+injury+alters+the+severity+of+autonomic+dysreflexia.">Genetic manipulation of intraspinal plasticity after spinal cord injury alters the severity of autonomic dysreflexia.</a> J. Neurosci. 26, 2923-2932 (2006).</li><li>Passini, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS-targeted+gene+therapy+improves+survival+and+motor+function+in+a+mouse+model+of+spinal+muscular+atrophy.">CNS-targeted gene therapy improves survival and motor function in a mouse model of spinal muscular atrophy.</a> The Journal of Clinical Investigation. 120, 1253-1264 (2010).</li><li>Lutz, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postsymptomatic+restoration+of+SMN+rescues+the+disease+phenotype+in+a+mouse+model+of+severe+spinal+muscular+atrophy.">Postsymptomatic restoration of SMN rescues the disease phenotype in a mouse model of severe spinal muscular atrophy.</a> The Journal of Clinical Investigation. 121, 3029-3041 (2011).</li><li>Chen, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dsAAV+type+2-mediated+gene+transfer+of+MORS196A-EGFP+into+spinal+cord+as+a+pain+management+paradigm.">dsAAV type 2-mediated gene transfer of MORS196A-EGFP into spinal cord as a pain management paradigm.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 20096-20101 (2007).</li><li>South, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conditional+deletion+of+the+NR1+subunit+of+the+NMDA+receptor+in+adult+spinal+cord+dorsal+horn+reduces+NMDA+currents+and+injury-induced+pain.">A conditional deletion of the NR1 subunit of the NMDA receptor in adult spinal cord dorsal horn reduces NMDA currents and injury-induced pain.</a> J. Neurosci. 23, 5031-5040 (2003).</li><li>Tappe, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+scaffolding+protein+Homer1a+protects+against+chronic+inflammatory+pain.">Synaptic scaffolding protein Homer1a protects against chronic inflammatory pain.</a> Nat. Med. 12, 677-681 (2006).</li><li>Colle, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+intracerebral+delivery+of+AAV5+vector+encoding+human+ARSA+in+non-human+primate.">Efficient intracerebral delivery of AAV5 vector encoding human ARSA in non-human primate.</a> Human Molecular Genetics. 19, 147-158 (2010).</li><li>Carbajal, K. S., Weinger, J. G., Whitman, L. M., Schaumburg, C. S., Lane, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+Transplantation+of+Mouse+Neural+Stem+Cells+into+the+Spinal+Cords+of+Mice+Infected+with+Neurotropic+Mouse+Hepatitis+Virus.">Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus.</a> J. Vis. Exp. (53), e2834 (2011).</li><li>Snyder, B. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+adeno-associated+viral+vector+serotypes+for+spinal+cord+and+motor+neuron+gene+delivery.">Comparison of adeno-associated viral vector serotypes for spinal cord and motor neuron gene delivery.</a> Hum. Gene Ther. 22, 1129-1135 (2011).</li><li>Towne, C., Pertin, M., Beggah, A. T., Aebischer, P., Decosterd, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+adeno-associated+virus+serotype+6+(rAAV2/6)-mediated+gene+transfer+to+nociceptive+neurons+through+different+routes+of+delivery.">Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery.</a> Mol. Pain. 5, 52 (2009).</li><li>Kaplitt, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+gene+expression+and+phenotypic+correction+using+adeno-associated+virus+vectors+in+the+mammalian.">Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian.</a> 8, 148-154 (1994).</li></ol>

Los autores declaran que no tienen intereses en conflicto financieros.

La inyección del vector estereotáxica permite dirigir las neuronas de médula espinal para aplicaciones tales como mapeo de la red neuronal basada en virus transsynaptic propagación 6,7 o disección optogenético 8, guiado de los axones durante la regeneración de lesiones 9,10, o la terapia génica para la prevención o el tratamiento de la neurodegeneración 11, 12. Los vectores virales han sido utilizados para la manipulación de genes en la médula espinal para estudiar...

Las tecnologías avanzadas de manipulación de genes en el ratón condicional permitir que los enfoques multifacéticos para la exploración de las vías sinápticas y las conexiones funcionales en el sistema nervioso central. Los transgenes pueden ser reguladas por efectores de moléculas pequeñas tales como doxiciclina que actúan sobre un transactivador controlado por tetraciclina, que puede ser diseñado para funcionar como un represor o un activador de la transcripción de genes, o tamoxifeno reconociendo un ligando mutado dominio de unión del receptor de estrógeno 1 . Modificación irreversible transgén se consigue comúnmente por el ácido desoxirribonucleico (ADN) recombinasas. Cre (recombinación causas) y FLP (enzima flipasa recombinación) catalizan la escisión, la inversión o translocación de fragmentos de ADN que están flanqueadas por loxP (locus de cruce sobre x, P1) o frontales (objetivo flipasa reconocimiento) los sitios, respectivamente 1. Las aplicaciones incluyen la activación de genes o el silenciamiento y inducible por ácido ribonucleico (ARN) interferencia <sup&gt; 2. Expresión condicional de reporteros fluorescentes o enzimáticos, tales como β-galactosidasa o fosfatasa alcalina se puede utilizar para etiquetar las neuronas y examinar su organización tópica y conectividad 3. Proyectos a gran escala de mutagénesis en América del Norte ( <a href="http://www.norcomm.org/index.htm" target="_blank">http://www.norcomm.org/index.htm</a> ) y Europa ( <a href="http://www.knockoutmouse.org/about/eucomm" target="_blank">http://www.knockoutmouse.org/about/eucomm</a> ) están produciendo bibliotecas de clones de embriones de ratón con células madre objetivos condicionales de genes y trampas que eventualmente se cubren todo el genoma del ratón. Los ratones generados a partir de estos clones se pueden cruzar con un número creciente de líneas de ratón que expresan recombinasas de ADN bajo promotores o loci específicos para una población particular de neuronas para la manipulación selectiva de genes ( <a href="http://nagy.mshri.on.ca/cre_new/index.php" target="_blank">http://nagy.mshri.on.ca/cre_new/index . php</a> ). <p class= &quot;Jove_content&quot;&gt; Sin embargo, la restricción de manipulación génica para diferentes poblaciones de neuronas o regiones particulares de interés no puede lograrse mediante modificación genética dirigida solo si un promotor específico para la población de neuronas de interés no se conoce o no se expresan por todas las neuronas en la región de interés. En la médula espinal, los diseños experimentales pueden requerir restricción espacial de la manipulación genética de uno o dos segmentos craneocaudal. Inyección estereotáxica de un vector viral que expresa Cre o Flp permite limitar la recombinación de genes para las regiones en la médula espinal de ratones en los que los fragmentos de ADN están flanqueados por sitios FRT o loxP, los llamados alelos floxed o flrted. A diferencia de la reordenación del ADN constitutiva, que sería el resultado de cruzamiento de los animales con ratones que expresan recombinasa, esta estrategia también proporciona un control temporal sobre la activación de genes o el silenciamiento. Los vectores virales que codifican floxed o coqueteó transgenes ofrecen una opción inversa de la manipulación genética en ratones que expresan el corresponding aguas abajo de un promotor de recombinasa específica de neuronas. Varios vectores recombinantes con afinidad a las neuronas están disponibles 4. De alta capacidad (gutless) adenovirus, virus adeno-asociados, virus del herpes simple y lentivirus son comúnmente utilizados vectores neurotrópicos. Selección del virus apropiado para una pregunta de investigación es una parte crucial del diseño experimental. Tamaño del transgén, vía de administración, la especificidad de la infección a las neuronas frente a las células gliales, infección de eficacia, los efectos secundarios inflamatorios y tóxicos necesitan ser considerados 4. Aquí se describe la inyección estereotáxica de un vector viral en el asta dorsal de la médula espinal, una técnica que se emplea para la regulación génica condicional en nuestra investigación sobre la neurobiología del dolor. El cuerno dorsal recibe impulsos aferentes primarios de neuronas somatosensoriales incluyendo las neuronas nociceptivas. Interneuronas locales procesar la información antes de transmitirla neuronas de proyección deel cuerno dorsal al cerebro 5. Se demuestra que la infección de las neuronas del asta dorsal a nivel espinal segmentaria L4 con un neurotrópico recombinante adeno-associated virus (rAAV) que expresa aumento de proteína verde fluorescente (EGFP) bajo un promotor de citomegalovirus constitutivamente activo.

<table border="1"><tbody><tr><td> Nombre del Material </td><td> Empresa </td><td> Número de catálogo </td><td></td></tr><tr><td> Placa base Espinal </td><td> David Kopf Instruments </td><td> 912 </td><td></td></tr><tr><td> Pequeños animales instrumento estereotáxico </td><td> David Kopf Instruments </td><td> 900 </td><td></td></tr><tr><td> Gas Ratón anestesia cabeza titular </td><td> David Kopf Instruments </td><td> 923-B </td><td></td></tr><tr><td> Soportes ajustables de base </td><td> David Kopf Instruments </td><td> 982 </td><td></td></tr><tr><td> V muesca picos </td><td> David Kopf Instruments </td><td> 987 </td><td></td></tr><tr><td> Temperatura pequeño sistema de control de animales </td><td> David Kopf Instruments </td><td> TCAT-2LV </td><td></td></tr><tr><td> Adson fórceps </td><td> Herramientas Artes Ciencias </td><td> 11006-12 </td><td></td></tr><tr><td> Fórceps laminectomía </td><td> Herramientas Artes Ciencias </td><td> 11223-20 </td><td></td></tr><tr><td> UltraMicroPump (uno) con SYS-Micro4 Controller </td><td> World Precision Instruments </td><td> UMP3-1 </td><td></td></tr><tr><td> Microjeringa 65RN </td><td> Hamilton </td><td> 7633-01 </td><td></td></tr><tr><td> RN accesorio de compresión, 1 mm </td><td> Hamilton </td><td> 55750-01 </td><td></td></tr><tr><td> Vidrio borosilicato capilares </td><td> World Precision Instruments </td><td> 1B100F-4 </td><td></td></tr><tr><td> Microgrinder </td><td> Narishige </td><td> EG-44 </td><td></td></tr></tbody></table>

stereotaxic injection of a viral vector for conditional gene manipulation in the mouse spinal cord

Inyección intraparenquimatosa de un vector viral permite la manipulación génica condicional en distintas poblaciones de neuronas o regiones particulares del sistema nervioso central. Se demuestra una técnica de inyección estereotáxica que permite la expresión del gen objetivo o el silenciamiento en el cuerno dorsal de la médula espinal de ratón. El procedimiento quirúrgico es breve. Se requiere laminectomía de una sola vértebra, proporcionando para la recuperación rápida del animal y de la motilidad irreprochable de la columna vertebral. Inyección controlada de un volumen pequeño vector suspensión a baja velocidad y el uso de una microjeringa con cánula de cristal biselado minimizar la lesión del tejido. La respuesta inmune local al vector depende de las propiedades intrínsecas del virus empleados, en nuestra experiencia, es menor y de corta duración cuando un recombinante de virus adeno-asociado se utiliza. Un gen informador tal como la proteína verde fluorescente mejorada facilita la distribución de seguimiento espacial del vector, y la eficacia y la especificidad celularficidad de la transfección.

El éxito de los rendimientos de transfección de expresión génica robusto en las neuronas del asta dorsal de inyección (Figura 1), evitando que el cuerno dorsal del lado contralateral, el cuerno ventral y los ganglios de la raíz dorsal. <img src="/files/ftp_upload/50313/50313fig1.jpg" alt="Figura 1" /> Figura 1. Transfección de neuronas del asta dorsal. (A) Expresión de la EGFP reportero fluorescente (verde) en el cuerno izquierdo dorsal de la m?...

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Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Los vectores virales permiten la manipulación dirigida de genes. Se demuestra un método para la expresión génica condicional o ablación en la médula espinal de ratón, usando inyección estereotáxica de un vector viral en el asta dorsal, un sitio prominente de contacto sináptico entre las vías aferentes somatosensoriales y las neuronas del sistema nervioso central.

Inyección estereotáxica de un vector viral para la manipulación genética condicional en la médula espinal de ratón

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

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30.1K vistas.  Centre National de la Recherche Scientifique (CNRS). Los vectores virales permiten la manipulación dirigida de genes. Se demuestra un método para la expresión génica condicional o ablación en la médula espinal de ratón, usando inyección estereotáxica de un vector viral en el asta dorsal, un sitio prominente de contacto sináptico entre las vías aferentes somatosensoriales y las neuronas del sistema nervioso central.

Video: Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Médula Espinal Electrofisiología II: Fabricación de electrodos de aspiración extracelular

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Investigación

Neurociencias

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

Imágenes in vivo de la médula espinal de ratón Utilizando microscopía de dos fotones

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

La expansión de las células madre embrionarias y adultas neurales por<em&gt; In Utero</em&gt; La electroporación o inyección estereotáxica Viral

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

Inyección Viral Intracerebroventricular de Neonatología del ratón Cerebro de transducción neuronal persistente y generalizada

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

Un procedimiento para el implante Cámara Espinal para Longitudinal<em&gt; En Vivo</em&gt; Imágenes de la médula espinal de ratón

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

De dos fotones de imágenes de Dinámica Celular en la médula espinal de ratón

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

Cirugía estereotáctica para manipulación genética en las células estriatales de cerebro de ratón Neonatal

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

Laminectomía e implantación de la ventana de la médula espinal en el ratón

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

Inyección directa de un Vector lentivirales destaca múltiples caminos del Motor en la médula espinal de rata

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Entrega intra-arterial de células madre neuronales a la rata y el cerebro del ratón: aplicación a la isquemia cerebral

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...