We thank the imaging platform of the Institut de la Vision. We acknowledge the French Muscular Dystrophy Association (AFM) for a PhD fellowship to O.V. and Allergan INC. This work performed in the frame of the LABEX LIFESENSES [reference ANR-10-LABX-65] was supported by French state funds managed by the ANR. We thank Peggy Barbe, and M&#233;lissa Desrosiers for technical assistance with AAV preparations. We are grateful to St&#233;phane Fouquet for excellent technical assistance in confocal microscopy and his expert input with the interpretation of the results.

Todos los animales utilizados en este estudio fueron atendidos y manejados de acuerdo a la Declaración de ARVO para el uso de animales en Oftálmica y Vision Research. 1. Producción de AAV recombinante (rAAV) por transfección transitoria de células HEK-293 17,18 NOTA: Ver McClure C, JoVE (2011) 19. <ol><li> Se purifica una preparación de plásmido a gran escala (por lo menos 1 mg / ml) de los plásmidos vectores de AAV. Utilice 3 plásmidos. El plásmido auxiliar AAV que lleva los genes de replicación y de la cápsida sin el terminal invertida de AAV ITRs se repite, el casete de expresión del transgén que lleva el AAV ITR conoce como el plásmido de transferencia, y el plásmido suministro de los genes auxiliares de adenovirus E2, E4, y los genes VA RNA que se hace referencia como pHELPER. </li><li> Transfectar células 293 con estos 3 plásmidos usando polietilenimina (u otro reactivo de transfección). Por encima de 80% de eficiencia de transfección es necesario para una buena virendimientos RAL. </li><li> Purificar AAV recombinante a partir de lisados ​​de células 293 en un gradiente de iodixanol. 15%, 25%, 40% y 60% iodixanol se utiliza para obtener el gradiente. Recoger la fracción de iodixanol 40% que contiene partículas de AAV, concentrar la fracción después de ultracentrifugación a 500.000 xg durante 1 hr y tampón de intercambio frente a PBS (tampón fosfato salino) con 0,001% de Pluronic. </li><li> Para determinar el título genómica viral 20, realizar una DNAsa proteinaseK compendio seguido por QPCR. Los cebadores directo e inverso amplificar la región de 62 pb situada en el AAV2 ITR. Para el estándar plásmido, la sonda está marcada con FAM y tiene un agujero negro extintor (BHQ). </li><li> Llevar a cabo qPCR (reacción en cadena de la polimerasa cuantitativa) en un volumen final de 25 l usando 340 nM cebador inverso ITR, 100 nM de cebador ITR hacia adelante, 100 nM de la sonda AAV2 ITR, 0,4 mM dNTPs, 2 mM MgCl2, 1x Platinum Taq Buffer, 1U Platinum Taq y plantilla 5 l (estándar, muestra y control sin molde). </li><li> Parael estándar, utilice el plásmido para la transfección en 7 x 10 veces diluciones seriadas (5 l de cada uno de 2 x 10 9 a 2 x 10 3 genomas / ml que resulta en una concentración final de 10 octubre-10 abril genomas / ml). </li><li> Comience el programa de una etapa de desnaturalización inicial a 95 ° C durante 15 min seguido de 40 ciclos de desnaturalización a 95 ° C en durante 1 min y recocido / extensión a 60 ° C durante 1 min. Almacenar a 4 ° C durante un almacenamiento a corto plazo oa -20 ° C para el almacenamiento prolongado. </li></ol> 2. inyección intravítrea de AAV <ol><li> Anestesiar a los ratones C57BL6J con ketamina (50 mg / kg) y xilazina (10 mg / kg) y verificar la pérdida del reflejo de enderezamiento, el reflejo de retirada y de pinzamiento del rabo de respuesta que indica una anestesia adecuada. Dilatar los alumnos mediante la aplicación de la córnea de neosinefrina 5% y 0,5% mydriaticum gotas para los ojos. Use ungüento veterinario en los ojos para evitar la sequedad mientras que bajo anestesia. </li><li> Aprobar un ultrafino 30 G disposabLe aguja a través de la línea ecuatorial y al lado del limbo, en la cavidad vítrea (ver Chiu K, JoVE (2007) 21). Inyectar 1 l de stock contiene de 1 a 4 x 10 11 vp de ShH10-GFP o AAV5-GFP con la observación directa de la aguja en el centro de la cavidad vítrea (Figura 1). Utilice un ojo como un control para los experimentos de ILM. Inyectar ambos ojos para experimentos BRB. Aplicar un tratamiento tópico antiinflamatorio y antibacteriano en los ojos inyectados. </li><li> Dilatar alumnos con neosinefrina y cae mydriaticum ojo para la imagen. Realizar exámenes de fondo de ojo con una cámara de fondo de ojo para seguir GFP (Green Fluorescent Protein) expresión 1 semana, 2 semanas, 1 mes y 2 meses después de la inyección intravítrea (Figura 1). Sacrificar los ratones por inhalación de CO2 1 a 2 meses después de la inyección. </li></ol> 3. La inmunohistoquímica <ol><li> Para cryosections de la retina (Figura 1), diseccionar ojos enucleados para eliminar lens y la córnea, y la inmersión en solución 4% de paraformaldehído durante 1 hr. <ol><li> Cryoprotect los ojos previamente fijados en 10% de sacarosa durante 1 hora a temperatura ambiente, 20% de sacarosa para otra hora a temperatura ambiente. Cryoprotect en solución de sacarosa al 30%, durante la noche a 4 ° C. Congelar y incrustar ojeras en la incrustación de resina tal como Cryo-matriz. Hacer 10 micras cryosections y montar en portaobjetos tratados especialmente para las secciones de tejido congelado y que mejoran la adherencia del tejido durante la tinción. </li><li> Permeabilizar secciones con 0,1% de Triton X100 en PBS pH 7,4 durante 5 min. Lavar 2x en PBS y el bloque durante 1 hora a temperatura ambiente en PBS con 1% de BSA, 0,1% de Tween 20. </li></ol></li><li> Para flatmounts de la retina (Figura 1), fijar los ojos enucleados en 4% de paraformaldehído durante 15 min para facilitar la disección. <ol><li> En 15-30 min, diseccionar los ojos para eliminar la córnea y el cristalino. Se separa la retina del epitelio pigmentario de la retina (RPE) y la esclerótica cortando alrededor de la ora serrata yel nervio óptico. Sumergir en paraformaldehído al 4% durante otros 30 min para fijar la retina y la proteína fluorescente en las células de la retina transducidas (fijación no debe exceder de 24 hr). Lavar 5 min en PBS estéril, pH 7,4. </li><li> Cambiar el PBS a tampón de bloqueo (PBS con 1% de BSA, 2% de suero normal de cabra o de burro, 0,5% Triton X-100), e incubar en tampón de bloqueo, ya sea para 4 hr a temperatura ambiente o 4 ° C durante la noche. </li></ol></li><li> Incubar los tejidos con anticuerpos primarios en tampón de bloqueo, ya sea para 4 horas a temperatura ambiente o a 4 ° C durante la noche. Lavar 3 veces con PBS. NOTA: Los anticuerpos utilizados son los siguientes: anti-laminina (1 / 1.000) marcar la ILM, clon anti-rodopsina 4D2 (1/500) varillas de etiquetado, (1 / 1.500) de etiquetado células de Müller clon sintetasa anti-glutamina GS-6 , PNA lectina (1/40) conos de etiquetado. </li><li> Incubar los tejidos con dilución 1: 500 de anticuerpos secundarios conjugados Alexa Fluor en tampón de bloqueo durante 1 hora (cryosections) o 2 hr (flatmounts) a tempe habitacióntura y protegido de la luz. Lavar 3 veces con PBS. </li><li> Hacer el alivio de los recortes a la retina y flatmount sobre un portaobjetos de vidrio, ya sea con el fotorreceptor o el lado de células ganglionares de la retina (RGC) mirando hacia arriba. Añadir mountingmedium acuosa, aplique cubreobjetos y se almacena a 4 ° C. </li><li> Realizar la microscopía confocal de escaneo láser confocal microscopio. Adquirir imágenes de forma secuencial, línea por línea, para reducir la excitación y la diafonía de emisiones. Definir tamaño de paso de acuerdo con el teorema de muestreo de Nyquist-Shannon. Utilice los ajustes de exposición que minimicen píxeles sobresaturados en las imágenes finales. Proceso de imágenes de 12 bits con FIJI, proyecto Z-secciones en un solo plano utilizando máxima intensidad bajo la función Z-proyecto y, finalmente, convertirse al modo de color de 8 bits RGB. </li></ol> 4. Análisis de PCR de muestras de sangre de ratón <ol><li> Inyectar 100 l de stock contiene de 1 a 4 x 10 11 partículas de un AAV-GFP en la vena del pene de ratones C57BL6J anestesiados (5% de isofluranopara la inducción en una cámara de cierre y 2,5% de isoflurano a través de un cono de la nariz durante la cirugía) usando una jeringa de insulina con una aguja de ultra-fina 6 mm. Este animal servirá como control positivo de la circulación de partículas de AAV. </li><li> Muestra de sangre de la cola del ratón antes de la inyección y 3 horas, 24 horas, 2 días y 3 días después de la inyección intravítrea ShH10 o después de la inyección intrapeniles (Figura 1). Acerca de 10-20 l se recoge en tubos de heparina. Sacrificar los ratones por inhalación de CO 2 después de la finalización del procedimiento. </li><li> Extraer el ADN genómico de las muestras de sangre utilizando el kit de extracción de su elección. </li><li> Realizar la amplificación por PCR de ADN genómico. Para este protocolo utilice el siguiente par de cebadores: GFP, sentido 5&#39;-CGACACAATCTGCCCTTTCG-3 &#39;, 5&#39;-antisentido CATGGACGAGCTGTACAAGGGA-3&#39;. El siguiente programa de PCR se realizó: 95 ° C 2 min (1 ciclo); 95 ° C 45 seg, 55 ° C 1 min, 72 ° C 45 seg (30 ciclos); 72 ° C 5 min (1 ciclo);4 ° C espera. </li></ol> 5. Opcional Evans Método Azul NOTA: Cuantificar la permeabilidad vascular mediante la medición de la albúmina de fuga de los vasos sanguíneos en la retina utilizando el método de azul de Evans 10. <ol><li> Preparar colorante azul de Evans por disolución en una solución salina normal (6 mg / ml), sonicación durante 5 min en un limpiador ultrasónico, y filtración a través de un filtro de 5 micras. </li><li> Anestesiar ratones C57BL6J (inhalación isoflurano, consulte el paso 4.1), comprobar la pérdida de los reflejos, el reflejo de retirada y la respuesta pizca de cola que indica una anestesia adecuada, y se inyecta azul de Evans (45 mg / kg) a través de la vena del pene (Figura 1). Compruebe que las patas, el hocico y las orejas de ratones se vuelven claramente azul después de la inyección azul de Evans, lo que confirma la captación y distribución del colorante. </li><li> Anestesiar a los ratones C57BL6J 3 hr después de la inyección del colorante con ketamina (50 mg / kg) y xilazina (10 mg / kg) y verificar la pérdida de righting reflex que indica una anestesia adecuada. Muestra intracardially aproximadamente 500 l de sangre en tubos de heparina y perfundir los ratones para 2 min a través del ventrículo izquierdo con un tampón de citrato (0,05 M, pH 3,5; disolver 7,8 g de ácido cítrico y 3,8 g de citrato de sodio en 1 L de agua destilada) pre-calentado a 37 ° C para evitar la vasoconstricción. Los ratones se sacrificaron a través de la perfusión. </li><li> Después de la perfusión, enucleate y cuidadosamente diseccionar los dos ojos bajo un microscopio quirúrgico para recoger las retinas (ver sección 3.2). Retinas secos en un evaporador centrífugo durante la noche, pesan, y extraer el colorante azul de Evans incubando retinas con 100 l de formamida de 18 horas a 65 ° C con agitación (100 xg). Muestras Centrífuga retina en 30K tubos de filtro omega en 20,798.5 xg durante 2 horas a 4 ° C y las muestras de sangre se centrifuga a 20,798.5 xg durante 15 min a 4 ° C. </li><li> Use ambos sobrenadantes para medir la absorbancia. Determinar la absorbancia de cada muestra sustrayendo la absorbancia máxima for Evans azul a 620 nm al mínimo la absorbancia a 740 nm. Calcular la concentración de azul de Evans en el plasma y la retina de una curva patrón de azul de Evans en formamida. Expresar BRB permeabilidad en microlitros de azul de Evans por gramo de retina seco por hora (l plasma / g de peso seco de la retina / hr). </li></ol> 6. Opcional angiografía con fluoresceína NOTA: Las fugas de los vasos sanguíneos de la retina en ratones puede ser visualizado por inyección intraperitoneal de fluoresceína de sodio. <ol><li> Anestesiar ratones C57BL6J (inhalación isoflurano, consulte el paso 4.1) y dilatar pupilas con gotas para los ojos (neosinefrina y mydriaticum). Use ungüento veterinario en los ojos para evitar la sequedad mientras que bajo anestesia. </li><li> Inyectar por vía intraperitoneal 0,01-0,02 ml de 10% de fluoresceína sódica en 0,1 ml de solución salina estéril para la angiografía con fluoresceína. </li><li> Recoge imágenes de ambos ojos después de la inyección utilizando una cámara de fondo de ojo. Sacrificar los ratones por inhalación de CO2 después de la proprocedimiento. NOTA: 20 segundos son necesarios para los vasos de la retina que se hacen visibles en la angiografía. Las imágenes tienen que ser capturado entre 3-10 min máximo después de la inyección de fluoresceína. Los puntos de fuga (si existe) se observan después de 2,5 min. En los puntos de tiempo posteriores la calidad de la imagen se pierde debido a la fluoresceína sódica difundirse en los así sólo imágenes vítreas, poco después de obtenidos se utilizan para el análisis. </li></ol>

<ol>
	<li>Halfter, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+the+retinal+basal+lamina+during+early+embryonic+development+leads+to+a+retraction+of+vitreal+end+feet,+an+increased+number+of+ganglion+cells,+and+aberrant+axonal+outgrowth.">Disruption of the retinal basal lamina during early embryonic development leads to a retraction of vitreal end feet, an increased number of ganglion cells, and aberrant axonal outgrowth.</a> J Comp Neurol. 397 (1), 89-104 (1998).</li><li>Halfter, W., Dong, S., Balasubramani, M., Bier, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporary+disruption+of+the+retinal+basal+lamina+and+its+effect+on+retinal+histogenesis.">Temporary disruption of the retinal basal lamina and its effect on retinal histogenesis.</a> Dev Biol. 238 (1), 79-96 (2001).</li><li>Halfter, W., Willem, M., Mayer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basement+membrane-dependent+survival+of+retinal+ganglion+cells.">Basement membrane-dependent survival of retinal ganglion cells.</a> Invest Ophthalmol Vis Sci. 46 (3), 1000-1009 (2005).</li><li>Abdelkader, E., Lois, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internal+limiting+membrane+peeling+in+vitreo-retinal+surgery.">Internal limiting membrane peeling in vitreo-retinal surgery.</a> Surv Ophthalmol. 53 (4), 368-396 (2008).</li><li>Dalkara, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inner+limiting+membrane+barriers+to+AAV-mediated+retinal+transduction+from+the+vitreous.">Inner limiting membrane barriers to AAV-mediated retinal transduction from the vitreous.</a> Mol Ther. 17 (12), 2096-2102 (2009).</li><li>Bringmann, 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=Muller+cells+in+the+healthy+and+diseased+retina.">Muller cells in the healthy and diseased retina.</a> Prog Retin Eye Res. 25 (4), 397-424 (2006).</li><li>Eichler, W., Kuhrt, H., Hoffmann, S., Wiedemann, P., Reichenbach, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF+release+by+retinal+glia+depends+on+both+oxygen+and+glucose+supply.">VEGF release by retinal glia depends on both oxygen and glucose supply.</a> Neuroreport. 11 (16), 3533-3537 (2000).</li><li>Kaur, C., Foulds, W. S., Ling, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-retinal+barrier+in+hypoxic+ischaemic+conditions:+basic+concepts,+clinical+features+and+management.">Blood-retinal barrier in hypoxic ischaemic conditions: basic concepts, clinical features and management.</a> Prog Retin Eye Res. 27 (6), 622-647 (2008).</li><li>Kaur, C., Sivakumar, V., Foulds, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+response+of+neurons+and+glial+cells+to+hypoxia+in+the+retina.">Early response of neurons and glial cells to hypoxia in the retina.</a> Invest Ophthalmol Vis Sci. 47 (3), 1126-1141 (2006).</li><li>Xu, Q., Qaum, T., Adamis, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+blood-retinal+barrier+breakdown+quantitation+using+Evans+blue.">Sensitive blood-retinal barrier breakdown quantitation using Evans blue.</a> Invest Ophthalmol Vis Sci. 42 (3), 789-794 (2001).</li><li>Amato, R., Wesolowski, E., Smith, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+visualization+of+the+retina+by+angiography+with+high-molecular-weight+fluorescein-labeled+dextrans+in+the+mouse.">Microscopic visualization of the retina by angiography with high-molecular-weight fluorescein-labeled dextrans in the mouse.</a> Microvasc Res. 46 (2), 135-142 (1993).</li><li>Yang, G. 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=Virus-mediated+transduction+of+murine+retina+with+adeno-associated+virus:+effects+of+viral+capsid+and+genome+size.">Virus-mediated transduction of murine retina with adeno-associated virus: effects of viral capsid and genome size.</a> J Virol. 76 (15), 7651-7660 (2002).</li><li>Li, W., 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=Gene+therapy+following+subretinal+AAV5+vector+delivery+is+not+affected+by+a+previous+intravitreal+AAV5+vector+administration+in+the+partner+eye.">Gene therapy following subretinal AAV5 vector delivery is not affected by a previous intravitreal AAV5 vector administration in the partner eye.</a> Mol Vis. 15, 267-275 (2009).</li><li>Koerber, J. T., 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=Molecular+evolution+of+adeno-associated+virus+for+enhanced+glial+gene+delivery.">Molecular evolution of adeno-associated virus for enhanced glial gene delivery.</a> Mol Ther. 17 (12), 2088-2095 (2009).</li><li>Klimczak, R. R., Koerber, J. T., Dalkara, D., Flannery, J. G., Schaffer, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+adeno-associated+viral+variant+for+efficient+and+selective+intravitreal+transduction+of+rat+Muller+cells.">A novel adeno-associated viral variant for efficient and selective intravitreal transduction of rat Muller cells.</a> PLoS One. 4 (10), e7467 (2009).</li><li>Vacca, O., 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=AAV-mediated+gene+delivery+in+Dp71-null+mouse+model+with+compromised+barriers.">AAV-mediated gene delivery in Dp71-null mouse model with compromised barriers.</a> Glia. , (2013).</li><li>Choi, V. W., Asokan, A., Haberman, R. A., Samulski, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+recombinant+adeno-associated+viral+vectors.">Production of recombinant adeno-associated viral vectors.</a> Curr Protoc Hum Genet. 12 (Unit 12 19), (2007).</li><li>Choi, V. W., Asokan, A., Haberman, R. A., Samulski, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+recombinant+adeno-associated+viral+vectors+for+in+vitro+and+in+vivo+use.">Production of recombinant adeno-associated viral vectors for in vitro and in vivo use.</a> Curr Protoc Mol Biol. 16 (Unit 16 25), (2007).</li><li>McClure, C., Cole, K. L., Wulff, P., Klugmann, M., Murray, 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=Production+and+titering+of+recombinant+adeno-associated+viral+vectors.">Production and titering of recombinant adeno-associated viral vectors.</a> J Vis Exp. (57), e3348 (2011).</li><li>Aurnhammer, C., 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=Universal+real-time+PCR+for+the+detection+and+quantification+of+adeno-associated+virus+serotype+2-derived+inverted+terminal+repeat+sequences.">Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences.</a> Hum Gene Ther Methods. 23 (1), 18-28 (2012).</li><li>Chiu, K., Chang, R. C., So, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravitreous+injection+for+establishing+ocular+diseases+model.">Intravitreous injection for establishing ocular diseases model.</a> J Vis Exp. (8), 313 (2007).</li><li>Kolstad, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+adeno-associated+virus-mediated+gene+delivery+in+retinal+degeneration.">Changes in adeno-associated virus-mediated gene delivery in retinal degeneration.</a> Hum Gene Ther. 21 (5), 571-578 (2010).</li><li>Sene, 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=Functional+implication+of+Dp71+in+osmoregulation+and+vascular+permeability+of+the+retina.">Functional implication of Dp71 in osmoregulation and vascular permeability of the retina.</a> PLoS One. 4 (10), e7329 (2009).</li><li>Benard, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+New+Quantifiable+Blood+Retinal+Barrier+Breakdown+Model+In+Mice.">A New Quantifiable Blood Retinal Barrier Breakdown Model In Mice.</a> ARVO Annual Meeting. , (2011).</li></ol>

The authors have nothing to disclose.

El BRB regula el intercambio de moléculas entre la sangre y la retina. Su composición se asocia con varias enfermedades tales como la retinopatía diabética o la degeneración macular relacionada con la edad (AMD). Recientemente, hemos demostrado que en un ratón distrofina ronda, que muestra permeable BRB, la retina se vuelve más permisiva para la entrega de genes mediada por vectores virales adeno-asociados (AAV). Sin embargo, a pesar de BRB partículas de AAV permeabilidad intraocular inyectado mantenerse confina...

Las células de Müller son el principal componente glial de la retina. Morfológicamente, que abarcan la retina radial y su endfeet, en contacto con el vítreo, se enfrentan a la ILM y componentes secretos de este último. El ILM es una membrana basal compuesta de unos diez diferentes proteínas de la matriz extracelular (laminina, agrina, perlecan, nidogen, colágeno y varios proteoglicanos de sulfato de heparina). Durante el desarrollo, su presencia es indispensable para la histogénesis de la retina, la navegación de axones ópticos, y la supervivencia de las células ganglionares de 1-3. Sin embargo, ILM no es esencial en la retina de adultos y se puede extirpar quirúrgicamente en ciertas patologías sin causar daño en la retina 4. En la terapia génica, esta membrana se convierte en una barrera física para la transducción eficiente de la retina utilizando AAV mediante inyección intravítrea 5. A través de la extensa arborización de sus procesos, las células de Müller proporcionan suppo nutricional y regulatoriort a ambas neuronas de la retina y células vasculares. Las células de Müller también están involucrados en la regulación de la homeostasis de la retina, en la formación y mantenimiento de la BRB 6. Las uniones estrechas entre las células endoteliales de los capilares de la retina, las células de Müller, astrocitos y pericitos forman la BRB. BRB evita ciertas sustancias entren en los retina.In muchas enfermedades como la retinopatía diabética, oclusión venosa de la retina y las enfermedades respiratorias, la hipoxia de la retina provoca fugas a través de la BRB 7-9. Esta ruptura se asocia con un aumento de la permeabilidad vascular que conduce a edema vasogénico, desprendimiento de retina y daño en la retina. Las células de Müller están estrechamente asociados con los vasos sanguíneos y la membrana basal, jugando un papel importante tanto en la integridad BRB y ILM. En consecuencia, el etiquetado de las células gliales de Müller es particularmente relevante para el estudio del estado físico de estas barreras de la retina. Clásicoaliado, BRB permeabilidad se mide utilizando el ensayo de azul de Evans que consiste en la inyección sistémica de colorante azul de Evans, que se une no covalentemente a la albúmina plasmática. Este ensayo mide la pérdida de albúmina (proteína de tamaño intermedio, ~ 66 kDa) de los vasos sanguíneos en la retina (ver Protocolos Sección 5) 10. Alternativamente, la fuga vascular puede ser visualizado por la angiografía de fluorescencia de la retina en el que conste de fuga de fluoresceína (molécula pequeña, ~ 359 Da; véase Protocolos Sección 6) 11. Sin embargo, ambos métodos permiten la evaluación de la permeabilidad BRB a pequeñas moléculas y proteínas, pero que no proporcionan información sobre la integridad ILM. Por lo tanto, para estudiar BRB permeabilidad, se utilizó un método basado en AAV que da información sobre la permeabilidad BRB a moléculas más grandes (por ejemplo, partículas de AAV, 25 nm de diámetro). De hecho, nuestro método puede detectar la presencia de transgen AAV en la sangre, lo que sugeriría que ~ partículas de 25 nm de diámetro haríaser capaces de infiltrarse en el torrente sanguíneo. Este método también proporciona información sobre la estructura de la ILM y las proteínas de la matriz extracelular en condiciones patológicas. Dos variantes de AAV son útiles para tal estudio: AAV5 y ShH10. Subretinally inyectado, AAV5 tiene un tropismo natural para los fotorreceptores y el epitelio pigmentario de la retina 12 pero no se puede conseguir a través de la retina externa cuando se administra en el vítreo en las retinas de tipo salvaje con ILM intacta 5,13. ShH10 es una variante de AAV que ha sido diseñado para dirigirse específicamente a las células gliales más de neuronas 14,15. ShH10 etiquetas selectivamente las células de Müller en ambas retinas sanas y enfermas con una mayor eficiencia en las retinas con barreras comprometidas 16. Estas herramientas virales junto con immuhistochemistry y el análisis de ADN de sangre proporcionan información sobre el estado de las barreras de la retina y su implicación en la enfermedad (Figura 1).

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using adeno-associated virus as a tool to study retinal barriers in disease

Las células de Müller son las principales células gliales de la retina. Sus pies finales forman los límites de la retina en las membranas limitantes exterior e interior (ILM), y en conjunción con los astrocitos, los pericitos y células endoteliales que establecen la barrera sangre-retina (BRB). BRB limita el transporte de material entre el torrente sanguíneo y la retina mientras que la ILM actúa como una membrana basal que define histológicamente la frontera entre la retina y la cavidad vítrea. Etiquetado de las células de Müller es particularmente relevante para estudiar el estado físico de las barreras de la retina, ya que estas células son una parte integral de la BRB y ILM. Tanto BRB y ILM se alteran con frecuencia en enfermedades de la retina y son responsables de síntomas de la enfermedad. Hay varios métodos bien establecidos para estudiar la integridad de la BRB, tales como el ensayo de azul de Evans o la angiografía con fluoresceína. Sin embargo, estos métodos no proporcionan información sobre el grado de permeabilidad BRB to moléculas más grandes, en el rango nanométrico. Además, no proporcionan información sobre el estado de otras barreras de la retina tales como la ILM. Para estudiar la permeabilidad BRB junto ILM de la retina, se utilizó un método basado en AAV que proporciona información sobre la permeabilidad de BRB a moléculas más grandes, mientras que indica el estado de las proteínas de ILM y de la matriz extracelular en estados de enfermedad. Dos variantes de AAV son útiles para tal estudio: AAV5 y ShH10. AAV5 tiene un tropismo natural para fotorreceptores pero no puede conseguir a través de la retina externa cuando se administra en el vítreo cuando la ILM está intacto (es decir, en las retinas de tipo salvaje). ShH10 tiene un fuerte tropismo hacia las células gliales y etiquetará selectivamente células de Müller en ambas retinas sanas y enfermas. ShH10 proporciona la entrega de genes más eficiente en retinas donde se ve comprometida ILM. Estas herramientas virales, junto con el análisis de inmunohistoquímica y de ADN de sangre arrojan luz sobre el estado de las barreras de la retina en la enfermedad.

Nos esperar un aumento de la transducción de la retina de las células gliales de Müller utilizando ShH10 si el modelo animal muestra perturbaciones en la estructura de la ILM (Figura 2A - B). Por ejemplo, hemos demostrado que, en ausencia de DP71, objetivos ShH10 específicamente, pero de manera más eficiente Müller células gliales por inyección intravítrea, lo que indica aumento de la permeabilidad de la ILM en esta línea de ratón en comparación con ratones de tipo salvaje <...

Watch this Scientific Journal Video about Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease at JoVE.com

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

Utilizando adeno-asociado virus como una herramienta para estudiar las barreras de retina en la enfermedad

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

using-adeno-associated-virus-as-tool-to-study-retinal-barriers

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14.9K vistas.  Sorbonne Universtés, UPMC Univ Paris 06, UMR_S 968. To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

Video: Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.
However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.
In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

De alta eficiencia de transducción de las células de cáncer de hígado por recombinante serotipo del virus adeno-asociado 3 Vectores

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

Investigación

Medicina

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

Cariotipo espectral para el Estudio de anomalías cromosómicas en humanos y ratones con la enfermedad renal poliquística

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

Osteopathic Manipulative Treatment as a Useful Adjunctive Tool for Pneumonia

Pneumonia, the inflammatory state of lung tissue primarily due to microbial infection, claimed 52,306 lives in the United States in 20071 and resulted in the hospitalization of 1.1 million patients2. With an average length of in-patient hospital stay of five days2, pneumonia and influenza comprise significant financial burden costing the United States $40.2 billion in 20053. Under the current Infectious Disease Society of America/American Thoracic Society guidelines, standard-of-care recommendations include the rapid administration of an appropriate antibiotic regiment, fluid replacement, and ventilation (if necessary). Non-standard therapies include the use of corticosteroids and statins; however, these therapies lack conclusive supporting evidence4. (Figure 1)

Osteopathic Manipulative Treatment (OMT) is a cost-effective adjunctive treatment of pneumonia that has been shown to reduce patients&#8217; length of hospital stay, duration of intravenous antibiotics, and incidence of respiratory failure or death when compared to subjects who received conventional care alone5. The use of manual manipulation techniques for pneumonia was first recorded as early as the Spanish influenza pandemic of 1918, when patients treated with standard medical care had an estimated mortality rate of 33%, compared to a 10% mortality rate in patients treated by osteopathic physicians6. When applied to the management of pneumonia, manual manipulation techniques bolster lymphatic flow, respiratory function, and immunological defense by targeting anatomical structures involved in the these systems7,8, 9, 10.

The objective of this review video-article is three-fold: a) summarize the findings of randomized controlled studies on the efficacy of OMT in adult patients with diagnosed pneumonia, b) demonstrate established protocols utilized by osteopathic physicians treating pneumonia, c) elucidate the physiological mechanisms behind manual manipulation of the respiratory and lymphatic systems. Specifically, we will discuss and demonstrate four routine techniques that address autonomics, lymph drainage, and rib cage mobility: 1) Rib Raising, 2) Thoracic Pump, 3) Doming of the Thoracic Diaphragm, and 4) Muscle Energy for Rib 1.5,11

Pneumonia is one of the top ten leading causes of death in the U.S. Osteopathic manipulative techniques (OMT) may be utilized as an adjunctive therapy in the treatment of pneumonia to enhance biomechanical and immune function; and ultimately, to reduce hospital stay, duration of antibiotics, incidence of respiratory failure, and mortality. In this video-article, we will review randomized controlled studies on the use of OMT in pneumonia patients, as well as demonstrate specific hands-on techniques that are routinely practiced by osteopathic physicians when treating pulmonary infections.

Manipulación Osteopática como herramienta complementaria útil para la Neumonía

Pneumonia, the inflammatory state of lung tissue primarily due to microbial infection, claimed 52,306 lives in the United States in 20071 and resulted in ...

Fundus Photography as a Convenient Tool to Study Microvascular Responses to Cardiovascular Disease Risk Factors in Epidemiological Studies

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an important role in maintaining cardiovascular health. The retina is a tissue that lines the interior of the eye and it is the only tissue that allows for a non-invasive analysis of the microvasculature. Nowadays, high-quality fundus images can be acquired using digital cameras. Retinal images can be collected in 5 min or less, even without dilatation of the pupils. This unobtrusive and fast procedure for visualizing the microcirculation is attractive to apply in epidemiological studies and to monitor cardiovascular health from early age up to old age.
Systemic diseases that affect the circulation can result in progressive morphological changes in the retinal vasculature. For example, changes in the vessel calibers of retinal arteries and veins have been associated with hypertension, atherosclerosis, and increased risk of stroke and myocardial infarction. The vessel widths are derived using image analysis software and the width of the six largest arteries and veins are summarized in the Central Retinal Arteriolar Equivalent (CRAE) and the Central Retinal Venular Equivalent (CRVE). The latter features have been shown useful to study the impact of modifiable lifestyle and environmental cardiovascular disease risk factors.
The procedures to acquire fundus images and the analysis steps to obtain CRAE and CRVE are described. Coefficients of variation of repeated measures of CRAE and CRVE are less than 2% and within-rater reliability is very high. Using a panel study, the rapid response of the retinal vessel calibers to short-term changes in particulate air pollution, a known risk factor for cardiovascular mortality and morbidity, is reported. In conclusion, retinal imaging is proposed as a convenient and instrumental tool for epidemiological studies to study microvascular responses to cardiovascular disease risk factors.

Retinal image analysis is an unobtrusive procedure for visualizing the microcirculation. The impact of cardiovascular disease risk factors can result in changes of retinal vessel calibers. The procedures to acquire fundus images and the steps for calculating the vessel calibers are described.

Fotografía del fondo como una herramienta conveniente para estudiar las respuestas microvasculares a factores de riesgo cardiovascular en los estudios epidemiológicos

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an ...

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research. Successful outcomes are dependent upon three critical components: test chambers that permit self-feeding while standing unrestrained in a confined space, recipes that mask the aversive taste/odor of commercially-available oral contrast agents, and a step-by-step test protocol that permits quantification of swallow physiology. Elimination of one or more of these components will have a detrimental impact on the study results. Moreover, the energy level capability of the fluoroscopy system will determine which swallow parameters can be investigated. Most research centers have high energy fluoroscopes designed for use with people and larger animals, which results in exceptionally poor image quality when testing mice and other small rodents. Despite this limitation, we have identified seven VFSS parameters that are consistently quantifiable in mice when using a high energy fluoroscope in combination with the new murine VFSS protocol. We recently obtained a low energy fluoroscopy system with exceptionally high imaging resolution and magnification capabilities that was designed for use with mice and other small rodents. Preliminary work using this new system, in combination with the new murine VFSS protocol, has identified 13 swallow parameters that are consistently quantifiable in mice, which is nearly double the number obtained using conventional (i.e., high energy) fluoroscopes. Identification of additional swallow parameters is expected as we optimize the capabilities of this new system. Results thus far demonstrate the utility of using a low energy fluoroscopy system to detect and quantify subtle changes in swallow physiology that may otherwise be overlooked when using high energy fluoroscopes to investigate murine disease models.

This study successfully adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research.

Adaptación videofluoroscópicos Swallow Métodos de Estudio Humanos para detectar y caracterizar disfagia en modelos murinos de enfermedades

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

Development of an Alpha-synuclein Based Rat Model for Parkinson&#039;s Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

Desarrollo de un modelo basado en la rata alfa-sinucleína para la enfermedad de Parkinson mediante inyección estereotáctica de un vector viral adeno-asociado recombinante

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

Usando proyección de imagen retiniana para el estudio de la demencia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.

Here we provide a detailed protocol to carry out in vivo cardiac gene editing in mice using recombinant Adeno-Associated Virus(rAAV)-mediated delivery of CRISPR. This protocol offers a promising therapeutic strategy to treat dystrophic cardiomyopathy in Duchenne muscular dystrophy and can be used to generate cardiac-specific knockout in postnatal mice.

Entrega de mediada por Virus adeno-asociado de CRISPR para Gene cardiaco en ratones

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

The Dyspepsia Educational Tool As a Novel Aid in Dyspepsia Management

Digital educational tools have a well-established role in current healthcare. In particular, disorders that are managed non-pharmacologically benefit from this development, as it enables patient engagement in self-management. Dyspepsia is a condition thought to arise from gastric and duodenal perturbations, brain-gut axis disturbances, and dietary factors. Behavioral interventions are a major part of dyspepsia treatment, hence patient engagement and motivation through education is essential. Protocols that describe the development process of such educational tools are scarce. We provide a methodology describing development of a dyspepsia educational tool. Assessment of users' needs is the first step, followed by a literature search. The content is developed based on the main themes and entered into a content management system, to build the program. Final adjustments are made after a pilot test of the tool. The presented protocol can be used as a guide for development of a digital dyspepsia educational tool or as a tool for similar situations.

This protocol describes the development process of a digital dyspepsia educational tool. Assessment of unmet needs and literature, content development, and building of the tool are presented. The methodology can be used as a guide for future development of digital educational tools.

La herramienta educativa Dyspepsia como una ayuda novedosa en la gestión de la dispepsia

Digital educational tools have a well-established role in current healthcare. In particular, disorders that are managed non-pharmacologically benefit from ...

Subconjunctival Administration of Adeno-associated Virus Vectors in Small Animal Models

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ease of accessibility via multiple administration routes. Subconjunctival (SCJ) injections offer advantages over other intraocular administration routes as they are simple, safe, and usually performed in an outpatient setting. SCJ injections in small animals usually require the assistance of an operating microscope due to the size of the eye. Previous work has demonstrated that SCJ injection of specific adeno-associated virus (AAV) serotypes is a valid gene delivery strategy for targeted transduction of the ocular surface, eye muscle, cornea, and optic nerve, providing a potential approach for the treatment of many ocular diseases.
Herein, a detailed protocol is presented for SCJ injections in a mouse model using an injection system consisting of a programmable infusion/withdrawal syringe pump (which allows for consistent and precise injection speed and pressure) and a gastight removable syringe coupled with microinjection needles. The injection system is also adaptable for other intraocular administration routes such as intrastromal, intracameral, intravitreal, and subretinal injections in small animals. Although the delivery of adeno-associated viral vectors for ocular gene therapy studies is described, the protocol herein can also be adapted for a variety of ophthalmic solutions in small animal models. The key practical steps in the administration route, setup for the injection platform, preparation of the injection, and tips from direct experience will be discussed in detail. In addition, common validation techniques for AAV delivery confirmation to the desired tissues will also be briefly discussed.

In this manuscript, subconjunctival injection is demonstrated as a valid vector delivery method for ocular tissues in mice using an injection system consisting of an infusion/withdrawal syringe pump and a gastight removable syringe coupled with microinjection needles. This injection system is also adaptable for other intraocular administration routes.

Administración subconjuntival de vectores de virus adenoasociados en modelos de animales pequeños

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ...