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

这项工作得到了来自 NIDCD 司内研究/NIH (DC000082-02 到 W.W.C., 以及 DC000081 先进成像核心) 的资金支持。我们感谢 NIDCD 动物设施的工作人员照顾我们的动物。

所有动物程序均经国家耳聋和其他传播疾病研究所 (NIDCD ASP1378-15) 动物护理和使用委员会批准。
1. 程序设置和准备
<ol><li>实验开始时用环氧乙烷消毒所有仪器。动物之间, 清洁的仪器使用珠杀菌。</li>
	<li>将含有病毒基因治疗的溶液载入微注射器的微中。本研究使用的病毒载体是 AAV2/8-whirlin (每毫升 1 x 1013基因组拷贝, 见材料表)。 注意: 通常, 1.1 µL 总卷加载到微中。</li>
</ol>2. 麻醉
注: 本研究使用的鼠标应变是 whirler 鼠。使用了纯合突变体 (Whrnwi-fi) 和异型窝 (Whrn+/无线)。
<ol><li>把母亲放在一个独立的笼子里 (与垃圾分开)。</li>
	<li>将含有垃圾 (P0 P5 幼崽) 的家庭笼子放在循环热垫上 (设置在37.5 摄氏度) 以保持老鼠的体温。</li>
	<li>切掉一只乳胶手套的拇指部分, 然后在里面放一只小狗。</li>
	<li>将小狗用乳胶手套拇指放入一桶冰中2分钟。</li>
	<li>将麻醉的小狗放在一个大广场商业塑料冷冻包装上, 幼崽和包表面之间有 4 "x 4" 纱布。</li>
	<li>用碎冰填充一只重型乳胶手套, 并将冰手套放在幼崽周围。</li>
	<li>检查小狗是否有充分的麻醉, 完全缺乏对各种刺激的反应 (包括坚定的脚趾捏)。在手术期间把幼崽留在冰袋上 (大约 5-10 分钟)。 注意: 我们建议在手术期间将幼崽放在冰包上不超过15分钟。</li>
</ol>3. 手术方法 (图 1)
<ol><li>当动物被麻醉后, 用碘擦拭和酒精擦拭来清洁耳后的皮肤。</li>
	<li>用微剪刀在耳后2毫米耳后切口, 用微剪刀将胸锁乳突肌分开。</li>
	<li>识别脸部神经和泡。泡在这个年龄是软骨和半透明的, 它位于面神经的内侧。stapedial 动脉可以通过泡在这个年龄看到, 这是一个有用的里程碑。</li>
	<li>遵循面部神经优和向后定位后半圆管 (PSCC)。用微剪刀去除后半规管上的肌纤维和软组织。 注意: PSCC 在这个年龄是软骨的。</li>
	<li>在微喷油器上使用玻璃微 (直径10微米) 穿透 PSCC。</li>
	<li>将病毒基因治疗注入内耳。 注: 通常, 20 注射 49 nL 的基因治疗被交付到后半圆运河超过四十年代 (总容量 ~ 1 µL)。使用的病毒滴度为每毫升 1 x 1013基因组拷贝。</li>
	<li>用 5-0 polyglactin 缝合闭合皮肤切口。</li>
</ol>4. 术后护理
<ol><li>将小狗放在暖垫上, 以恢复从麻醉中恢复正常体温, 经常手动刺激/滚动与戴手套的人的手指。</li>
	<li>一旦小狗醒了, 把它放回它的笼子里。</li>
	<li>爱抚每一个小狗, 棉花拭子已经暴露在家庭笼床上用品。 注意: 这样做的目的是让老鼠的气味, 因为他们在手术前, 这增加了母亲再次接受她的垃圾后手术的可能性。如果可能的话, 母亲的尿液可以用棉签来收集和揉在幼崽身上, 以进一步降低拒绝的可能性。</li>
	<li>将矿物油应用到母亲的鼻子上, 脱敏她的 8, 并将母亲重新引入家庭笼子.</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>

作者没有相关的披露。

几种手术方法被描述为进入啮齿动物内耳。Cochleostomy 和圆形窗口的方法是最常用的进入耳蜗, 而后半圆管和淋巴囊方法通常用于访问前庭器官1。在最近的一项研究中, 我们发现, 后半规管注射的病毒基因治疗导致高效率的头发细胞转导的前庭器官和耳蜗5。事实上, 耳蜗毛细胞转导是通过后半圆管注射更高, 当与圆形窗口注射相比, 同一鼠遗传性听力损失和前庭功?...

内耳基因治疗是一个快速发展的研究领域。它已应用于各种动物模型, 以对抗耳毒性, 噪音损伤和遗传性听力损失1。最近的几项研究表明, 在内耳基因治疗后突变小鼠听力和平衡功能的功能恢复2,3,4,5,6,7. 确定内耳基因治疗成功与否的关键因素之一, 是用于进入内耳的手术方法。理想的做法是, 手术方法很容易执行, 解剖标志将是一致的, 易于辨认, 并由此产生的定向细胞类型的转导是很高的。
在最近的一项研究中, 我们表明, 当病毒基因治疗是通过 whirler 突变小鼠的后半规管注射 (一种听觉丧失和前庭功能障碍模型), 在前庭中看到的感觉毛细胞的有效传导器官并且在耳蜗5。感觉毛细胞转导的高效性导致了这些突变小鼠听觉和前庭功能的改善。
在本文中, 我们详细描述了后半圆管的方法, 以获得新生小鼠内耳。

<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

内耳基因治疗提供了一个伟大的承诺, 作为一种潜在的治疗听力损失和头晕。内耳基因治疗成功的关键决定因素之一是找到一种分娩方法, 从而使靶细胞类型的传导效率保持在最小, 同时最大限度地减少听力损失。在本研究中, 我们描述了后半规管方法作为一种可行的方法, 为内耳基因分娩的新生小鼠。我们表明, 通过后半规管的基因传递能够灌注整个内耳。简单的解剖鉴定的后半圆管, 以及最低限度的操作所需的颞骨, 使这种手术方法是一个有吸引力的选择内耳基因传递。

注射 AAV8-whirlin 基因治疗新生儿 whirler 小鼠通过后半规管导致 whirlin 表达 (绿色) 在 utricular 毛细胞 (图 2), 整体感染效率为 53.1% (SD 38.1, n = 28)5.转基因毛细胞有拉长的张图片 (红色) 与对侧非注射耳朵的毛细胞 (5.35 @ 2.11 µm vs 3.20 @ 0.34 µm, 分别) 5.
AAV8-whirlin 后半圆管注射液?...

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

在本研究中, 我们描述了后半圆管方法作为一种可靠的方法, 为内耳基因分娩的新生小鼠。我们表明, 通过后半规管的基因传递能够灌注整个内耳。

后半圆管法在新生小鼠内耳基因传递中的研究

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 次观看.  National Institutes of Health. 在本研究中, 我们描述了后半圆管方法作为一种可靠的方法, 为内耳基因分娩的新生小鼠。我们表明, 通过后半规管的基因传递能够灌注整个内耳。

视频: 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.

小鼠胚胎植耳蜗的文化和基因转移通过电穿孔

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

科研

发育生物学

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 为成人和新生小鼠内耳局部药物传递的外科治疗方法

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

遗传学

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.

一种新的手术方法气管内注射生物活性剂在胎鼠模型

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

医学

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.

转基因小鼠内耳的发展<em&gt;在体内</em&gt;电穿孔

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 ...

神经科学

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.

通过圆窗膜病毒介导的基因传递给鼠标内耳手术方法

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.

在鼠背耳蜗核的听觉通路的光遗传学刺激直接可视化

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.

新生儿鼠耳蜗外科手术技术<em&gt;体外</em&gt;听力研究筛选工具

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.

一种非人类灵长类常见狨中内耳药物的外科手术方法 (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.

针对鼠标内耳给药方法的比较研究:Bullostomy<em&gt;对战</em&gt;鼓室注射

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. ...

生物学

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.

耳道造口术用于将局部药物输送到成年小鼠的内耳

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

后半圆管法在新生小鼠内耳基因传递中的研究

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