Name: posterior semicircular canal approach for inner ear gene delivery in neonatal mouse
Uploaded: 2018-03-02T14:00:00.000+00:00
Duration: 3 min 52 s
Description: Watch this Scientific Journal Video about Posterior Semicircular Canal Approach for Inner Ear Gene Delivery in Neonatal Mouse at JoVE.com

Este trabajo fue apoyado por fondos de la NIDCD División de intramuros investigación /NIH (DC000082-02 para W.W.C.), así como DC000081 a base de proyección de imagen avanzada. Estamos agradecidos por el personal de Animalario NIDCD para el cuidado de nuestros animales.

Todos los procedimientos animales fueron aprobados por el cuidado Animal y uso en el Instituto Nacional en sordera y otros trastornos de la comunicación (NIDCD ASP1378-15).
1. preparación y configuración del procedimiento
<ol><li>Esterilizar todos los instrumentos de óxido de etileno en el inicio del experimento. Entre los animales, limpiar instrumentos usando esterilización del grano.</li>
	<li>Cargar la solución con terapia génica viral en una micropipeta en el inyector de micro. Los vectores virales utilizados en este estudio fue AAV2/8-whirlin (1 x 1013 copias de genoma por mililitro, consulte la tabla de materiales). Nota: Típicamente, volumen total de 1,1 μl se carga en la micropipeta.</li>
</ol>2. anestesia
Nota: La cepa de ratón utilizada en este estudio es el ratón whirler. Mutantes homocigóticos (Whrnwi/wi) y hermanos de camada heterozigóticos (Whrn+ wi) fueron utilizados.
<ol><li>Lugar de la madre en una jaula separada (separada de la basura).</li>
	<li>Coloque la jaula casera que contiene basura (P0 - P5 PUP) sobre una almohadilla de calor recirculación (set a 37,5 ° C) para mantener calientes los ratones.</li>
	<li>Cortar la parte del pulgar de un guante de látex y colocar un cachorro en el mismo.</li>
	<li>Ponga el cachorro en pulgar guante de látex en un cubo de hielo durante 2 minutos.</li>
	<li>Coloque el cachorro anestesiado en un paquete de congelación plástico plaza comercial grande con una gasa de 4 "x 4" entre el cachorro y la superficie del paquete.</li>
	<li>Llenar un guante de látex resistente con hielo picado y coloque el guante de hielo alrededor de la cría.</li>
	<li>Verifique si el cachorro está adecuadamente anestesiado por la completa falta de respuesta a diversos estímulos (incluyendo una pizca del dedo del pie firme). Dejar el cachorro en el paquete de hielo para la duración de la cirugía (aproximadamente 5-10 min). Nota: Se recomienda dejar los cachorros en el paquete de hielo para no más de 15 minutos durante la cirugía.</li>
</ol>3. vía de abordaje (figura 1)
<ol><li>Limpiar la piel detrás del oído con un paño de yodo y un paño con alcohol una vez que el animal está anestesiado.</li>
	<li>Haga una incisión postauricular ~ 2 mm detrás de la oreja usando micro tijeras y dividir el músculo esternocleidomastoideo con micro tijeras.</li>
	<li>Identificar el nervio facial y la bulla. La bulla es cartilaginosa y semitransparente a esta edad y se encuentra medial al nervio facial. La arteria stapedial puede verse a través de la bulla a esta edad, que es un punto de referencia útil.</li>
	<li>Seguir el nervio facial superior y posteriorly para localizar el canal semicircular posterior (PSCC). Quitar las fibras musculares y tejido blando que cubre el canal semicircular posterior con micro tijeras. Nota: La PSCC es cartilaginosa a esta edad.</li>
	<li>Penetrar la PSCC utilizando una micropipeta de vidrio (~ 10 μm de diámetro) en el inyector de micro.</li>
	<li>Inyectar la terapia génica viral en el oído interno. Nota: Por lo general, un total de 20 inyecciones de 49 nL de la terapia del gene se entregan en el canal semicircular posterior más de ~ 40 s (μL volumen total ~ 1). El título viral utilizado era 1 x 1013 copias de genoma / mL.</li>
	<li>Cerrar la incisión de piel con una sutura de poligalactina 5-0.</li>
</ol>4. posoperatorio cuidado
<ol><li>Colocar el cachorro sobre una almohadilla calentadora para restablecer la temperatura normal del cuerpo durante la recuperación de la anestesia con el manual constante estimulación/balanceo con guantes dedos humanos.</li>
	<li>Una vez que el cachorro está despierto, colocar en su jaula casera.</li>
	<li>Acariciar cada cachorro con un hisopo de algodón que ha sido expuesto a las camas jaula casera. Nota: El propósito de esto es que los ratones huelen como lo hicieron antes de la cirugía, lo que aumenta la probabilidad de que la madre vuelva a aceptar después de la cirugía de su camada. Si es posible, orina de la madre puede ser recogido y frotado en los cachorros con un hisopo de algodón para disminuir aún más la probabilidad de rechazo.</li>
	<li>Aplicar aceite mineral al nariz de la madre a desensibilizar su8y reintroducir a la madre en la jaula de la casa.</li>
</ol>

<ol>
	<li>Chien, W. W., Monzack, E. L., McDougald, D. S., Cunningham, L. L. <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+sensorineural+hearing+loss.">Gene therapy for sensorineural hearing loss.</a> Ear Hear. 36, 1-7 (2015).</li><li>Askew, 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=Tmc+gene+therapy+restores+auditory+function+in+deaf+mice.">Tmc gene therapy restores auditory function in deaf mice.</a> Sci Transl Med. 7, 295 (2015).</li><li>Akil, 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=Restoration+of+hearing+in+the+VGLUT3+knockout+mouse+using+virally+mediated+gene+therapy.">Restoration of hearing in the VGLUT3 knockout mouse using virally mediated gene therapy.</a> Neuron. 75, 283-293 (2012).</li><li>Pan, B., 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+restores+auditory+and+vestibular+function+in+a+mouse+model+of+Usher+syndrome+type+1c.">Gene therapy restores auditory and vestibular function in a mouse model of Usher syndrome type 1c.</a> Nat Biotechnol. 35, 264-272 (2017).</li><li>Isgrig, 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=Gene+Therapy+Restores+Balance+and+Auditory+Functions+in+a+Mouse+Model+of+Usher+Syndrome.">Gene Therapy Restores Balance and Auditory Functions in a Mouse Model of Usher Syndrome.</a> Mol Ther. 25, 780-791 (2017).</li><li>Lentz, J. J., 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=Rescue+of+hearing+and+vestibular+function+by+antisense+oligonucleotides+in+a+mouse+model+of+human+deafness.">Rescue of hearing and vestibular function by antisense oligonucleotides in a mouse model of human deafness.</a> Nat Med. 19, 345-350 (2013).</li><li>Shibata, S. B., 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=RNA+Interference+Prevents+Autosomal-Dominant+Hearing+Loss.">RNA Interference Prevents Autosomal-Dominant Hearing Loss.</a> Am J Hum Genet. 98, 1101-1113 (2016).</li><li>Van Sluyters, R. C., Obernier, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+care+and+use+of+mammals+in+neuroscience+and+behavioral+research.">Guidelines for the care and use of mammals in neuroscience and behavioral research.</a> Contemp Top Lab Anim. 43, 48 (2004).</li><li>Chien, W. 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+Restores+Hair+Cell+Stereocilia+Morphology+in+Inner+Ears+of+Deaf+Whirler+Mice.">Gene Therapy Restores Hair Cell Stereocilia Morphology in Inner Ears of Deaf Whirler Mice.</a> Mol Ther. 24, 17-25 (2016).</li><li>Hirose, K., Hartsock, J. J., Johnson, S., Santi, P., Salt, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+lipopolysaccharide+compromises+the+blood-labyrinth+barrier+and+increases+entry+of+serum+fluorescein+into+the+perilymph.">Systemic lipopolysaccharide compromises the blood-labyrinth barrier and increases entry of serum fluorescein into the perilymph.</a> J Assoc Res Otolaryngol. 15, 707-719 (2014).</li><li>Okada, H., 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+transfer+targeting+mouse+vestibule+using+adenovirus+and+adeno-associated+virus+vectors.">Gene transfer targeting mouse vestibule using adenovirus and adeno-associated virus vectors.</a> Otol Neurotol. 33, 655-659 (2012).</li><li>Suzuki, J., Hashimoto, K., Xiao, R., Vandenberghe, L. H., Liberman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+gene+therapy+with+ancestral+AAV+in+adult+mice:+complete+transduction+of+inner+hair+cells+without+cochlear+dysfunction.">Cochlear gene therapy with ancestral AAV in adult mice: complete transduction of inner hair cells without cochlear dysfunction.</a> Sci Rep. 7, 45524 (2017).</li></ol>

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Se han descrito varios abordajes quirúrgicos para tener acceso a roedores interior de orejas. Cochleostomy y enfoques de ventanas redondas son más utilizados para acceder a la cóclea, mientras que el canal semicircular posterior y el saco endolinfático enfoques se suelen utilizar para acceder a los órganos vestibulares1. En un estudio reciente, mostramos canal semicircular posterior inyecciones de terapia génica viral dio lugar a la alta eficacia de transducción de la célula de pelo en los órganos vestibulares y la cóclea5. De hecho, la transducción de células ciliadas cocleares fue mayor a través de las inyecciones del canal semicircular posterior, en comparación con las inyecciones de la ventana redonda en el mismo modelo de ratón de hipoacusia hereditaria y disfunción vestibular5,9. Dada la proximidad anatómica entre la ventana redonda y la cóclea, parece paradójico que las inyecciones de la ventana redonda pueden conducir a la transducción de células ciliadas cocleares inferior en comparación con las inyecciones del canal semicircular posterior. Este hallazgo puede explicarse por el hecho de que la ventana redonda está situada cerca del acueducto coclear. Por lo tanto, cuando la terapia génica viral se inyecta a través de la ventana redonda, su concentración puede ser diluida por el líquido cefalorraquídeo desde el acueducto coclear10.
El hallazgo de que los resultados del canal semicircular posterior inyección en la transducción de células ciliadas cocleares y vestibulares también se han descrito otros estudios11,12. En el estudio de Okada et al., informó de transduction de las células de pelo cocleares y vestibulares por AAV-GFP. Sin embargo, no se realizó la cuantificación numérica. El estudio realizado por Suzuki et al., reportó altos niveles de transducción de células ciliadas cocleares y vestibulares con el AAV-Anc80-GFP utilizando el enfoque del canal semicircular posterior en ratones adultos. En ratones adultos, canulación del canal semicircular posterior con un pequeño catéter es preferible, ya que la cubierta huesuda del canal semicircular posterior es totalmente osificado12. Cateterización del conducto semicircular posterior en ratones adultos puede ayudar a minimizar el reflujo de la inyección, que puede disminuir la eficacia de transducción de señales. Este paso no es necesario en ratones neonatales, ya que la cubierta huesuda del canal semicircular posterior es aún cartilaginosa a esa edad.
Uno de los inconvenientes de la inyección del canal semicircular posterior es el hecho de que uno no puede ser cierto si la terapia del gene inyectado se entrega en la perilinfa o endolinfa. A pesar de esta carencia, el canal semicircular posterior es anatómicamente fácil de localizar y requiere mínima manipulación del hueso temporal para su identificación. Esto disminuye la probabilidad de daño del oído interno causada por trauma quirúrgico. La inyección del canal semicircular posterior es una opción atractiva para la entrega de terapia de gen de oído interno.

Oído interno la terapia génica es un campo de rápido desarrollo de la investigación. Se ha aplicado en varios modelos de animales para combate ototoxicidad, traumatismo de ruido y de pérdida de audición hereditaria1. Varios estudios recientes han demostrado la recuperación funcional de la audición y las funciones de balance en ratones mutantes después de oído interno gene terapia entrega2,3,4,5,6, 7. uno de los principales factores en determinar el éxito de la terapia génica de oído interno es el método quirúrgico utilizado para acceder el oído interno. Idealmente, el acercamiento quirúrgico sería fácil de realizar, los hitos anatómicos sería consistente y fácil de identificar y la transducción resultante de tipos celulares específicos sería alta.
En un estudio reciente, demostró que cuando la terapia génica viral fue inyectada a través del canal semicircular posterior del ratón mutante whirler (un modelo de pérdida de la audición y la disfunción vestibular), transducción eficiente de células de pelo sensoriales fue visto en el vestibular órganos, así como en la cóclea5. La alta eficiencia de transducción de la célula de pelo sensorial dio lugar a la mejora de las funciones auditivas y vestibulares en estos ratones mutantes.
En este artículo, describimos en detalle el enfoque del canal semicircular posterior para tener acceso al oído interno de ratón neonatal.

<table><tbody><tr><td>Operating microscope</td><td>Zeiss</td><td></td><td>OPMI Pico ENT microscope. Other dissection microscopes would also work.</td></tr><tr><td>Micro-forcepts</td><td>Fine Science Tools</td><td>11251-10, 11295-51</td><td>#5 and #55 Dumont</td></tr><tr><td>Micro-scissors</td><td>Fine Science Tools</td><td>15002-08</td><td></td></tr><tr><td>Nanoliter2000 microinjector</td><td>World Precision Instruments</td><td></td><td></td></tr><tr><td>Heating pad</td><td>Mastex</td><td>Model 500/600</td><td></td></tr><tr><td>5-0 vicryl sutures</td><td>Ethicon</td><td></td><td></td></tr><tr><td>AAV8-whirlin</td><td>Vector Biolabs</td><td></td><td></td></tr><tr><td>Glass pipette</td><td>Sutter Instruments</td><td>B100-75-10</td><td>Borosilicate glass</td></tr></tbody></table>

posterior semicircular canal approach for inner ear gene delivery in neonatal mouse

Oído interno la terapia génica ofrece gran promesa como tratamiento potencial para la pérdida de oído y mareos. Uno de los determinantes críticos del éxito de la terapia génica de oído interno debe encontrar un método de entrega que se traduce en eficiencia de transducción consistente de tipos celulares específicos y reducir al mínimo pérdida de la audición. En este estudio, describimos el enfoque del canal semicircular posterior como un método viable para la entrega del gene de oído interno en ratones neonatales. Mostramos que entrega del gene a través del canal semicircular posterior es capaz de inundar todo oído interno. La fácil identificación anatómica del canal semicircular posterior, así como mínima manipulación del hueso temporal necesario, hacer este quirúrgico acercarse una opción atractiva para la entrega del gene de oído interno.

Inyección de whirlin AAV8 terapia génica en ratones neonatales whirler a través del canal semicircular posterior dio lugar a whirlin expresión (verde) en células de pelo utricular (figura 2), con la eficiencia global de la infección del 53,1% (SD 38.1, n = 28)5 . Células transduced había alargado estereocilios (rojo) en comparación con las células de pelo de orejas no contralaterales (5.35 μm ± 2.11 vs 3,20 ± 0.34 μm, respectivamente)5.
Canal semicircular posterior inyección de whirlin AAV8 también resultó en la transducción de células de pelo cocleares en los ratones whirler (figura 3). La eficiencia de infección de la célula de pelo interna promedio fue de 77.1% (SD 12,7, n = 8)5. Células transduced expresó whirlin (verde) en las puntas de los estereocilios y había alargado estereocilios (rojo) en comparación con las células de pelo de orejas no contralaterales (5,04 μm ± 0,72 vs 1.01 ± 0.08 μm en el ápex coclear, respectivamente)5.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56648/56648fig1.jpg"> Figura 1 : Imágenes intraoperatorias. Imágenes intraoperatorias mostrando acceso quirúrgico al canal semicircular posterior (PSCC) en un ratón de P0. El oído izquierdo se muestra. La PSCC está delineado en las líneas punteadas de negro. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56648/56648fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56648/56648fig2.jpg"> Figura 2 : Células de pelo vestibulares son transduced mediante entrega del gene PSCC. Whirlin AAV8 entregados vía la PSCC acercamiento dio lugar a altos niveles de infección de la célula de pelo utricular. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56648/56648fig2large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56648/56648fig3.jpg"> Figura 3 : Células de pelo cocleares son transduced mediante entrega del gene PSCC. Whirlin AAV8 entregados vía la PSCC acercamiento dio lugar a altos niveles de infección de células ciliadas cocleares. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56648/56648fig3large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>

Watch this Scientific Journal Video about Posterior Semicircular Canal Approach for Inner Ear Gene Delivery in Neonatal Mouse at JoVE.com

Posterior Semicircular Canal Approach for Inner Ear Gene Delivery in Neonatal Mouse

En este estudio, describimos el enfoque del canal semicircular posterior como un método confiable para la entrega del gene de oído interno en ratones neonatales. Mostramos que entrega del gene a través del canal semicircular posterior es capaz de inundar todo oído interno.

Enfoque del Canal Semicircular posterior para la entrega del Gene de oído interno en ratón Neonatal

Inner ear gene therapy offers great promise as a potential treatment for hearing loss and dizziness. One of the critical determinants of the success of ...

posterior-semicircular-canal-approach-for-inner-ear-gene-delivery

Nanyang Technological University

Research

JoVE Journal

Medicine

9.7K vistas.  National Institutes of Health. En este estudio, describimos el enfoque del canal semicircular posterior como un método confiable para la entrega del gene de oído interno en ratones neonatales. Mostramos que entrega del gene a través del canal semicircular posterior es capaz de inundar todo oído interno.

Video: Posterior Semicircular Canal Approach for Inner Ear Gene Delivery in Neonatal Mouse

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Cultura de embriones de ratón explantes cocleares y Gene Transfer por electroporación

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Investigación

Biología del Desarrollo

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) has been shown to be a useful approach to local drug delivery into the inner ear. The goal of this article is to describe, in detail, the surgical techniques involved in canalostomy in both adult and neonatal mice. As indicated by fast-green dye and adeno-associated virus serotype 8 with the green fluorescent protein gene, the canalostomy facilitated broad distribution of injected reagents in the cochlea and vestibular end-organs with minimal damage to hearing and vestibular function. The surgery was successfully implemented in both adult and neonatal mice; indeed, multiple surgeries could be performed if required. In conclusion, canalostomy is an effective and safe approach to drug delivery into the inner ears of adult and neonatal mice and may be used to treat human inner ear diseases in the future.

Here we describe canalostomy procedure which allows local drug delivery into the inner ears of adult and neonatal mice through the semicircular canal with minimum damage to hearing and vestibular function. This method can be used to inoculate viral vectors, pharmaceuticals, and small molecules into the mouse inner ear.

Canalostomy como un abordaje quirúrgico a la entrega del Local de la droga en los oídos interiores de ratones adultos y Neonatal

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

Genética

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Un nuevo enfoque quirúrgico para la administración intratraqueal de agentes bioactivos en un modelo de ratón fetal

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Medicina

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

Transferencia de genes a la oreja del ratón en desarrollo por el interior<em&gt; En Vivo</em&gt; La electroporación

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

Neurociencias

Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and genetic mechanisms that contribute to hearing loss. Introduction of vectors into the inner ear must be done in a way that widely distributes the agent throughout the cochlea while minimizing injury to the existing structures. This manuscript describes a post-auricular surgical approach that can be used for mouse cochlear therapy using molecular, pharmacologic, and viral delivery to mice postnatal day 10 and older via the round window membrane (RWM). This surgical approach enables rapid and direct delivery into the scala tympani while minimizing blood loss and avoiding animal mortality. This technique involves negligible or no damage to essential structures of the inner and middle ear as well as neck muscles while wholly preserving hearing. To demonstrate the efficacy of this surgical technique, the vesicular glutamate transporter 3 knockout (VGLUT3 KO) mice will be used as an example of a mouse model of congenital deafness that recovers hearing after delivery of VGLUT3 to the inner ear using an adeno-associated virus (AAV-1).

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

Método quirúrgico para la entrega de genes mediada por virus de la oreja del ratón Interior a través de la ventana de membrana Ronda

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...

Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. Few studies have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem, which contains second order neurons of the auditory pathway, is a potential site for gene transfer. In this protocol, a technique for direct and maximal exposure of the murine DCN via a posterior fossa approach is demonstrated. This approach allows for either acute or survival surgery. Following direct visualization of the DCN, a host of experiments are possible, including injection of opsins into the cochlear nucleus and subsequent stimulation by an optical fiber coupled to a blue light laser.&#160;Other neurophysiology experiments, such as electrical stimulation and neural injector tracings are also feasible.&#160;The level of visualization and the duration of stimulation achievable make this approach applicable to a wide range of experiments.

The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.

La visualización directa de la murino Dorsal coclear Núcleo de optogenética La estimulación de la vía auditiva

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

Técnica de Explante Coclear Murino Neonatal como<em&gt; In Vitro</em&gt; Herramienta de detección en la investigación de la audición

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset (Callithrix jacchus)

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset (Callithrix jacchus) is a small, easy-to-handle New World monkey which has a similar anatomy of the temporal bone, including the middle ear ossicular chains and inner ear to humans, than in comparison with that of rodents. Here, we report a reproducible, safe, and rational surgical approach to the cochlear round window niche for the drug delivery to the inner ear of the common marmoset. We adopted posterior tympanotomy, a procedure used clinically in human surgery, to avoid manipulation of the tympanic membrane that may cause conductive hearing loss. This surgical procedure did not lead to any significant hearing loss. This approach was possible due to the large bulla structure of the common marmoset, although the lateral semicircular canal and vertical portion of the facial nerve should be carefully considered. This surgical method allows us to perform the safe and accurate administration of drugs without hearing loss, which is of great importance in obtaining pre-clinical proof of concept for translational research.

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset Callithrix jacchus

We report a surgical method to administrate drugs to the inner ear of a non-human primate, the common marmoset (Callithrix jacchus), via the round window membrane.

Un procedimiento quirúrgico para la administración de medicamentos para el oído interno en una no humanos primates común tití (Callithrix jacchus)

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset ...

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. The first procedure consists of perforation of the tympanic bulla, termed bullostomy; the second one is a transtympanic injection. Both emulate human clinical intratympanic procedures.
Chitosan-glycerophosphate (CGP) and Ringer&#180;s Lactate buffer (RL) were used as biocompatible vehicles for local drug delivery. CGP is a nontoxic biodegradable polymer widely used in pharmaceutical applications. It is a viscous liquid at RT&#160;but it congeals to a semi solid phase at body temperature. RL is an isotonic solution used for intravenous administrations in humans. A small volume of this vehicle is precisely placed on the Round Window (RW) niche by means of a bullostomy. A transtympanic injection fills the middle ear and allows less control but broader access to the inner ear.
The safety profiles of both techniques were studied and compared by using functional and morphological tests. Hearing was evaluated by registering the Auditory Brainstem Response (ABR) before and several times after microsurgery. The cytoarchitecture and preservation level of cochlear structures were studied by conventional histological techniques in paraformaldehyde-fixed and decalcified cochlear samples. In parallel, unfixed cochlear samples were taken and immediately frozen to analyze gene expression profiles of inflammatory markers by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR).
Both procedures are suitable as drug delivery methods into the mouse middle ear, although transtympanic injection proved to be less invasive compared to bullostomy.

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

Two microsurgery approaches for local drug delivery to the inner ear are described here and compared in terms of impact on hearing parameters, cochlear cytoarchitecture and expression of inflammatory markers.

Estudio comparativo de los métodos de entrega de fármacos dirigidos a la oreja del ratón interno: Bullostomy<em&gt; vs.</em&gt; Inyección transtimpánicos

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. ...

Biología

Canalostomy for Local Drug Delivery into the Inner Ear of an Adult Mouse

Source: Guo, J. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57351">Canalostomy as a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice.</a>&#160;J. Vis. Exp. (2018)
This video showcases canalostomy, a procedure that allows local drug delivery into the inner ear of a mouse through the semicircular canal with minimal damage to hearing and vestibular function. This method enables the inoculation of viral vectors, pharmaceuticals, and small molecules into the mouse&#39;s inner ear.

Canalostomía para la administración local de fármacos en el oído interno de un ratón adulto

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Device ...

Enfoque del Canal Semicircular posterior para la entrega del Gene de oído interno en ratón Neonatal

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