Bu eser NIDCD bölümü, Intramural araştırma /NIH (DC000082-02 W.W.C.) yanı sıra DC000081 fonlarından için gelişmiş görüntüleme çekirdek tarafından desteklenmiştir. Bizim hayvanlar için bakım için NIDCD hayvan tesis personel için sana şükrediyoruz.

Tüm hayvan yordamları hayvan bakım ve kullanım Komitesi Ulusal Enstitüsü sağırlık ve diğer iletişim bozuklukları (NIDCD ASP1378-15) tarafından kabul edildi.
1. yordam kurulum ve hazırlık
<ol><li>Etilen oksit denemenin başında tarafından tüm aletleri sterilize. Hayvanlar arasında boncuk sterilizasyon kullanarak aletleri temiz.</li>
	<li>Bir micropipette üzerinde mikro-enjektör içine viral gen terapisi içeren çözüm yükleyin. Bu çalışmada kullanılan viral vektör AAV2/8-whirlin yapıldı (1 x 1013 genom kopya sayısı mililitre, malzemelerin tabloya bakın). Not: Genellikle, 1.1 µL Toplam hacim micropipette yüklenir.</li>
</ol>2. anestezi
Not: Bu çalışmada kullanılan fare zorlanma whirler faredir. Homozigoz mutantlar (okulwi/kablosuz) ve heterozigoz littermates (okul+/ wi) kullanılmıştır.
<ol><li>Anne ayrı kafese (çöp ayrıdır) yerleştirin.</li>
	<li>Fareler sıcak tutmak için bir dolaşım ısı yastık (set 37,5 ° c) üzerinde çöp (P0 - P5 pups) içeren ev kafes yerleştirin.</li>
	<li>Lateks eldiven başparmak kısmı keser ve bir yavru içine yerleştirir.</li>
	<li>Yavru bir kova dolusu buz ~ 2 min için lateks eldiven başparmak yerleştirin.</li>
	<li>İmzalat yavru bir 4 "x 4" gazlı bez pup ve pack'ın yüzeyi arasında büyük kare ticari plastik donma paketiyle yerleştirin.</li>
	<li>Ağır bir lateks eldiven ezilmiş buz ile doldurun ve buz eldiveni yavru fok çevresinde yer.</li>
	<li>Yavru fok (bir firma ayak çimdik dahil) çeşitli uyaranlara karşı bir yanıt tam eksikliği ile yeterli anestezi Eğer görmek için kontrol edin. Yavru fok buz torbası (yaklaşık 5-10 dk) ameliyat süresince bırakın. Not: Belgili tanımlık pups ameliyat sırasında en fazla 15 dakika buz torbası açık bırakmak öneririz.</li>
</ol>3. cerrahi yaklaşım (şekil 1)
<ol><li>Hayvan anestezi bir kez deri bir iyot temizleme işlemi ve bir alkol silme ile kulak arkasında temiz.</li>
	<li>Postauricular kesik ~ 2 mm mikro-makas kullanarak kulağının arkasında olun ve mikro-makas ile boyun kas bölün.</li>
	<li>Yüz sinir ve bulla tanımlar. Bulla kıkırdak ve yarı saydam bu yaşta ve bu yüz sinir medial yatıyor. Stapedial arter bulla ile yararlı bir dönüm noktası olduğunu bu yaşta görülebilir.</li>
	<li>Yüz sinir konumlanmıştır ve özafagusu izleyin posterior semisirküler kanal (bundan) bulmak için. Kas lifleri ve yumuşak doku posterior semisirküler kanal mikro-makas kullanarak örten kaldırın. Not: Bundan bu yaşta kıkırdak.</li>
	<li>Bir cam micropipette (~ 10 mikron çapında) mikro-enjektörü kullanarak bundan nüfuz.</li>
	<li>Viral gen terapisi iç kulak enjekte. Not: Genellikle, Toplam 49 20 enjeksiyonlari nL gen tedavisi teslim edilir posterior semisirküler kanal 40 ~ s (Toplam hacim ~ 1 µL). Kullanılan viral titresi mL başına 1 x 1013 genom kopya oldu.</li>
	<li>5-0 polyglactin dikiş kullanarak cilt kesi kapatın.</li>
</ol>4. ameliyat sonrası bakım
<ol><li>Yavru fok anestezi sürekli manuel stimülasyon/haddeleme ile eldivenli insan parmak ile kurtarma sırasında normal vücut ısısı geri yüklemek için Isınma bir yastık yerleştirin.</li>
	<li>Yavru fok uyanık olduğunda, geri onun ev kafesin içine yerleştirin.</li>
	<li>Her yavru için eve kafes yatak maruz bir pamuk bez ile okşadı. Not: Bunun amacı onu çöp ameliyat sonrası yeniden kabul anne olasılığını artırır ameliyat öncesi yaptıkları gibi kokuyorsun fareler sahip olmaktır. Mümkünse, idrar anneden toplanan ve daha fazla reddedilme olasılığını azaltmak için bir pamuklu çubukla kullanarak pups ovuşturdu.</li>
	<li>Onun8duyarsızlaştırmak için annenin Burun madeni yağ uygulayın ve ev kafesin içine annesinin reintroduce.</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>

Yazarlar yapmak için hiçbir ilgili açıklamalar var.

Birkaç cerrahi yaklaşım kemirgen iç kulak erişmek için tarif edilmistir. Posterior semisirküler kanal ve endolymphatic kesesi yaklaşımlar genellikle Vestibüler organları1erişmek için kullanılır, ancak Cochleostomy ve yuvarlak pencere yaklaşımlar en sık koklea, erişmek için kullanılır. Bir son çalışmada, o posterior semisirküler kanal enjeksiyonları viral gen tedavisi saç hücre iletim Vestibüler organ veya koklea5yüksek verimliliği sonuçland?...

İç kulak gen terapisi araştırma çalışmaları hızla gelişmekte olan bir alandır. Bu savaş ototoksisite, gürültü travma ve kalıtsal işitme kaybı1çeşitli hayvan modellerinde uygulanmıştır. Birkaç son çalışmalar mutant farelerde iç kulak gen terapisi teslim2,3,4,5,6sonra, işitme ve denge işlevlerini fonksiyonel iyileşme göstermiştir 7. bir anahtar faktörler iç kulak gen terapisi başarı iç kulak erişmek için kullanılan cerrahi yaklaşım olduğunu belirleme. İdeal olarak, cerrahi yaklaşım gerçekleştirmek kolay olurdu, anatomik işaretlerini tutarlı ve kolay tanımlamak olurdu ve hedeflenen hücre tiplerinin elde edilen iletim yüksek olacaktır.
Bir son çalışmada, viral gen terapisi whirler mutant fare (işitme kaybı ve Vestibüler disfonksiyon bir model) posterior semisirküler kanalından enjekte edildiğinde duyusal saç hücrelerinin verimli iletim Vestibüler içinde görülmüş gösterdi organları de olduğu gibi koklea5. Duyusal saç hücre iletim yüksek verimlilik iyileştirme bu mutant farelerde işitsel ve Vestibüler fonksiyonların sonuçlandı.
Bu makalede, Yenidoğan fare iç kulak erişmek için posterior semisirküler kanal yaklaşım biz ayrıntılı olarak tarif.

<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

İç kulak gen tedavisi büyük söz işitme kaybı ve baş dönmesi için potansiyel bir tedavi olarak sunmaktadır. İç kulak gen terapisi başarısı önemli belirleyicileri işitme kaybı en aza indirerek tutarlı iletim verimliliği hedef hücre tiplerinin sonuçları bir teslimat yöntemi bulmak için biridir. Bu çalışmada, iç kulak gen teslim yenidoğan farelerde için uygun bir yöntem olarak posterior semisirküler kanal yaklaşım tarif. Gen teslim posterior semisirküler kanal aracılığıyla tüm iç kulakta sıvı mümkün olduğunu göstereceğiz. Posterior semisirküler kanal kolay anatomik tanımlanması, hem de en az gerekli temporal kemik manipülasyon, iç kulak gen teslim için cazip bir seçenek yapmak bu cerrahi yaklaşım.

Enjeksiyon AAV8 whirlin gen tedavisi yenidoğan whirler fareler posterior semisirküler kanal aracılığıyla içine %53.1 genel enfeksiyon verimliliği ile sonuçlandı (yeşil) ifadesinde utricular saç hücreleri (Resim 2), whirlin (SD 38,1, n = 28)5 . Transduced saç hücreleri uzamış kontralateral sigara enjekte kulaklarından saç hücrelerine göre stereocilia (kırmızı) (5.35 ± 2.11 µm 3.20 ± 0.34 µm, vs sırasıyla)<s...

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

Bu çalışmada, iç kulak gen teslim yenidoğan farelerde için güvenilir bir yöntem olarak posterior semisirküler kanal yaklaşım tarif. Gen teslim posterior semisirküler kanal aracılığıyla tüm iç kulakta sıvı mümkün olduğunu göstereceğiz.

İç kulak gen teslim yenidoğan Mouse için posterior semisirküler kanal yaklaşım

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 Görüntüleme.  National Institutes of Health. Bu çalışmada, iç kulak gen teslim yenidoğan farelerde için güvenilir bir yöntem olarak posterior semisirküler kanal yaklaşım tarif. Gen teslim posterior semisirküler kanal aracılığıyla tüm iç kulakta sıvı mümkün olduğunu göstereceğiz.

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.

Elektroporasyon tarafından Embriyonik Fare Koklear Eksplantlarından Kültür ve Gen Transferi

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

Gelişim Biyolojisi

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 yerel ilaç dağıtım yetişkin ve Neonatal farelerin iç kulaklarına bir cerrahi yaklaşım olarak

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.

Bir Fetal Fare Modelinde Biyoaktif Ajan intratrakeal Yönetimi İçin Yeni Bir Cerrahi Yaklaşım

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

Tıp

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.

Tarafından geliştirilmesi Fare İç Kulak için Gen Transferi<em&gt; In Vivo</em&gt; Elektroporasyon

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 ...

Nörobilim

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.

Yuvarlak Pencere Membran aracılığıyla Fare İç Kulak için virally ve Gen Teslimat Cerrahi Yöntemi

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.

İşitsel Yolu ve optogenetic Stimülasyon için Murin Dorsal Koklear Çekirdek Doğrudan Görselleştirme

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.

Yenidoğan Murin Koklear Explant Tekniği<em&gt; In vitro</em&gt; İşitme Araştırmalarında Tarama Aracı

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.

Bir insan olmayan primatların ortak Marmoset (Callithrix jacchus) iç kulak ilaçlara Yönetim için bir cerrahi prosedür

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.

Fare İç Kulak Hedefli İlaç Dağıtım Yöntemleri Karşılaştırmalı İncelemesi: Bullostomy<em&gt; Karşı</em&gt; Transtimpanik Enjeksiyon

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. ...

Biyoloji

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.

Yetişkin Bir Farenin İç Kulağına Lokal İlaç Verilmesi için Kanalostomi

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

İç kulak gen teslim yenidoğan Mouse için posterior semisirküler kanal yaklaşım

Özet

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