Este estudio fue apoyado por la Universidad Nacional de Seúl Beca de Investigación (800-20140542), el Programa de Investigación de Pioneer NRF / MEST (2012-0009544), y el Programa de Bio-Signal Analysis Innovación Tecnológica de la NRF / MEST (2009-0090895), y la concesión de NRF / MEST (2015M3A9E6028949).

Todos los experimentos con animales se realizaron de acuerdo con la Asociación para la Investigación en Visión y Oftalmología para el Uso de Animales en Oftálmica y Vision Research, y las directrices y normas establecidas por el Comité Nacional de Seúl Cuidado de Animales institucional y el empleo de la Universidad Nacional de Seúl y Universidad Comité de Bioseguridad del hospital. 1. Preparación Kit de inyección y virales Vectores <ol><li> Prepare la jeringa microlitro equipado con una aguja roma 33 G esterilizada utilizando gas de óxido de etileno. Diluir los vectores virales en PBS durante la titulación adecuada en el tubo micro (es decir, 1 x 10 6 TU / l). Enjuague la jeringa varias veces con vectores virales para eliminar cualquier espacio muerto en la jeringa. </li></ol> 2. La inyección subretiniana de vectores virales <ol><li> Anestesiar los ratones adultos (es decir, 6 - 8 semanas de edad) con una inyección intraperitoneal de la mezcla de tiletaminane y zolazepam (1: 1, 2,25 mg / kg de peso corporal) y clorhidrato de xilazina (0,7 mg / kg de peso corporal) o un régimen de anestesia adecuada alternativa. </li><li> Dilatar los alumnos con un colirio de fenilefrina al 0,5% y tropicamida 0,5%. </li><li> Prepare la jeringa microlitro cargando con 1,5-2 l de vectores virales. </li><li> Abra el párpado y el ojo sobresalga para exponer el ecuador para inyección conveniente y centrarse en bajo el microscopio operativo. Mantener la posición del ojo sobresalía hasta terminar la inyección, o el desplazamiento de la aguja puede ocurrir durante la inyección. Para mantener el globo ocular con firmeza, coloque los dedos fuera del reborde orbitario. </li><li> Aplicar una gota de solución oftálmica viscoelástica a la superficie corneal. </li><li> Coloque un pequeño tobogán cubierta redonda en la parte superior de la córnea para visualizar la retina. </li><li> Perforar un pequeño agujero en ligero detrás del limbo utilizando un estéril 30 G 1/2 aguja para la inyección subretiniana más. Hacer el agujero inferior for el ojo derecho, y superior para el ojo izquierdo por la comodidad. NOTA: Si el agujero se hace en temporal o nasal, es difícil ser cubierto por el párpado después de la inyección. Punción inicial debe hacerse ligeramente por detrás del limbo para evitar los vasos del limbo que se ejecutan a lo largo del limbo y se pueden reconocer fácilmente. Tenga cuidado de no golpear el objetivo con la aguja al hacer la punción inicial. No inserte todo el bisel de la aguja para evitar la punción de la lente. </li><li> Coloque la aguja roma 33 G de la jeringa microlitro a través del orificio pre-perforado y acercarse a la aguja en el espacio subretiniano, hasta el punto en que se sienta resistencia leve. NOTA: Para la inyección subretiniana, el mejor ángulo de aproximación de la aguja es de unos 45 grados contra plano del iris, y la aguja roma debería ser empujado posteriormente hacia el área peripapilar. Cuadrada punteada indica la vía aguja sugerido a través de la cavidad vítrea para el injectio subretinianan en la Figura 1. NOTA: No habrá resistencia sintió cuando la perforación (o paso) las capas de la retina, ya que son muy suaves. Así, el primer sentimiento de resistencia leve indica que la aguja ya ha tocado la capa de RPE y la inserción de la aguja debe ser detenido. Tener cuidado de no penetrar en el tejido escleral con una presión excesiva debido a que la aguja debe ser colocado en el potencial espacio subretiniano. Si la aguja perfora el tejido de la esclerótica por potencia excesiva, entra en los espacios orbitales extraoculares sin resistencia. (Este paso es crítico) </li></ol><img alt="Figura 1" src="/files/ftp_upload/53030/53030fig1.jpg" /> Figura 1. Esquema de la inyección subretiniana. H &amp; E manchadas sección transversal del ojo del ratón que representa las estructuras con una vía de aguja para la inyección subretiniana marcada (un cuadrado de puntos). Ampliación: 40X, barra de escala:<html> 500 micras. <a href="https://www.jove.com/files/ftp_upload/53030/53030fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <ol start="9"><li> Inyectar los vectores virales (por ejemplo, 1 x 10 6 TU / l) suavemente en el espacio subretiniano sin temblor de evitar daños en el tejido no deseado y retirar la aguja suavemente. Sostenga firmemente el globo ocular durante la inyección como se describe en 2.4. </li><li> Observar la formación de la ampolla subretiniana después de la inyección bajo microscopio quirúrgico para asegurarse de que no hay hemorragia retiniana. </li></ol><img alt="Figura 2" src="/files/ftp_upload/53030/53030fig2.jpg" /> Figura 2. Subretinal BELB Formación sin retiniana Hemorragia. Vista microscópica de BELB subretiniana después de la inyección subretiniana bajo microscopio quirúrgico muestra la formación de vesículas éxito sin hemorragia retiniana.m / files / ftp_upload / 53030 / 53030fig2large.jpg &quot;target =&quot; _ blank &quot;&gt; Haga clic aquí para ver una versión más grande de esta figura. <ol start="11"><li> Cierre suavemente el párpado para cubrir el lugar de la inyección para la auto-sellado. Devolver los ratones para jaula y mantenerse con vida hasta la evaluación. NOTA: Después de que los investigadores se acostumbran a los procedimientos, que pueden obtener los resultados repetibles con procedimientos rápidos saltarse estos pasos por (2.5, 6 y 10) </li></ol> 3. Evaluación de la eficacia de la entrega de genes en el epitelio pigmentario de la retina <ol start="1"><li> Sacrificar los ratones en la cámara de CO 2. Enuclear los ojos con unas tijeras. </li><li> Fijar los ojos en la solución de paraformaldehído al 4% a 4 ° C durante un mínimo de 1 hora a varios días. </li><li> Coge la córnea con unas pinzas finas y perforar la córnea con micro-tijeras. Retire la córnea después de tijera a lo largo del limbo. Retire la lente con unas pinzas. </li><li> Recorte el cuerpo ciliar con mICRO-tijeras que permiten el desprendimiento de retina. Tire suavemente toda la retina de la / coroides / esclerótica complejo EPR. Cortar el RPE / coroides / complejo escleral radialmente desde la periferia hacia el centro, cerca del nervio óptico varias veces para asegurarse de 4 a 8 hojas para el montaje plana. </li><li> Da la vuelta al RPE / coroides / complejo escleral para hacer lateral RPE abajo, y recortar todos los tejidos que quedan en la parte escleral incluyendo los músculos, la conjuntiva y el nervio óptico. Este paso es importante hacer una RPE / coroides / complejo escleral plana porque cualquier tejido restante hace que el complejo desigual. </li><li> Incubar el complejo con el anticuerpo primario y secundario para sus fines específicos de investigación (opcional, consulte las referencias para los métodos detallados). Piso-montar las RPE / coroideo / complejos escleral en el portaobjetos de vidrio y absorber el PBS con una esponja absorbente. </li><li> Añadir 30 l de solución de montaje y coloque la tapa deslizante. Observar la muestra de la eficacia de la prestación de vector bajo el microscopio fluoresceína. Mantener las muestras enel refrigerador para su posterior observación. </li></ol>

<ol>
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(79), (2013).</li><li>Wert, K. J., Skeie, J. M., Davis, R. J., Tsang, S. H., Mahajan, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subretinal+injection+of+gene+therapy+vectors+and+stem+cells+in+the+perinatal+mouse+eye.">Subretinal injection of gene therapy vectors and stem cells in the perinatal mouse eye.</a> J Vis Exp. (69), (2012).</li><li>Eberle, D., Santos-Ferreira, T., Grahl, S., Ader, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subretinal+transplantation+of+MACS+purified+photoreceptor+precursor+cells+into+the+adult+mouse+retina.">Subretinal transplantation of MACS purified photoreceptor precursor cells into the adult mouse retina.</a> J Vis Exp. (84), e50932 (2014).</li><li>Sahel, J. A., Roska, B. <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+for+blindness.">Gene therapy for blindness.</a> Annu Rev Neurosci. 36, 467-488 (2013).</li><li>Allocca, 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=Novel+adeno-associated+virus+serotypes+efficiently+transduce+murine+photoreceptors.">Novel adeno-associated virus serotypes efficiently transduce murine photoreceptors.</a> J Virol. 81 (20), 11372-11380 (2007).</li><li>Takahashi, K., 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=Sustained+transduction+of+ocular+cells+with+a+bovine+immunodeficiency+viral+vector.">Sustained transduction of ocular cells with a bovine immunodeficiency viral vector.</a> Hum Gene Ther. 13 (11), 1305-1316 (2002).</li><li>Rolling, F., 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+therapeutic+prospects+in+early+onset+of+severe+retinal+dystrophy:+restoration+of+vision+in+RPE65+Briard+dogs+using+an+AAV+serotype+4+vector+that+specifically+targets+the+retinal+pigmented+epithelium.">Gene therapeutic prospects in early onset of severe retinal dystrophy: restoration of vision in RPE65 Briard dogs using an AAV serotype 4 vector that specifically targets the retinal pigmented epithelium.</a> Bull Mem Acad R Med Belg. 161 (10-12), 497-508 (2006).</li><li>Le Meur, 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=Restoration+of+vision+in+RPE65-deficient+Briard+dogs+using+an+AAV+serotype+4+vector+that+specifically+targets+the+retinal+pigmented+epithelium.">Restoration of vision in RPE65-deficient Briard dogs using an AAV serotype 4 vector that specifically targets the retinal pigmented epithelium.</a> Gene Ther. 14 (4), 292-303 (2007).</li><li>Alexander, J. 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The authors have nothing to disclose.

En este artículo de vídeo, describimos la técnica de inyección subretiniana limbo-enfoque en detalle con resultados representativos de RPE / coroides / esclerótica de montaje plana. Esta es una técnica fácil y conveniente para la inyección subretiniana de vectores virales en RPE. La visualización directa de la formación de la ampolla durante la inyección es un paso importante para la entrega exacta para los principiantes. Hay algunas técnicas subretinianas inyección introducidos en Journal of experimentos v...

En oftalmología, terapia génica ha emergido como la modalidad de tratamiento de retinopatías hereditarias monogénicas. Hay retinopatías hereditarias asociadas con genes en el epitelio pigmentario de la retina (EPR) incluyendo Leber amurosis congénita 1,2, la retinitis pigmentosa 3 y 4 coroideremia. El campo de la investigación de la terapia génica se está expandiendo en ambos estudios preclínicos y ensayos clínicos utilizando vectores virales como el virus adeno -Associated (AAV), lentivirus (LV) y adenovirus (Ad) 5. Diferentes vectores virales tienen diferente tropismo en la retina. Para una terapia génica seguros y eficaces, vectores virales deben ser seleccionados cuidadosamente de acuerdo con las células diana y los genes diana. La ruta de administración de genes también es importante para la administración de genes eficaz a las células diana, por lo tanto, debe elegirse cuidadosamente, también. Los dos métodos más comunes para la entrega intraocular de vectores virales se subretinal inyección y la inyección intravítrea 6. Este último, inyección intravítrea, ha sido ampliamente utilizado para la administración de fármacos para el tratamiento de la neovascularización coroidea en la degeneración húmeda macular relacionada con la edad (AMD) y edema macular en la retinopatía diabética 7. Ruta intravítrea proporciona una exposición de vectores virales a retina vítreo e interior, pero la difusión de los vectores de retina externa es limitado. Por otra parte, la ruta subretinal proporciona la entrega directa de vectores virales para el espacio potencial entre la retina y RPE, induciendo una ampolla localizada. Por lo tanto, la inyección subretiniana se considera actualmente una ruta más eficiente para atacar a las células fotorreceptoras y RPE. En términos de abordaje quirúrgico, pars plana es elegido como un lugar seguro para la inyección intravítrea de evitar daños a la retina en pacientes humanos. Simplemente modificando este enfoque a los ratones, podríamos inyectar vectores virales subretinally o intravireally través enfoque del limbo. En este videoartículo, demostramos un método fácil y conveniente de la inyección subretiniana de vectores virales en ratones RPE. Después de la punción única en posterior a limbo con un medio de aguja 30 G, 33 una aguja roma G microlitro equipado jeringa se inserta en el espacio subretiniano a través del sitio de la punción limbal. Los vectores virales de 1,5-2 l de volumen se inyectan al espacio potencial entre la retina y el RPE inducir ampollas subretinianas. Este procedimiento se puede realizar bajo visualización directa usando microscopio quirúrgico. La práctica repetida garantiza resultados reproducibles incluso sin la visualización directa de la formación de la ampolla. Esto ayudará a los investigadores a realizar experimentos precisos y de ahorro de tiempo para la entrega de genes en ratones subretiniano RPE.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>TWEEZERS DUMONT #5 11cm DUMOSTAR 0.1 x 0.06 mm TIPS</td>
			<td>WPI</td>
			<td>500233</td>
			<td></td>
		</tr>
		<tr>
			<td>VANNAS Scissors S/S, 105mm</td>
			<td>WPI</td>
			<td>555583S</td>
			<td></td>
		</tr>
		<tr>
			<td>33G Blunt needle</td>
			<td>WPI</td>
			<td>NF33BL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoFil Syringe, 10 microliter&#160;</td>
			<td>WPI</td>
			<td>NANOFIL</td>
			<td></td>
		</tr>
		<tr>
			<td>RPE-KIT</td>
			<td>WPI</td>
			<td>RPE-KIT</td>
			<td>For easy one hand injection</td>
		</tr>
		<tr>
			<td>30Gx1/2 (0.3mmx 13mm) BD PrecisionGlideTM Needle</td>
			<td>BD</td>
			<td>305107</td>
			<td>Initial puncture for subretinal injection</td>
		</tr>
		<tr>
			<td>Microscope Cover Glasses (No. 1 3 mm diameter)</td>
			<td>Warner Instruments</td>
			<td>64-0720&#160; (CS-3R)</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica operating microscope</td>
			<td>Leica</td>
			<td>LM M80</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoresecein microscope</td>
			<td>Nikon</td>
			<td>Eclipse 80i</td>
			<td></td>
		</tr>
		<tr>
			<td>Lentivirus</td>
			<td>Thermo scientific</td>
			<td>TMO.LV-Ctr</td>
			<td>Used to dilute vectors</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010-015</td>
			<td>Used to dilute vectors</td>
		</tr>
		<tr>
			<td>Troperin (Phenylephrin 0.5%-Tropicamide 0.5%)</td>
			<td>Hanmi</td>
			<td></td>
			<td>For dilation</td>
		</tr>
		<tr>
			<td>Proparacaine Hydrochloride Ophthalmic Solution USP, 0.5% (Sterile)</td>
			<td>Bausch&#38;Lomb</td>
			<td></td>
			<td>For topical anesthesia</td>
		</tr>
		<tr>
			<td>Healon GV OVD</td>
			<td>Abbott Medical Optics Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zoletil 50 (tiletamine hypochloride and zolazepam hypochloride)</td>
			<td>Virbac</td>
			<td></td>
			<td>For general anesthesia</td>
		</tr>
		<tr>
			<td>Rompun&#174; injection (Xylazine HCl)</td>
			<td>Bayer</td>
			<td></td>
			<td>For general anesthesia</td>
		</tr>
	</tbody>
</table>

limbal approach-subretinal injection of viral vectors for gene therapy in mice retinal pigment epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Para evaluar la eficacia de la inyección subretiniana en viral transducción génica mediante este protocolo, hemos utilizado vectores LV disponibles en el mercado con el promotor CMV que expresa tanto las buenas prácticas agrarias y RFP para el indicador. Los ojos fueron enucleados después de que el período de tiempo adecuado de acuerdo con el propósito de la investigación. Para los resultados representativos, los ojos fueron enucleados 10 semanas y 20 semanas después de la inyección subretiniana. Después de l...

Watch this Scientific Journal Video about Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium at JoVE.com

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

Limbal Inyección-Enfoque Subretinal de vectores virales para terapia génica en ratones epitelio pigmentario de la retina

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

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JoVE Journal

Neuroscience

20.9K vistas.  Seoul National University College of Medicine. Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

Video: Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

Modelos experimentales para el estudio de la fisiología epiteliales del pigmento retiniano y fisiopatología

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

Investigación

Neurociencias

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

Inyección intracraneal de adeno-asociado vectores virales

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

Disección de un ojo del ratón de un monte entero del epitelio pigmentario de la retina

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

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

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

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 ...

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 Protocol for Transcranial Photobiomodulation Therapy in Mice

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide range of neurological and psychiatric disorders, and memory enhancement in age-related cognitive decline and neurodegenerative diseases. We describe a laboratory protocol for transcranial photobiomodulation therapy (PBMT) in mice. Aged BALB/c mice (18 months old) are treated with a 660 nm laser transcranially, once daily for 2 weeks. Laser transmittance data shows that approximately 1% of the incident red light on the scalp reaches a 1 mm depth from the cortical surface, penetrating the dorsal hippocampus. Treatment outcomes are assessed by two methods: a Barnes maze test, which is a hippocampus-dependent spatial learning and memory task evaluation, and measuring hippocampal ATP levels, which is used as a bioenergetics index. The results from the Barnes task show an enhancement of the spatial memory in laser-treated aged mice when compared with age-matched controls. Biochemical analysis after laser treatment indicates increased hippocampal ATP levels. We postulate that the enhancement of memory performance is potentially due to an improvement in hippocampal energy metabolism induced by the red laser treatment. The observations in mice could be extended to other animal models since this protocol could potentially be adapted to other species frequently used in translational neuroscience, such as rabbit, cat, dog, or monkey. Transcranial photobiomodulation is a safe and cost-effective modality which may be a promising therapeutic approach in age-related cognitive impairment.

Photobiomodulation therapy is an innovative noninvasive modality for the treatment of a wide range of neurological and psychiatric disorders and can also improve healthy brain function. This protocol includes a step-by-step guide to performing brain photobiomodulation in mice by transcranial light delivery, which can be adapted for use in other laboratory rodents.

Un protocolo de Transcranial Photobiomodulation terapia en ratones

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide ...

In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative cell replacement therapies while also creating tools to study cell fate in situ. To date, it has been possible to obtain neurons via in vivo reprogramming, but the precise phenotype of these neurons or how they mature has not been analyzed in detail. In this protocol, we describe a more efficient conversion and cell-specific identification of the in vivo reprogrammed neurons, using an AAV-based viral vector system. We also provide a protocol for functional assessment of the reprogrammed cells&#8217; neuronal maturation. By injecting flip-excision (FLEX) vectors, containing the reprogramming and synapsin-driven reporter genes&#160;to specific cell types in the brain that serve as the target for cell reprogramming. This technique allows for the easy identification of newly reprogrammed neurons. Results show that the obtained reprogrammed neurons functionally mature over time, receive synaptic contacts and show electrophysiological properties of different types of interneurons. Using the transcription factors Ascl1, Lmx1a and Nurr1, the majority of the reprogrammed cells have properties of fast-spiking, parvalbumin-containing interneurons.

This protocol aims at generating directly reprogrammed interneurons in vivo, using an AAV-based viral system in the brain and a FLEX synapsin-driven GFP reporter, which allows for cell identification and further analysis in vivo.

En Vivo Reprogramación Directa de Células Gliales Residentes en Interneuronas por Inyección Intracerebral de Vectores Virales

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative ...

Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of the mouse neuroretina&#160;with intact retinal pigment epithelium (RPE). Depending on the research question, retinas can be isolated from wild-type animals or from disease models, to study, for instance, diabetic retinopathy or hereditary retinal degeneration. Eyes from early postnatal day 2&#8211;9 animals are enucleated under aseptic conditions. They are partially digested in proteinase K to allow for a detachment of the choroid from the RPE. Under the stereoscope, a small incision is made in the cornea creating two edges from where the choroid and sclera can be gently peeled off from the RPE and neuroretina. The lens is then removed, and the eyecup is cut in four points to give it a four-wedged shape resembling a clover leaf. The tissue is finally transferred in a hanging drop into a cell culture insert holding a polycarbonate culturing membrane. The cultures are then maintained in R16 medium, without serum or antibiotics, under entirely defined conditions, with a medium change every second day.
The procedure described enables the isolation of the retina and the preservation of its normal physiological and histotypic context for culturing periods of at least 2 weeks. These features make organotypic retinal explant cultures an excellent model with high predictive value, for studies into retinal development, disease mechanisms, and electrophysiology, while also enabling pharmacological screening.

The protocol describes organotypic explants of mouse neuroretina, cultivated together with its retinal pigment epithelium (RPE), in R16 defined medium, free of serum and antibiotics. This method is relatively simple to perform, less expensive, and time-consuming when compared to in vivo experiments, and can be adapted to numerous experimental applications.

A largo plazo, Cultivo Sin Suero De Explantes Organotípicos De La Retina De Ratón Con Epitelio Pigmento Retiniano Intacto

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of ...

Viral Construct (AAV) Injection in Mouse Superior Colliculus for Calcium Imaging

Begin by securing the anesthetized mouse onto a stereotaxic frame with ear bars. Apply ophthalmic ointment on the mouse's eyes to prevent drying, and maintain the body temperature of the mouse at 37 degrees Celsius with a thermostatic heating pad. After shaving the fur and sterilizing the surgical site, inject lidocaine, tilidine, and meloxicam.Once the skin over the SC is removed, clean the skull with a cotton swab. Adjust the stereotaxic frame until the skull surface is flat. Using a drill, create a whole 0.5 millimeter lateral, and 0.42 millimeter anterior to the lambda, until the dura is exposed.Inject an adeno-associated virus, or AAV, expressing GCaMP6m, at depths of one millimeter and 1.6 millimeters from the Lambda using a beveled glass micro-pipette. After the injection, slowly withdraw the injector and proceed with the implantation of the head plate.

Inyección de construcción viral (AAV) en el colículo superior de ratón para obtener imágenes de calcio

Punción lumbar de AAV9 en ratas juveniles para terapia génica del SNC

Intrathecal Vector Delivery in Juvenile Rats via Lumbar Cistern Injection

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a toxic gene, or provide a gene whose product can modulate the biology of the disease. The delivery method for the therapeutic vector can take many forms, ranging from intravenous infusion for systemic delivery to direct injection into the target tissue. For neurodegenerative disorders, it is often desirable to skew transduction towards the brain and/or spinal cord. The least invasive approach to target the entire central nervous system involves injection into the cerebrospinal fluid (CSF), allowing the therapeutic to reach a large fraction of the central nervous system. The safest approach to deliver a vector into the CSF is the lumbar intrathecal injection, where a needle is introduced into the lumbar cistern of the spinal cord. This technique, also known as a lumbar puncture, has been widely used in neonatal and adult rodents and in large animal models. While the technique is similar across species and developmental stages, subtle differences in size, structure, and elasticity of tissues surrounding the intrathecal space require accommodations in the approach. This article describes a method for performing lumbar puncture in juvenile rats to deliver an adeno-associated serotype 9 vector. Here, 25-35 &#181;L of vector were injected into the lumbar cistern, and a green fluorescent protein (GFP) reporter was used to evaluate the transduction profile resulting from each injection. The benefits and challenges of this approach are discussed.

Autor destacado: Evaluación de la eficacia de la terapia génica intratecal en ratas juveniles

A surgical procedure is described to perform injections into the lumbar cistern of the juvenile rat. This approach has been used for the intrathecal delivery of gene therapy vectors, but it is anticipated that this approach can be used for a variety of therapeutics, including cells and drugs.

Administración intratecal de vectores en ratas juveniles mediante inyección en cisterna lumbar

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a ...