Name: dissection of adult mouse utricle and adenovirus-mediated supporting-cell infection
Uploaded: 2012-03-28T12:00:00
Description: Watch this Scientific Journal Video about Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection at JoVE.com

这项工作得到了国家耳聋与其他交流障碍研究所院内研究司。 NIDCD的5R01 DC007613提供了额外的支持。

1。囊解剖和文化<ol><li>安乐死的成年人（4周龄以上）鼠标使用批准的协议和斩首。 </li><li>剪掉头部两侧外耳道，并向前拉往鼻子的皮肤。开张头从后到前，消除双方的大脑揭示骨迷路。 </li><li>远离骨耳蜗和修剪颅骨骨迷路（包括泡）转移到组织文化引擎盖配备在解剖显微镜（注：腺病毒的工作应在II级生物安全柜中进行）。 </li><li>在引擎盖，每个耳蜗转移到一个35mm的组织培养皿含无菌夹层动...

<ol>
	<li>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=The+adult+mouse+utricle+as+an+in+vitro+preparation+for+studies+of+ototoxic-drug-induced+sensory+hair+cell+death.">The adult mouse utricle as an in vitro preparation for studies of ototoxic-drug-induced sensory hair cell death.</a> Brain Res. , (2006).</li><li>Schmitt, N. C., Rubel, E. W., Nathanson, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cisplatin-induced+hair+cell+death+requires+STAT1+and+is+attenuated+by+epigallocatechin+gallate.">Cisplatin-induced hair cell death requires STAT1 and is attenuated by epigallocatechin gallate.</a> J. Neurosci. 29, 3843-3851 (2009).</li><li>Ou, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+FDA-approved+drugs+and+bioactives+that+protect+hair+cells+in+the+zebrafish+(Danio+rerio)+lateral+line+and+mouse+(Mus+musculus)+utricle.">Identification of FDA-approved drugs and bioactives that protect hair cells in the zebrafish (Danio rerio) lateral line and mouse (Mus musculus) utricle.</a> J. Assoc. Res. Otolaryngol. 10, 191-203 (2009).</li><li>Chiu, 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=Using+the+zebrafish+lateral+line+to+screen+for+ototoxicity.">Using the zebrafish lateral line to screen for ototoxicity.</a> J. Assoc. Res. Otolaryngol. 9, 178-190 (2008).</li><li>Sugahara, K., Rubel, E. W., 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=JNK+signaling+in+neomycin-induced+vestibular+hair+cell+death.">JNK signaling in neomycin-induced vestibular hair cell death.</a> Hear Res. , 221-221 (2006).</li><li>Matsui, J. I., Cotanche, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+hair+cell+death+and+regeneration:+two+halves+of+the+same+equation.">Sensory hair cell death and regeneration: two halves of the same equation.</a> Curr. Opin. Otolaryngol. Head Neck Surg. 12, 418-4125 (2004).</li><li>Ryals, B. M., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+cell+regeneration+after+acoustic+trauma+in+adult+Coturnix+quail.">Hair cell regeneration after acoustic trauma in adult Coturnix quail.</a> Science. 240, 1774-176 (1988).</li><li>Corwin, J. T., Cotanche, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+sensory+hair+cells+after+acoustic+trauma.">Regeneration of sensory hair cells after acoustic trauma.</a> Science. 240, 1772-174 (1988).</li><li>Shang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supporting+cell+division+is+not+required+for+regeneration+of+auditory+hair+cells+after+ototoxic+injury+in+vitro.">Supporting cell division is not required for regeneration of auditory hair cells after ototoxic injury in vitro.</a> J. Assoc. Res. Otolaryngol. 11, 203-222 (2010).</li><li>Stone, J. S., Cotanche, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+cell+regeneration+in+the+avian+auditory+epithelium.">Hair cell regeneration in the avian auditory epithelium.</a> Int. J. Dev. Biol. 51, 6-7 (2007).</li><li>Lahne, M., Gale, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Damage-induced+activation+of+ERK1/2+in+cochlear+supporting+cells+is+a+hair+cell+death-promoting+signal+that+depends+on+extracellular+ATP+and+calcium.">Damage-induced activation of ERK1/2 in cochlear supporting cells is a hair cell death-promoting signal that depends on extracellular ATP and calcium.</a> J. Neurosci. 28, 4918-4928 (2008).</li><li>Cunningham, L. L., Cheng, A. G., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase+Activation+in+Hair+Cells+of+the+Mouse+Utricle+Exposed+to+Neomycin.">Caspase Activation in Hair Cells of the Mouse Utricle Exposed to Neomycin.</a> Journal of Neuroscience. 22, 8532-8540 (2002).</li><li>Matsui, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase+inhibitors+promote+vestibular+hair+cell+survival+and+function+after+aminoglycoside+treatment+in+vivo.">Caspase inhibitors promote vestibular hair cell survival and function after aminoglycoside treatment in vivo.</a> J. Neurosci. 23, 6111-6122 (2003).</li><li>Matsui, J. I., Ogilvie, J. M., Warchol, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+caspases+prevents+ototoxic+and+ongoing+hair+cell+death.">Inhibition of caspases prevents ototoxic and ongoing hair cell death.</a> J. Neurosci. 22, 1218-1227 (2002).</li><li>Nakagawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+technique+for+inducing+local+inner+ear+damage.">A novel technique for inducing local inner ear damage.</a> Hear Res. 176, 1-2 (2003).</li><li>Forge, A., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+death+of+hair+cells+in+mammalian+vestibular+sensory+epithelia.">Apoptotic death of hair cells in mammalian vestibular sensory epithelia.</a> Hear Res. 139, 1-2 (2000).</li><li>Liu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase+inhibitors+prevent+cisplatin-induced+apoptosis+of+auditory+sensory+cells.">Caspase inhibitors prevent cisplatin-induced apoptosis of auditory sensory cells.</a> Neuroreport. 9, 2609-2614 (1998).</li><li>Taleb, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsp70+inhibits+aminoglycoside-induced+hearing+loss+and+cochlear+hair+cell+death.">Hsp70 inhibits aminoglycoside-induced hearing loss and cochlear hair cell death.</a> Cell Stress Chaperones. 14, 427-437 (2009).</li><li>Taleb, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsp70+inhibits+aminoglycoside-induced+hair+cell+death+and+is+necessary+for+the+protective+effect+of+heat+shock.">Hsp70 inhibits aminoglycoside-induced hair cell death and is necessary for the protective effect of heat shock.</a> J. Assoc. Res. Otolaryngol. 9, 277-289 (2008).</li><li>Francis, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Celastrol+Inhibits+Aminoglycoside-Induced+Ototoxicity+via+Heat+Shock+Protein+32+(HSP32).">Celastrol Inhibits Aminoglycoside-Induced Ototoxicity via Heat Shock Protein 32 (HSP32).</a> Cell Death and Disease. , (2011).</li><li>Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase+inhibitors,+but+not+c-Jun+NH2-terminal+kinase+inhibitor+treatment,+prevent+cisplatin-induced+hearing+loss.">Caspase inhibitors, but not c-Jun NH2-terminal kinase inhibitor treatment, prevent cisplatin-induced hearing loss.</a> Cancer Res. 64, 9217-9224 (2004).</li><li>Bird, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supporting+cells+eliminate+dying+sensory+hair+cells+to+maintain+epithelial+integrity+in+the+avian+inner+ear.">Supporting cells eliminate dying sensory hair cells to maintain epithelial integrity in the avian inner ear.</a> J. Neurosci. 30, 12545-12556 (2010).</li><li>Sato, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+fractalkine+receptor+CX3CR1+on+cochlear+macrophages+influences+survival+of+hair+cells+following+ototoxic+injury.">Expression of fractalkine receptor CX3CR1 on cochlear macrophages influences survival of hair cells following ototoxic injury.</a> J. Assoc. Res. Otolaryngol. 11, 223-234 (2010).</li></ol>

感觉毛细胞受到的各种压力，包括老龄化，噪声外伤，和接触，包括氨基糖甙类抗生素和抗癌药顺铂的耳毒性药物引起的死亡。在哺乳动物毛细胞，导致永久性听力丧失和/或平衡紊乱导致死亡。 在体外模型系统研究旨在确定的细胞和分子机制的毛细胞死亡，以及那些旨在防止或扭转毛细胞死亡的关键工具。不同于成年哺乳动物耳蜗毛细胞，毛细胞的囊良好的生存文化。囊毛细胞是敏感的?...

A correction was made to <a href="http://www.jove.com/video/3734">Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection</a>. There was an error with a figure, the authors middle names were omitted, and the acknowledgements were updated.

Erratum: Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt;试剂</strong</td&gt;<td<strong&gt;公司</strong</td&gt;<td<strong&gt;产品目录号</strong</td&gt;</tr&gt;<tr&gt;<td&gt;中等199</td&gt;<td&gt; Gibco公司/ Invitrogen公司</td&gt;<td&gt; 12350</td&gt;</tr&gt;<tr&gt;<td&gt;的DMEM/F12</td&gt;<td&gt; Gibco公司/ Invitrogen公司</td&gt;<td&gt; 11320</td&gt;</tr&gt;<tr&gt;<td&gt;精细镊子（＃55）</td&gt;<td&gt;精细的科学工具</td&gt;<td&gt; 11255-20</td&gt;</tr&gt;<tr&gt;<td&gt;精细镊子（＃5）</td&gt;<td&gt;精细的科学工具</td&gt;<td&gt; 11252-30</td&gt;</tr&gt;<tr&gt;<td&gt;钳（＃3）</td&gt;<td&gt;精细的科学工具</td&gt;<td&gt; 11231-30</td&gt;</tr&gt;<tr&gt;<td&gt;青霉素G</td&gt;<td&gt;西格玛</td&gt;<td&gt; P3032</td&gt;</tr&gt;<tr&gt;<td&gt;胎牛血清</td&gt;<td&gt; Invitrogen公司</td&gt;<td&gt; 10082-1​​39</td&gt;</tr&gt;<tr&gt;<td&gt; NUNC小托盘</td&gt;<td&gt;费舍尔</td&gt;<td&gt; 12-565-68</td&gt;</tr&gt;<tr&gt;<td&gt;腺病毒GFP</td&gt;<td&gt;矢量Biolabs公司</td&gt;<td&gt; 1060 *</td&gt;</tr&gt;<tr&gt;<td&gt;加州前decalcifier</td&gt;<td&gt;费舍尔</td&gt;<td&gt; CS510-1D</td&gt;</tr&gt;<tr&gt;<td&gt;硼氢化钠</td&gt;<td&gt;西格玛</td&gt;<td&gt; 452882</td&gt;</tr&gt;<tr&gt;<td&gt;抗肌球蛋白7A（多克隆）</td&gt;<td&gt;变形Biosciences公司</td&gt;<td&gt; 25-6790</td&gt;</tr&gt;<tr&gt;<td&gt;抗肌球蛋白7A（单克隆）</td&gt;<td&gt;发展研究杂交瘤细胞银行</td&gt;<td&gt; MYO7A 138-1</td&gt;</tr&gt;<tr&gt;<td&gt;抗-钙调蛋白</td&gt;<td&gt;西格玛</td&gt;<td&gt;ç3545</td&gt;</tr&gt;<tr&gt;<td&gt;反，钙结合蛋白</td&gt;<td&gt; Chemicon公司</td&gt;<td&gt; AB1778</td&gt;</tr&gt;<tr&gt;<td&gt; FluoromountĞ</td&gt;<td南方生物技术公司</td&gt;<td&gt; 0100-01</td&gt;</tr&gt;</tbody&gt;</table&gt;<p class="jove_content"<strong&gt;表1</strong&gt;。在囊文化和腺病毒感染的试剂和使用的工具。</p&gt;<p class="jove_content"&gt; *注：通常是由AD-GFP载体Biolabs公司提供股票滴度在有1x10<sup&gt; 10</sup&gt; PFU / ml的。我们要求定制，以提供股票病毒滴度为1.2×10放大<sup&gt; 11</sup&gt; PFU / ml的。</p&gt;

dissection of adult mouse utricle and adenovirus-mediated supporting-cell infection

听力损失和平衡紊乱往往造成mechanosensory毛细胞是内耳的受体细胞死亡。由于没有细胞行圆满代表了哺乳动物的毛细胞，毛细胞的研究依赖于基层机关文化。最好具有成熟的哺乳动物毛细胞在体外模型系统，利用从成年鼠（ 图1）1-6椭圆囊器官培养。囊是前庭器官，以及椭圆囊毛细胞的听觉器官，Corti器毛细胞结构和功能相似。成年小鼠的囊准备成熟的感觉上皮细胞的生存，动态平衡，这些细胞死亡的分子信号调节的研究。 哺乳动物耳蜗毛细胞的终末分化，当他们失去了，不能再生。在非-MAM其次是强大的再生，恢复听力和平衡功能7，8，马里脊椎动物，听觉或前庭毛细胞死亡。再生毛细胞介导的胶质状的支持细胞，毛细胞的基底表面的接触中感觉上皮9，10。支持细胞是重要的调停毛细胞生存和死亡的11。我们最近开发出一种技术，支持在使用培养椭圆囊腺病毒的细胞感染。利用腺病毒5型（dE1/E3）提供含有CMV启动子控制下的绿色荧光蛋白的转基因，我们发现，腺病毒特别有效地支持细胞的感染。支持细胞的感染效率约为25-50％，并没有感染毛细胞（ 图2）。重要的是，我们发现，不支持细胞的腺病毒感染导致毛细胞或支持细胞的毒性，因为在AD-GFP INF细胞计数ected椭圆囊相当于在非疫区的椭圆囊（ 图3）。因此，在支持细胞培养椭圆囊腺病毒介导的基因表达提供了一个功能强大的工具，研究支持细胞作为调解人毛细胞生存，死亡和再生的作用。

Watch this Scientific Journal Video about Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection at JoVE.com

Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection

mechanosensory毛细胞是内耳的受体细胞。最好的特点<em&gt;在体外</em成熟的哺乳动物毛细胞模型系统利用从成年鼠的椭圆囊器官培养。我们目前的成年小鼠的囊的剥离，我们证明腺病毒介导的支持细胞在体外培养椭圆囊的感染。

解剖成年小鼠的椭圆囊和腺病毒介导的支撑细胞的感染

Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear.  Since there ...

Research

JoVE Journal

Neuroscience

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

多鼠神经解剖学磁共振成像

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

科研

神经科学

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

解剖鼠标眼的视网膜色素上皮的全山

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

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

成年鼠脑铸造三维成像和形态分析

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

小鼠多巴胺和三维胶原基质检测纹状体外植体的解剖和文化

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

幼虫，蛹和成人的解剖和免疫组化<em&gt;果蝇</em&gt;视网膜

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

全山解剖和成年小鼠耳蜗的免疫

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

一个简单的一步解剖协议成人全安装准备<em&gt;果蝇</em&gt;脑

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection

成年小鼠背根节外植体和分离细胞模型调查可塑性和对包括病毒感染在内的环境侮辱的反应

This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory ...

Cochlear Surface Preparation in the Adult Mouse

成年小鼠中的科利耳表面制备

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...

Cryo-section Dissection of the Adult Subependymal Zone for Accurate and Deep Quantitative Proteome Analysis

冷冻切片解剖成人室管膜下区，用于准确和深入的定量蛋白质组分析

The subependymal neurogenic niche consists of a paraventricular ribbon of the lateral ventricular wall of the lateral ventricle. The subependymal zone ...