This work is supported by an R21 grant from the National Institutes of Health and by a grant from Hearing Research, Incorporated.

注：所有的程序和动物处理符合NIH道德准则和加州大学的机构动物护理和使用委员会，旧金山批准的协议要求。 1.准备动物外科手术<ol><li>开展外科手术在干净的专用空间。高压灭菌所有的手术器械，消毒与手术前玻璃珠灭菌。 注：在这个协议中，使用日龄10-12（P10-12）FVB小鼠。不同年龄和小鼠的菌株可用于满足特定项目的需要。年纪比P40小鼠是具有挑战性的，因为泡骨头变得更难。 </li><li>麻醉小鼠腹膜内注射盐酸氯胺酮（100毫克/千克），盐酸赛拉嗪（10毫克/千克），以及乙酰丙嗪（2mg / kg的）的混合物。开始外科准备后，才动物不再响应疼痛刺激，如脚趾捏。如果necessarY，施用麻醉剂鸡尾酒的加强剂量（五分之一原剂量），以恢复原来的麻醉平面。 注：请注意在这一步，因为大多数在这个年龄观察到的死亡率是由麻醉剂过量。 </li><li>涵盖了动物的眼睛有保护眼药膏在麻醉过程中，保持眼睛湿润，抑制动物的眨眼反射。 </li><li>将鼠标与颈部伸展在整个手术过程中的加热垫，直到鼠标完全清醒，防止麻醉后体温过低。 </li><li>剃左侧耳后区域与推剪和消毒用70％乙醇和碘伏手术操作之前。 </li></ol> 2.手术过程和载体注射<ol><li>使用耳后的办法，以暴露鼓泡。切开小剪刀皮下组织以露出耳后肌肉。缩回后脂肪组织为th切口电子后侧，分离的肌肉向右侧和左侧垂直于切口以暴露颞骨。确保此切口足够长以提供足够的可视舒适地工作。 </li><li>穿孔鼓室泡与地下25针和通过剥离骨背面以允许接近耳蜗的底转扩大孔根据需要用钳子，然后扩大到足以显现镫骨动脉和圆窗膜（RWM） 。 </li><li>轻轻刺破RWM在中心用硼硅毛细吸管。观察通过RWM在这一点上的流体流出;这是正常的。等到流出稳定（从耳蜗effluxed流体用无菌滤纸干燥）。 注：小心保持和推进玻璃针，使得RWM穿孔是尽可能小时。根据所使用的显微镜​​，持有使用显微操作或手工移液器用吸管持有人。 </li><li>准备使用移液管拉拔器相同的方式膜片钳移液器制备的移液器用于注射。确保该顶端直径较大（大约15微米直径），以便吸取病毒和缩小是很容易做到。 </li><li>制定1〜2微升AAV1-VGLUT3，AAV1-GFP或AAV2-GFP的到注射吸管。后的流出被稳定（5至10分钟），microinject这个固定体积入鼓阶通过同一孔先前在RWM制成。 </li><li>拉出吸管与一个小插件肌肉后迅速密封该RW利基并与一小滴组织粘合剂放置在肌肉，以避免从圆窗泄漏固定。 注意：密封该孔很好针后都与一个小插件肌肉的删除，以防止从耳蜗-此步骤的任何外淋巴泄漏是非常关键的保护听力。未能完全密封的RWM会导致听力下降一段时间。 </li><li>注射后，覆盖孔在听泡与脂肪组织，返回耳后的肌肉和脂肪组织到它们的正常位置，并缝合在层中的卷绕有6-0或更小的可吸收铬酸缝合。 </li><li>消毒用碘伏伤口。 </li></ol> 3.术后护理<ol><li>小鼠放置在一个温暖的笼子里，不随意放置，直到他们完全康复然后再回到与母亲。建议把幼崽回来的母亲之前，请从笼子里的男性。 </li><li>施用皮下卡洛芬（2毫克/千克）对镇痛术后且每隔24小时后3天，以管理炎症和疼痛。每天监测动物遇险，异常消瘦，疼痛，或感染的迹象。通常由手术所有小鼠通常应作用后的第3天。如果遇险或疾病的任何迹象的第3天之后，出现在一个鼠标，考虑安乐死吨他鼠标按机构的指导方针。 </li></ol>耳蜗功能4.评估继病毒输送用听性脑干反应（ABR）录音注：听性脑干反应（ABR）是一个听觉诱发电位经由头皮下放置电极从大脑中的正在进行的电活动中提取和记录。将动物刺激的声音。将所得记录包括五个波反映在听觉通道的连续点中的第一个10毫秒的电活动开始的听觉刺激后。 <ol><li>如在该协议的第1.2节中所述，除了没有乙酰丙嗪麻醉小鼠腹腔注射盐酸氯胺酮和盐酸赛拉嗪的混合物。 </li><li>把鼠标放在整个听力测试一个加热垫，直到鼠标完全清醒，防止麻醉后体温过低。 注：使用下面的声音刺激在这个实验中：点击（5毫秒的持续时间; 31赫兹展示率）。 </li><li>放置皮下针状电极在顶点，左耳（参考）的耳廓的下方，下方的对侧耳朵（地）和如先前在一个隔音室27,28描述开始记录的ABR。记录ABR波形，以5 dB声压级间隔时间从最大幅度下降。 </li><li>确定ABR阈病毒分娩后术后早在4天 注：阈值被定义为最低的刺激水平，在该响应的波峰值的I-V显然并目测反复出现。 </li><li>测量波我来分析活动从耳蜗神经。能产生可检测的ABR波形的最低的刺激电平被定义为阈值。 </li></ol> 5.转基因耳蜗蛋白表达使用免疫<ol><li>通过intrap的过量麻醉小鼠eritoneal注射盐酸氯胺酮和盐酸赛拉嗪的混合物在本协议第1.2描述，除了没有乙酰丙嗪。 </li><li>斩首后只动物不再响应疼痛刺激，如脚趾捏的头部。 </li><li>解剖耳蜗出来，过程整个安装免疫所描述的26。耳蜗全挂载免疫荧光使用抗VGLUT3和抗肌球蛋白抗体7A的详细协议阿基洛夫等人 ，到2013年29说明。 </li><li>的GFP标记，过夜孵育耳蜗全坐骑在4℃下用兔抗GFP抗体以1：250的稀释。 </li><li>冲洗耳蜗两次，每次10分钟，用PBS再孵育2小时在山羊抗兔IgG缀合的Cy2稀释1：4000于PBS中。 </li><li>冲洗用PBS耳蜗两次，每次10分钟，并孵育它们用DAPI 15分钟。 </li><li>安装在载玻片上的耳蜗，观察UND呃用免疫荧光共焦显微镜。 </li></ol>

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H., Kanzaki, S., Brown, N., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Functional+and+Structural+Outcome+of+Inner+Ear+Gene+Transfer+via+the+Vestibular+and+Cochlear+Fluids+in+Mice.">The Functional and Structural Outcome of Inner Ear Gene Transfer via the Vestibular and Cochlear Fluids in Mice.</a> Mol. Ther. 4 (6), 575-585 (2001).</li><li>Lalwani, A. K., Han, J. J., Walsh, B. J., Zolotukhin, S., Muzyczka, N., Mhatre, 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=Green+fluorescent+protein+as+a+reporter+for+gene+transfer+studies+in+the+cochlea.">Green fluorescent protein as a reporter for gene transfer studies in the cochlea.</a> Hear Res. 114 (1-2), 139-147 (1997).</li><li>Lalwani, A. 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=Long-term+in+vivo+cochlear+transgene+expression+mediated+by+recombinant+adeno-associated+virus.">Long-term in vivo cochlear transgene expression mediated by recombinant adeno-associated virus.</a> Gene Ther. 5 (2), 277-281 (1998).</li><li>Raphael, Y., Frisancho, J. C., Roessler, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral-mediated+gene+transfer+into+guinea+pig+cochlear+cells+in+vivo.">Adenoviral-mediated gene transfer into guinea pig cochlear cells in vivo.</a> Neurosci. Lett. 207 (2), 137-141 (1996).</li><li>Weiss, M. A., Frisancho, J. C., Roessler, B. J., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+mediated+gene+transfer+in+the+cochlea.">Viral mediated gene transfer in the cochlea.</a> Int. J. Dev. Neurosci. 15 (4=5), 577-583 (1997).</li><li>Pettis, R. M., Han, J. J., Mhatre, A. N., Lalwani, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracochlear+infusion+of+recombinant+adeno+associated+virus:+Analysis+of+its+dissemination+to+near+and+distant+tissues.">Intracochlear infusion of recombinant adeno associated virus: Analysis of its dissemination to near and distant tissues.</a> Assoc. Res. Otolaryngol. Abstr. 21, 673 (1998).</li><li>Konish, i. M., Kawamoto, K., Izumikawa, M., Kuriyama, H., Yamashita, T. <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+into+guinea+pig+cochlea+using+adeno-associated+virus+vectors.">Gene transfer into guinea pig cochlea using adeno-associated virus vectors.</a> J. Gene Med. 10 (6), 610-618 (2008).</li><li>Kaplitt, M. G., 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=Long-term+gene+expression+and+phenotypic+correction+using+adeno-associated+virus+vectors+in+the+mammalian+brain.">Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain.</a> Nature Genetics. 8 (2), 148-154 (1994).</li></ol>

No conflicts of interest declared.

在这项工作中，我们详细地描述了可用于耳蜗基因疗法，以恢复或抢救是受遗传缺陷损害正常听觉功能的目标的技术。因为它通常是无创伤，这种方法是安全的耳蜗的基因转移或其他潜在的分子疗法30。其他的方法对于耳蜗疗法已被描述，其中包括一个腹侧方法24，在小鼠和豚鼠内耳开窗31,32和内淋巴囊递送33。在我们的经验中，耳后的方法是更快，更简单，并具有...

基因治疗一直被建议作为对遗传性听力损失的潜在的治疗，但在这方面的成功一直难以实现1。迄今为止，病毒介导的方法学都占优势由于相对难以接近耳蜗内靶向特定细胞类型的理论能力。两个腺病毒（AV）和腺相关病毒（AAV）也已用于耳蜗基因递送。自动增值服务是在耳蜗为若干原因是有利的。它们是复制缺陷型病毒，并且可以有效的转基因的分子转移到不同类型的细胞，包括神经元，为一些听力损失的原因的一个重要目标。 AAV进入细胞是通过特异性受体介导2;因此，特定的血清型的选择必须是兼容的细胞类型，以被转导。自动增值服务可以有效地转染毛细胞3和掺入到宿主基因组中，导致稳定的，长期的TRA的表达nsgenic蛋白和该单元4的表型变化。而对于短期应用，例如毛细胞再生未必是有利的，长期表达是遗传缺陷稳定救援非常重要的。因为自动增值服务不与任何人疾病或感染相关的，并证明无耳毒性5,6,7，它们是在基因治疗中的用途为听力损失8的遗传形式的理想候选。 转移外源遗传物质导入用病毒载体的哺乳动物内耳已经研究在过去十年中，并成为一个有前途的技术，用于治疗遗传和获得性形式的听力损失9。耳蜗是潜在的基因治疗有几个原因的理想目标：1）它的体积小，就必须在有限的所需要的病毒量; 2）从它的其他器官系统的限制副作用相对隔离;及3）其流体填充腔促进病毒交付整个迷宫10，11,12,13,14，15。 先天性耳聋的小鼠模型允许使用的研究方法很多监测系统的，可复制的方式内耳的发展。虽然鼠标耳蜗的小尺寸确实存在一些手术难度，鼠标作为遗传性听力损失的研究具有极其重要的模式，有几个实验优于其他品种16。小鼠模型允许范围内通过遗传连锁分析，收集了详细的形态学观察，并模拟情景致病特点的评估;正因为如此，它们是很好的候选病毒介导的基因治疗。在小鼠中结合的技术进步的广泛的遗传研究已经使得有可能产生转基因小鼠在整个实验室17,18，19，20,21的可重现方式。 FurthermorE，有两个获得性和遗传听力损失表型小鼠，使严格的测试，在这个动物模型22，23,24存在众多的车型。因此，校正听力使用病毒介导的基因治疗在小鼠模型是在寻求一种治疗人类疾病的适当的第一步骤。 之前我们已经表明，缺乏的水泡谷氨酸转运体3（VGLUT3）转基因小鼠天生聋哑，由于缺乏谷氨酸释放的IHC带状突触25。由于此突变不导致感觉毛细胞的初级变性，这些突变小鼠有潜在其中测试耳蜗基因治疗先天性听力损失的优良模型。 迄今为止，一些病毒递送技术耳蜗基因治疗已被描述，包括圆窗膜扩散，圆窗膜注射，并且经由内耳开窗递送。有强大的IAL优点和每种方法9的缺点。 这里，我们报告为通过圆窗膜（RWM）病毒介导的基因递送到VGLUT3 KO小鼠内耳的外科方法。在耳后RWM注射方法是微创具有优良的听证会保留，并且是比较快的。如我们先前发表的，在为了恢复听力在该小鼠模型中，源自于AAV2载体携带VGLUT3基因（AAV1-VGLUT3）引入这些耳聋小鼠的耳蜗在出生后第12天（P！@），导致听26的恢复。听力在VGLUT3 KO小鼠经听性脑干反应（ABR）验证，而转基因蛋白的表达用免疫荧光（IF）的验证。这种方法因而表明病毒介导的基因疗法可以纠正一个遗传缺陷否则将导致耳聋。

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			<td height="40" style="height:40px;width:355px;">Acepromazine</td>
			<td style="width:177px;">Provided by UCSF LARC</td>
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			<td height="40" style="height:40px;width:355px;">Carprofen analgesia</td>
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			<td style="width:177px;">Betadine Puredue Pharma</td>
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			<td style="width:111px;"></td>
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			<td height="24" style="height:24px;width:355px;">dexamethasone ophthalmic ointment (TobraDex)</td>
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			<td height="24" style="height:24px;width:355px;">Heating pad</td>
			<td style="width:177px;">Braintree scientific, inc.</td>
			<td style="width:161px;"></td>
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			<td height="24" style="height:24px;width:355px;">25G needle</td>
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			<td height="40" style="height:40px;width:355px;">Borosilicate capillary pipette</td>
			<td style="width:177px;">World precision instruments, inc.</td>
			<td style="width:161px;">1B100F-4</td>
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			<td height="24" style="height:24px;width:355px;">Suture PDS*plus Antibacterial</td>
			<td style="width:177px;">Ethicon</td>
			<td style="width:161px;">PDP149</td>
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			<td height="24" style="height:24px;width:355px;">Tissue glue (Vetcode)</td>
			<td style="width:177px;">Butler Schein</td>
			<td align="right" style="width:161px;">31477</td>
			<td style="width:111px;"></td>
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			<td height="24" style="height:24px;width:355px;">Rabbit Anti-GFP antibody</td>
			<td style="width:177px;">Invitrogen</td>
			<td style="width:161px;">A11122</td>
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			<td height="20" style="height:20px;">Dissecting microscope&#160;&#160;&#160;&#160;&#160;</td>
			<td>Leica</td>
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			<td>MZ95</td>
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			<td height="20" style="height:20px;">Flaming/ Brown Micropipette&#160;&#160;&#160;&#160;&#160;</td>
			<td>Sutter Instrument Co</td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Puller Model P-97&#160;&#160;</td>
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			<td height="20" style="height:20px;">TDT BioSig III System&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>&#160;Tucker-Davis Technologies</td>
			<td></td>
			<td></td>
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surgical method for virally mediated gene delivery to the mouse inner ear through the round window membrane

基因疗法，用于实现由感觉神经性耳聋功能恢复，承诺给予更深入的了解，有助于听力损失潜在的分子和遗传机制。导入载体的进入内耳必须以一种方式在整个耳蜗损伤，同时最大限度地降低到现有结构即广泛分布的试剂来完成。这个手稿描述耳后的手术的方法，可以使用分子，药物，和病毒递送至小鼠10日龄和老年人通过圆窗膜（RWM）用于小鼠耳蜗疗法。这种手术方式，可以快速地直接供货到鼓阶，同时尽量减少失血，避免动物的死亡率。此技术涉及到内和中耳的基本的结构以及颈部肌肉，同时完全保留听力忽略的或没有损害。为了证明这一点的手术技术，囊泡glutam的功效吃转运3敲除（VGLUT3 KO）小鼠将被用作先天性耳聋，它回收输送VGLUT3到内耳后使用腺相关病毒（AAV-1）听力小鼠模型的一个例子。

为了验证该技术的特点和对耳蜗分子治疗耳后的方法实用，AAV1-VGLUT3，AAV1-GFP和AAV2-GFP分别通过RWM送入P10-12小鼠内耳。该方法证明了内毛细胞内成功的转基因表达（IHC）（VGLUT3 图1和GFP 图2和GFP 图3A），外毛细胞（OHC）（GFP 图2）和支持细胞（GFP 图2和图3A）26没有尔蒂伤害显著的器官。表达GFP不同细胞类型的观察看?...

Watch this Scientific Journal Video about Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane at JoVE.com

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

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 ...

surgical-method-for-virally-mediated-gene-delivery-to-mouse-inner-ear

Research

JoVE Journal

Neuroscience

15.1K 次观看.  University of California, San Francisco. 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. 

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

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

活体鼠脑微循环显微镜使用一个封闭的颅窗口

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

科研

神经科学

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

脊髓电二：外吸电极的制作

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

隔离LacZ基因表达的小鼠内耳组织用流式细胞仪检测细胞

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

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 ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

抛光和钢筋，颅骨变薄窗口，为长期的小鼠脑成像

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

膜片钳记录在内耳毛细胞的分离斑马鱼

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

成像pHluorin标记插入到质膜受体原代培养的小鼠神经元

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

采用组合运用各种方法，定义质膜交付在轴突分支和神经元形态发生中的作用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

鼠标显微注水技术进行有针对性的物质传递到中枢神经系统<i&gt;通过</i&gt;的颈内动脉

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

拉米内切手术和脊髓窗口植入小鼠

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...