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

Diese Arbeit wurde mit Mitteln aus der NIDCD Abteilung der intramuralen Forschung /NIH (DC000082-02 bis W.W.C.) sowie DC000081, erweiterte Bildgebung Kern unterstützt. Wir sind dankbar für die NIDCD Tierhaus Mitarbeiter für die Pflege unserer Tiere.

Alle tierische Verfahren stimmten die Animal Care und Use Committee am nationalen Institut auf Taubheit und anderen Kommunikationsstörungen (NIDCD ASP1378-15).
1. Verfahren einrichten und Vorbereitung
<ol><li>Sterilisieren Sie alle Instrumente von Ethylenoxid in den Beginn des Experiments. Zwischen Tieren reinigen Sie mit Wulst Sterilisation Instrumente.</li>
	<li>Laden Sie die Projektmappe mit viralen Gentherapie in einer Mikropipette auf dem Mikro-Injektor. Die viralen Vektoren, die in dieser Studie verwendet wurde AAV2/8-whirlin (1 x 1013 Genom Kopien pro Milliliter, siehe die Tabelle der Materialien). Hinweis: In der Regel ist 1,1 µL Gesamtvolumen in die Mikropipette geladen.</li>
</ol>(2) Anästhesie
Hinweis: In dieser Studie verwendete Maus-Stamm ist der Wirbler Maus. Reinerbige Mutanten (WhrnWLAN/wi) und heterozygote Wurfgeschwistern (Whrn+ / wi) dienten.
<ol><li>Legen Sie die Mutter in einem separaten Käfig (getrennt aus dem Wurf).</li>
	<li>Legen Sie die Heimat Käfig mit dem Wurf (P0 - P5 Welpen) auf einem umlaufenden Wärmekissen (Set bei 37,5 ° C) um die Mäuse warm zu halten.</li>
	<li>Schneiden Sie den Daumen Teil eines Latex-Handschuh, und entgegenbringen Sie einen Welpen.</li>
	<li>Platzieren Sie den Welpen in Latex-Handschuh Daumen in einen Eimer mit Eis für ~ 2 min.</li>
	<li>Platzieren Sie den narkotisierten Hund auf einem großen quadratischen kommerziellen Kunststoff Einfrieren-Pack mit einem 4 "x 4" Gaze zwischen den Welpen und das Pack Oberfläche.</li>
	<li>Einen schweren Latex-Handschuh mit crushed Ice füllen und den Eis-Handschuh rund um die Welpen zu platzieren.</li>
	<li>Überprüfen Sie, ob der Welpe ausreichend durch das völlige Fehlen von jeder Reaktion auf verschiedene Reize (einschließlich einer festen Fuß Klemme) betäubt ist. Lassen Sie den Welpen auf das Packeis für die Dauer der Operation (ca. 5-10 min). Hinweis: Wir empfehlen die Welpen auf das Packeis für nicht mehr als 15 min während der Operation.</li>
</ol>3. chirurgische Vorgehen (Abbildung 1)
<ol><li>Reinigen Sie die Haut hinter dem Ohr mit einer Jod-wischen und ein Alkohol abwischen, sobald das Tier betäubt ist.</li>
	<li>Machen Sie einen postauricular Schnitt ~ 2 mm hinter dem Ohr mit Mikro-Schere, und teilen Sie den sternocleidomastoideus-Muskel mit Mikro-Schere.</li>
	<li>Nervus facialis und die Bulla zu identifizieren. Die Bulla ist knorpelig und semi-transparent in diesem Alter und es liegt medial Nervus facialis. Die stapedial Arterie durch die Bulla in diesem Alter sehen, die ein nützliches Wahrzeichen ist.</li>
	<li>Nervus facialis zu folgen, souverän und nach hinten, die hinteren Semicircular Kanal (PSCC) zu finden. Entfernen Sie die Muskelfasern und Weichgewebe über den hinteren Semicircular Kanal mit Mikro-Schere. Hinweis: In diesem Alter ist die PSCC knorpelig.</li>
	<li>Dringen Sie die PSCC mit einem Glas Mikropipette (~ 10 μm Durchmesser) auf der Mikro-Injektor.</li>
	<li>Injizieren Sie virale Gentherapie in das Innenohr. Hinweis: In der Regel insgesamt 20 Injektionen von 49 nL der Gentherapie werden geliefert in den hinteren Semicircular Kanal über ~ 40 s (Gesamtvolumen ~ 1 µL). Die virale Titer verwendet wurde 1 x 10-13 -Genom-Kopien pro mL.</li>
	<li>Schließen Sie der Hautschnitt mit einem 5: 0 Polyglactin Naht.</li>
</ol>4. postoperative Pflege
<ol><li>Ort der Welpe auf eine wärmende Unterlage, um eine normale Körpertemperatur während der Wiederherstellung aus der Narkose mit konstanter manuelle Stimulation/Rollen mit behandschuhten Fingern menschlichen wiederherzustellen.</li>
	<li>Sobald der Welpe wach ist, legen Sie sie zurück in seine Heimat Käfig.</li>
	<li>Streicheln Sie jeder Welpe mit einem Wattestäbchen, das die Heimat Käfig Einstreu ausgesetzt worden ist. Hinweis: Der Zweck davon ist, haben die Mäuse riechen wie sie es vor der Operation, das erhöht die Wahrscheinlichkeit, dass die Mutter ihr Wurf nach der Operation wieder zu akzeptieren. Wenn möglich, kann Urin von der Mutter gesammelt und rieb auf die Welpen mit einem Wattestäbchen, um die Wahrscheinlichkeit einer Ablehnung weiter zu verringern.</li>
	<li>Nase, die Mutter um ihre8desensibilisieren Mineralöl zuweisen und Wiedereinführung der Muttergottes in den Käfig zu Hause.</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>

Die Autoren haben keine entsprechenden Angaben zu machen.

Mehrere chirurgische Ansätze sind beschrieben worden, um Nager inneren Ohren zu gelangen. Cochleostomie und Rundfenster Ansätze werden am häufigsten verwendet Zugriff die Cochlea, während die hinteren Semicircular Canal und endolymphatic Sac Ansätze in der Regel die vestibulären Organe1Zugriff auf verwendet werden. In einer aktuellen Studie haben wir gezeigt, dass posterior Semicircular Canal, die hohe Effizienz der Haarzelle Transduktion in der vestibulären Organe und die Schnecke<sup clas...

Innenohr Gentherapie ist ein sich rasant entwickelnden Gebiet der Untersuchung. Es wurde in verschiedenen Ausführungen der Tiere zu bekämpfen Ototoxizität, Lärm Trauma und erbliche Gehör Verlust1angewendet. Mehrere neuere Studien belegen funktionelle Erholung des Hörens und Gleichgewicht Funktionen mutierte Mäuse nach Innenohr Gen Therapie Lieferung2,3,4,5,6, 7. einer der wichtigsten Faktoren bei der Bestimmung der Erfolg des Innenohrs Gentherapie ist das chirurgische Vorgehen verwendet, um Zugriff auf das Innenohr. Im Idealfall das chirurgische Vorgehen wäre einfach durchzuführen, die anatomischen Landmarken wäre konsistent und leicht zu identifizieren, und die daraus resultierende Transduktion von gezielten Zelltypen wären hoch.
In einer aktuellen Studie haben wir gezeigt, dass bei viralen Gentherapie durch den hinteren Semicircular Kanal der Wirbler mutierte Maus (ein Modell der Hörverlust und vestibulären Dysfunktion) injiziert wurde, effizienten Transduktion von sensorischen Haarzellen in der vestibulären gesehen wurde auch Organe wie die Cochlea-5. Die hohe Effizienz der sensorischen Haarzellen Transduktion führte zu Verbesserung der auditorischen und vestibulären Funktionen in diesen mutierten Mäusen.
In diesem Artikel beschreiben wir ausführlich den hinteren Semicircular Canal Ansatz um die Neugeborenen Maus Innenohr gelangen.

<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

Innenohr Gentherapie bietet große Versprechung als potenzielle Behandlung von Schwerhörigkeit und Schwindel. Der wichtige Determinanten für den Erfolg des Innenohrs Gentherapie ist eine Lieferung Methode zu finden, die konsequente Transduktion Effizienz der gezielten Zelltypen führt bei gleichzeitiger Minimierung der Hörverlust. In dieser Studie beschreiben wir den hinteren Semicircular Kanal-Ansatz als eine praktikable Methode für Innenohr-gen Lieferung bei neugeborenen Mäusen. Wir zeigen, dass gen-Lieferung durch den hinteren Semicircular Kanal in der Lage, das gesamte Innenohr durchspülen. Die leicht anatomische Identifikation des hinteren Semicircular Kanals sowie minimale Manipulation des Schläfenbeins erforderlich, machen diesem chirurgischen Ansatz eine attraktive Option für Innenohr-gen Lieferung.

Injektion von AAV8 whirlin Gentherapie in Neugeborenen Wirbler Mäuse durch den hinteren Semicircular Kanal führte zu whirlin Ausdruck (grün) in utricular Haarzellen (Abbildung 2), mit der Infektion Gesamtwirkungsgrad von 53,1 % (SD 38,1, n = 28)5 . Haarzellen ausgestrahlt hatte Stereocilia (rot) im Vergleich zu Haarzellen von kontralateralen nicht injiziert Ohren länglich (5,35 ± 2.11 µm vs. 3,20 ± 0,34 µm bzw.)<sup class="xref...

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

In dieser Studie beschreiben wir den hinteren Semicircular Kanal-Ansatz als eine zuverlässige Methode für Innenohr-gen Lieferung bei neugeborenen Mäusen. Wir zeigen, dass gen-Lieferung durch den hinteren Semicircular Kanal in der Lage, das gesamte Innenohr durchspülen.

Posterior Semicircular Canal Ansatz für Innenohr-gen Lieferung in Neugeborenen Maus

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

Research

JoVE Journal

Medicine

9.7K Aufrufe.  National Institutes of Health. In dieser Studie beschreiben wir den hinteren Semicircular Kanal-Ansatz als eine zuverlässige Methode für Innenohr-gen Lieferung bei neugeborenen Mäusen. Wir zeigen, dass gen-Lieferung durch den hinteren Semicircular Kanal in der Lage, das gesamte Innenohr durchspülen.

Serie: 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.

Kultur von embryonalen Maus Cochlear Explantate und Gentransfer durch Elektroporation

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

Forschung

Entwicklungsbiologie

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 als ein chirurgisches Vorgehen zu Local Drug Delivery in das innere Ohr von Erwachsenen und Neugeborenen Mäusen

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

Genetik

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.

A Novel Surgical Ansatz intratracheale Verabreichung von Wirkstoffen in einem 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 ...

Medizin

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.

Gentransfer an sich entwickelnden Maus Innenohr<em&gt; In Vivo</em&gt; Die Elektroporation

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 ...

Neurowissenschaften

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.

Chirurgische Verfahren zum viral vermittelte Gentransfer in die Maus-Innenohr über die runde Fenstermembran

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.

Direkte Visualisierung der Murine Rücken Cochlear Nucleus für optogenetische Stimulation der Hörbahn

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.

Neonatal Murine Cochlear Explant Technik als<em&gt; In vitro</em&gt; Screening-Tool in der Hörforschung

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.

Ein chirurgischer Eingriff für die Verwaltung der Medikamente an das Innenohr in eine nicht-menschliche Primaten Weißbüschelaffe (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.

Eine vergleichende Studie von Drug-Delivery-Methoden Gezielte an die Maus Inner Ear: Bullostomy<em&gt; Versus</em&gt; Transtympanale 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. ...

Biologie

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

Source: Guo, J. et al., <a href="https://app.jove.com/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.

Kanalostomie zur lokalen Verabreichung von Medikamenten in das Innenohr einer erwachsenen Maus

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

Posterior Semicircular Canal Ansatz für Innenohr-gen Lieferung in Neugeborenen Maus

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