The authors thank Masaki Yoda and Elise Lamar for critical reading of the manuscript, Kazumasa Takenouchi for help with histology, Mari Fujiwara for help with microscopy and Makoto Morikawa for help in photographing human and mouse auditory ossicles.

在这项研究中进行的所有动物的程序是由庆应义塾大学机构动物护理和使用委员会（IACUC - 批准文号:09221），并按照庆应大学对动物实验的机构准则在研究中使用动物。人体标本是从捐献给解剖学，医学庆应义塾大学医学院系的尸体中分离，并按照规定的机构中使用。 1.听泡和胶囊隔离<ol><li>安乐死小鼠含有上述浸泡在异氟醚或七氟烷，直到呼吸停止通风超过一分钟以上纸巾一个平台，一个罐子，然后执行颈椎脱位。要小心，以避免浸泡过的纸巾小鼠的直接接触。 </li><li>做一个小横切口在颈部的背侧和拉开皮肤朝头部和用双手尾以暴露底层的颈部亩SCLE组织。 </li><li>在使用14厘米锋利手术剪颈椎区斩首老鼠。 </li><li>剥离皮肤完全对鼻子。用鼻子和门牙一起切断所有的皮肤。 </li><li>将剪刀放进嘴里，切两侧咬肌肌肉。 </li><li>小心打开下颚和去除舌头和下颌一起。 </li><li>用锋利的剪刀，分裂颅骨和颅底入沿矢状切面（ 图1A，B）两部分。 </li><li>使用镊子，取出大脑和小脑半球及脑干。听觉泡和胶囊的位于外侧小脑和脑干。注意，听觉泡是进一步横向于听觉胶囊（ 图1C，D）。 </li><li>解剖出泡和胶囊与周围颅骨（ 图1E）。 </li><li>转移样品至含有磷酸盐缓冲盐水（PBS）pH 7.4的在RT一个菜。 </li><li> ü一个升气管双目解剖显微镜，使用镊子拉开周围的骨骼和剪刀剪开绕泡及胶囊（ 图1F）松动的边界。除去周围的骨骼是basioccipital（腹边框），exoccipital（腹，后缘），枕骨（后缘），interparietal，顶叶（背境），鳞（后背成前缘），alisphenoid（前缘）和基蝶骨（安特罗腹境）的骨头。注意，styliform过程（SP），它支持耳咽管41的鼓膜开口，是从颞骨茎突不同。 </li></ol>锤骨，砧骨和镫骨:2听骨的分离<ol><li> 锤骨 <ol><li>同时使用小剪刀和镊子，取出外耳道横向于沟tympanicus的部分，以使鼓膜是可见的（ 图2A，B）。 </li><li>在腹侧（虚线）和后（＃）的壁（ 图2C）除去鼓膜和鼓室骨附近malleal突短（圆形隆起，见讨论），两者的一部分。锤骨和鼓膜张肌现在应该接触（ 图2D，E）。 </li><li>提起锤（ 图2F）和切断鼓膜张肌与27克针（ 图2G）的斜边。注意，malleal柄牢牢附着于鼓膜，如在其他哺乳动物中看到。 </li><li>从胸骨柄，这是脆弱的仔细分离鼓膜。取下鼓膜骨露出三个听小骨。 </li><li>脱臼从砧骨锤骨的听小骨关节（ 图2H）。 </li><li>通过在goni​​ale压裂前突隔离锤骨。 </li></ol></li><li> 砧骨和镫骨 <ol><li>隔离ŧ他砧通过切断砧骨的后韧带在短脚（ 图3A）。 </li><li>隔离通过切断镫骨附近的镫骨动脉用27克针（ 图3B，C）的斜边镫骨。如果有必要，切镫骨肌肌腱在与针镫骨肌过程。 </li><li>插入一个缝纫针（或标记针）到镫骨闭孔并提起镫骨。除去镫骨后，卵圆窗开口应清晰可见（ 图3D）。 </li></ol></li></ol> 3.听泡和胶囊包埋<ol><li> 在石蜡包埋块准备 <ol><li>如第1节隔离肺大泡和胶囊。 </li><li>切割泡（在styliform处理）关闭，用剪刀的前端，浸泡在PBS中的4％多聚甲醛（PFA）的泡和胶囊，在4℃C和允许固定剂进入到大疱。如果空气变得在泡捕集，用针和注射器将其删除。留在固定剂的泡和胶囊，在4℃的CO / N于管旋转。 注意:PFA是有毒的，应谨慎处理。 </li><li>用PBS洗一次。 </li><li>在10％的乙二胺四乙酸二钠盐二水合物脱钙泡和胶囊为一个星期，在4℃（EDTA-2Na盐），100毫摩尔Tris碱，pH 7.0，在2毫升管中。更改缓冲区隔日1次。 </li><li>用PBS洗一次。标本可在4℃储存在水中70％的乙醇。任选地，通过分级醇系列（30％，50％，在水中的70％）转移到70％的乙醇。 </li><li>上的组织处理器，脱水在等级系列的乙醇溶液的试样（70％，2×95％，3×100％，每1小时），清除在二甲苯（4倍，在40℃下各1小时），并渗入与标本熔化的石蜡42。或者，替代二甲苯与商业组织clearin摹解决方案（ 例如 ，组织相容-清除）。 </li><li>从处理器卸载标本，并从他们的磁带中删除。 </li><li>在一个组织包埋控制台系统，地方标本放入盛有熔化的石蜡模具。继续嵌入（第4节）。 </li></ol></li><li> 在冷冻块嵌入（川本的电影法）制备43 <ol><li>如第1节隔离肺大泡和胶囊。 </li><li>切割泡（在styliform处理）关闭，用剪刀的前端，浸泡在固定液大疱和胶囊，在4℃（2％或1％的PFA而非PBS中的4％至保留抗原性）。如果空气被截留在大疱，使用针和注射器将其删除。留在固定剂的泡和胶囊，在4℃的CO / N于管旋转。 </li><li>在PBS快速洗涤泡和胶囊以及液体冷冻包埋化合物在4℃立即浸入。 </li><li>重要提示:除去气泡，如果任何中间并通过抽吸外耳通过针，并通过添加用钳子嵌入化合物。继续嵌入（第4节）。 </li></ol></li></ol> 4.样品方向和嵌入注:全部泡和胶囊必须嵌入到切期望段期间被布置在一个特定的方向。下面概述的过程用于部分中不同取向的锤骨。 <ol><li> 锤骨纵向（矢状窦旁）切片 <ol><li>把泡或外耳道的侧面向下在温暖石蜡（或冷冻包埋化合物）。调整方向，使得锤骨的颈部和横向薄片平行于嵌入盘（ 图4A - C）的水平底部。需要注意的是鼓膜在小鼠头部（ 图4A以大约30°的角度倾斜于垂直;图59中的Kampen <suP&gt; 44）。 </li></ol></li><li> 锤骨水平切片 <ol><li>水平放置背嵴在温暖的石蜡（或低温嵌入化合物）。调整泡和胶囊的取向，使得锤骨的颈部和横向薄层垂直于嵌入碟（ 图4D - F）的底部。 </li></ol></li><li> 柄及鼓膜5的正面切片（横截面） <ol><li>放置在温暖石蜡（或冷冻包埋化合物）的malleal柄，使得其垂直于嵌入盘的底部。 </li></ol></li><li>冷却块到相应于一个组织灌封控制台系统上硬化石蜡温度（可替代地，使用冷冻包埋化合物在干冰/己烷浴）。 </li><li>流程组织块，用常规方法切段。例如，用苏木精和曙红（H＆＃染色石蜡切片38; E），番红O（软骨），或抗酒石酸酸性磷酸酶（TRAP）活性（为破骨细胞）3，或者通过免疫组化。脱钙冰冻切片都适合使用荧光染料14骨标签，茜素红染色钙和免疫42。 </li></ol>

The authors have nothing to disclose.

在这里，我们提出来隔离在出生后小鼠听泡和胶囊有用的方法。在此之前P12，组织是脆弱和孤立过程中可能会损坏。 P12之后，听觉泡和胶囊可以容易地从周围组织中分离。切片之前解剖从头部泡具有几个优点。首先，发生汽蚀产后和听泡的增长率从P6起最积极，是由P14 50完成。鼓膜和耳蜗壁之间的间充质组织是由空气通过气蚀过程所取代。在中耳腔所得空气可阻碍期间固定，脱钙和嵌入组织和液体之间的接触。这是比较容易通过切断的前端（styliform处理），而不是试图在未分离大疱这样做从分离的听觉泡除去空气。第二，锤骨（和鼓膜）的方向不垂直在头部。它是一节中所希望的平面内的锤骨嵌入分离的听觉泡和胶囊在给定的取向，因此更容易。 一旦分离，听泡和胶囊可用于多种分析非常有用。例如，高清晰度的X射线显微CT可以揭示骨微结构的形态，例如在锤骨14成骨毛细管。所述stereofluorescence解剖显微镜是可视化中评价表达在中耳或内耳33荧光蛋白报告小鼠结构的有力工具。此外，各种在体内或离体荧光标记的方法和整装免疫检测可以进行。光片荧光显微镜也用于三维分析51是有用的。虽然这里没有描述，与听觉泡和胶囊如外周神经，血管相关多样的解剖结构，并在中耳的鼓膜也可使用此协议进行评价。 注意，石蜡切片需要嵌入前骨组织脱钙，因此不允许矿化的分析。与此相反，可以在不脱钙进行用于制备冰冻切片的川本膜的方法43，适合使用体内骨标记技术或特殊染色，如茜素染色矿化研究。冰冻切片条件应根据基于鼠标的年龄进行优化。例如，在低温恒温室内的较少凉爽温度推荐为老年小鼠的标本尽量减少部分的损坏。 在鼠标的锤骨的突出的半球形突起正确的术语是"圆形隆起"。尽管如此，术语"突短"已被广泛地用于指示更多次的圆形隆起一个二十年，特别是在小鼠的发育生物学家16,20,22-25。 "短突"原指横向过程（突外侧），它从圆形隆起不同。在人类中，横向处理类似的轻微锥形突起形成附着于鼓膜总路线，从柄（未示于图6B中 ，内侧视图）延伸。在小鼠中，横向过程也是在相对端的壳顶48上的柄的投影。鼓膜的鼓膜松弛部是上述锤骨的横向过程。圆形隆起是不被人类锤骨显而易见。

中耳的三个听小骨，即锤骨，砧骨和镫骨，形成从鼓膜到内耳或耳蜗1,2-发送声音的哺乳动物特异性听觉链。听力功能可在小鼠中通过测量听性脑干反应（ABR）阈值3-6，和鼓膜可使用激光多普勒Vibrometry（LDV）7监视后面锤骨的振动来评价。通过结合ABR，LDV和畸变产物耳声发射（DPOAE）的测量，传导性听力损失可以从感减值8歧视。 耳条件动物模型都需要，给予听觉和耳部健康的患者的青睐福祉的重要性。例如，中耳炎是人类婴儿和儿童，以及严重，急性中耳炎看到一种极为常见的耳部感染，如果康迪其并发症可能会出现化是不是适当的抗生素治疗9。中耳炎小鼠模型可以证明在了解发病机制和开发治疗10,11有用。 鼠小骨，其中（除了锤骨的goniale部分）由软骨内骨化12,13形成的，是对骨代谢和形态发生的研究高度相关。首先，它们的小尺寸允许骨头高分辨率分析用X射线或荧光显微镜14一个完整的骨膜。其次，异常骨代谢，如过度或不足骨吸收或骨细胞15名受损的相互作用，可以分析作为一个潜在的贡献者听力损失3,4,7。第三，异常小骨形态发生报道在几个基因缺陷型小鼠，如缺乏HOXA2 16-19，MSX1 20-22 Prrx1 23，Goosecoid动物（GSC）24,25，Bapx1 13，Tshz1 26，DUSP6（Mkp3）27， 头蛋白 （ 木栓 ）28，29 FGFR1，甲状腺激素受体（THRA，THRB）5，Bcl2的 30和其他1,31或过表达的小鼠HOXA2 32。最后，尽管其体积小，听小骨与诸如肌肉33和关节34,35相关的结构进行访问。 鼠标小骨比人听小骨小，但值得注意的是，鼠标中耳不是其人力对应的微缩版。例如，在小鼠中，镫骨动脉，穿过镫骨的环，持续整个生命36，而在人类中，该胚胎镫骨动脉妊娠期间消失。此外，鼠标锤骨的形态不同于第的不同Ë人骨（ 见图6）。在小鼠中，听觉（鼓膜）泡包围充满空气的中耳腔，而在人类中，在颞骨小梁骨组成乳突气房容纳小骨，而不是一个大泡37。在这两个物种中，听觉胶囊（耳囊，骨迷路）包围耳蜗及内耳半规管。中耳的比较与进化生物学已经被广泛的审查38-40。 下面首先提供的协议描述了如何解剖分别出听泡和胶囊，其主要包括中耳和内耳。该协议还演示了如何隔离听觉泡锤骨，砧骨和镫骨。最后，它显示了如何定向听泡和胶囊制剂中嵌入的听骨组织切片。

<table><tbody><tr><td>&lt;strong&gt;Tools/Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Paper towel</td><td>Daio Paper Corporation</td><td>703347</td><td>can be purchased from other vendors</td></tr><tr><td>Glass Jar</td><td>Various</td><td></td><td>can be purchased from other vendors</td></tr><tr><td>14 cm surgical scissors</td><td>Fine Science Tools (F.S.T.)</td><td>91400-14</td><td>can be purchased from other vendors</td></tr><tr><td>Extra fine scissors-straight</td><td>Fine Science Tools (F.S.T.)</td><td>14084-08</td><td>can be purchased from other vendors</td></tr><tr><td>Fine Forceps Angled 45&lt;b&gt;&amp;deg;&lt;/b&gt;</td><td>Fine Science Tools (F.S.T.)</td><td>11063-07</td><td>can be purchased from other vendors</td></tr><tr><td>Dissecting microscope</td><td>Nikon</td><td>SMZ800N</td><td>for routine dissection</td></tr><tr><td>Dissecting microscope</td><td>Nikon</td><td>SMZ18</td><td>for movies&amp;nbsp;</td></tr><tr><td>Injection needle 27 G</td><td>TERUMO</td><td>NN-2719S</td><td></td></tr><tr><td>Syringe (1 mL)</td><td>TERUMO</td><td>SS-01T</td><td></td></tr><tr><td>Marking Pin</td><td>Various</td><td></td><td></td></tr><tr><td>Tube rotator RT-50</td><td>TAITEC</td><td>0000165-000</td><td>can be purchased from other vendors</td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM3050S</td><td>http://www.leicabiosystems.com/histology-equipment/cryostats/details/product/leica-cm3050-s/</td></tr><tr><td>TC-65 Tungsten blade</td><td>Leica</td><td>14021626379</td><td>for Kawamoto&#039;s firm method</td></tr><tr><td>Stainless containers</td><td>Leica</td><td></td><td>for Kawamoto&#039;s firm method</td></tr><tr><td>Cryofilm type IIC</td><td>Leica</td><td></td><td>for Kawamoto&#039;s firm method</td></tr><tr><td>Silane coated slide (New Silane II)</td><td>Muto Pure Chemicals</td><td>511617</td><td>can be purchased from other vendors</td></tr><tr><td>Cover glass</td><td>Matsunami</td><td></td><td>can be purchased from other vendors</td></tr><tr><td>Tissue processor</td><td>Sakura Finetek</td><td>VIP-5</td><td>can be purchased from other vendors</td></tr><tr><td>Tissue Embedding Console System</td><td>Sakura Finetek</td><td>Tissue-Tek TEC 5&amp;nbsp;</td><td>can be purchased from other vendors</td></tr><tr><td>Sliding microtome for paraffin</td><td>Yamato Kohki Industrial</td><td>REM-710</td><td>can be purchased from other vendors</td></tr><tr><td>Path Blade+pro for hard tissue</td><td>Matsunami</td><td>PB3503C</td><td>for paraffin section</td></tr><tr><td>Micro-CT</td><td>RIGAKU</td><td>R_mCT2</td><td>http://www.rigaku.com/en</td></tr><tr><td>Fluorescence microscope</td><td>KEYENCE</td><td>BZ-9000</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Maruishi pharmaceutical Co. Ltd</td><td></td><td></td></tr><tr><td>NaCl</td><td>wako</td><td>191-01665</td><td>for PBS</td></tr><tr><td>KCl</td><td>wako</td><td>285-14</td><td>for PBS</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; 12H&lt;sub&gt;2&lt;/sub&gt;O</td><td>wako</td><td>196-02835</td><td>for PBS</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>wako</td><td>287-21</td><td>for PBS</td></tr><tr><td>Paraformaldehyde (PFA, EM Grade)</td><td>TAAB</td><td>P001</td><td></td></tr><tr><td>EDTA-2Na</td><td>wako</td><td>15111-45</td><td></td></tr><tr><td>Trizma base</td><td>Sigma</td><td>T1503-1KG</td><td></td></tr><tr><td>Super Cryoembedding Medium</td><td>Leica</td><td></td><td>for Kawamoto&#039;s firm method</td></tr><tr><td>Dry Ice</td><td>Various</td><td></td><td>for Kawamoto&#039;s firm method</td></tr><tr><td>Hexane</td><td>wako</td><td>080-03423</td><td>for Kawamoto&#039;s firm method</td></tr><tr><td>Super Cryomouting Medium type R2</td><td>Leica</td><td></td><td>for Kawamoto&#039;s firm method</td></tr><tr><td>Paraffin</td><td>Sakura Finetek</td><td>781001A0107</td><td></td></tr><tr><td>Histo-Clear</td><td>NDS</td><td>HS-200</td><td></td></tr><tr><td>Calcein</td><td>DOJINDO</td><td>340-00433</td><td></td></tr><tr><td>Hematoxylin&amp;nbsp;</td><td>wako</td><td>131-09665</td><td></td></tr><tr><td>Eosin</td><td>wako</td><td>051-06515</td><td></td></tr></tbody></table>

dissection of the auditory bulla in postnatal mice: isolation of the middle ear bones and histological analysis

在大多数哺乳动物中，在中耳听小骨，包括锤骨，砧骨和镫骨，是最小的骨头。在小鼠中，骨性结构称为听觉泡容纳小骨，而听觉胶囊包封内耳，即耳蜗和半规管。鼠小骨是极大的兴趣在耳鼻喉科领域的研究人员和听觉因此，至关重要，但它们的代谢，发育和进化是其他领域密切相关。改变的骨代谢能影响听力功能在成年小鼠和各种基因缺陷小鼠显示在子宫内听小骨的形态发生变化。尽管鼠听小骨是微小的，它们的操作是可行的，如果人理解他们的解剖定位和三维结构。在这里，我们将介绍如何剖析听泡和出生后的小鼠胶囊，然后通过去除肺大泡的一部分分离单个听小骨。我们还讨论了如何EM床在不同的方向的泡和胶囊，以产生石蜡或冰冻切片适于制备锤骨的纵向，横向，或正面部的。最后，我们列举小鼠和人类听小骨之间的解剖学差异。这些方法将在分析听小骨的病理，发育和进化方面和小鼠中耳有用。

这个协议呈现分离从小鼠听泡小骨的方法。首先，将泡和胶囊被解剖出作为从头骨（ 图1）的单件。然后解剖泡用于制备锤骨（ 图2）和砧骨及镫骨（ 图3）。听泡和胶囊的地标在肺大泡，背嵴，前半规管，以及subarcuate窝（ 图1F）的前端的styliform过程。 Microcomputed断层扫描（CT）成像揭示听觉泡小骨，以及对那些听小骨的纵向和横向切片（ 图4）的最佳定向。 对于锤骨的纵向石蜡切片中，泡和胶囊是在EDTA中，在4℃脱钙一周，嵌入在巴在图4所示的一个方向拉芬块- C，切片在4微米，然后用H＆E染色。附着在听觉泡鼓膜锤骨揭示在P14（ 图5A）正在进行的软骨内骨化。为了形象化新骨形成，钙黄绿素（30微克/克体重），腹腔注入P20鼠标和大疱和胶囊均在P21隔离24小时后。未经脱钙样品包埋冷冻，然后在使用基于川本43的方法的粘合膜为6μm冰冻切片。用DAPI核染色（4&#39;，6-二脒基-2-苯基）后，在荧光显微镜下观察到的部分。钙黄绿素信号（绿色）揭示了在锤骨（米）的新骨形成，泡和胶囊（ 图5B）。对于锤骨的横切片，从5周龄小鼠中分离听觉泡未经脱钙嵌入冷冻（对于定向见图4D - F），在使用方法川本6微米冰冻切片，用H＆E染色。所述malleal突短（MPB）的横切片还示出了耳蜗（ 图5C）。 从P31小鼠中分离合适的听小骨的内侧视图显示鼠标锤的典型特征，即"滑翔，海鸥翼状"（或波斯剑状45）柄，一个突出的突短（圆形隆起见讨论），以及横向薄片（ 图6）。请注意，前突（前突）是围绕goniale解剖过程中的断裂，并从鼓膜环（ectotympanic）分离。这种代表性的样本表现出锤砧骨和之间的完整incudomalleolar关节，而砧镫关节脱臼。腱插入到malleal和镫骨肌过程是可检测的（ 图6A，星号）。 图6B以相同放大倍率比较小鼠和人的听小骨。品种不同，大小比其他的，包括以下内容。该malleal柄是翼状的小鼠，但俱乐部般的人类。解剖轴线（或旋转轴，通过锤骨和砧骨的短过程的前过程中的线）和柄之间的角度是在小鼠中小得多，在两个几乎平行的，而不是接近垂直在人类中6,46-48。在人类的听小骨，vibrometric研究揭示，incudo，踝关节移动，而不是固定的功能49。鼠标锤展品种类，薄，平整横向叶片在人类47并不明显。在小鼠中，突前保险丝膜质骨，即goniale和定音鼓C环，而在人类中突前减小到骨41的小毛刺。小鼠和人类的镫骨也不同:在小鼠中，前小腿是弯曲的，并且后小腿更直的，而在人类中，前小腿更直比后部小腿。值得一提的是，相对于身体大小的锤骨头在物种大量扩大诸如金摩尔，以"最小"筋骨48异速生长关系证明显著变性。 <img alt="图1" src="/files/ftp_upload/55054/55054fig1.jpg" /> 图1.听泡和胶囊的解剖。 （A）一P31鼠标的头骨被分成左右两半。 A，前壁; P，后路; L，左; R，右。 （B）中的等分的，皮肤的头部的右半的内侧表面上。 CX，大脑皮层; CB，小脑;烧烤，brainstem。 D，背;五，腹。大脑的（C）去除用钳子。 （D）在右侧颅骨听觉胶囊内侧视图。背嵴（箭头）位于颅中窝（MCF），后颅窝（PCF）与分离后背成前，听囊腹，后表面。比例尺，2毫米。 （五）听泡和胶囊（内侧视图）的高倍。有限公司耳蜗;七，面神经;八，听神经; AC，前（上级）半规管; SF，subarcuate窝，里面有小脑paraflocculus。比例尺，1毫米。孤立听泡和胶囊（内侧视图）（F）显微照片。 SP，styliform过程。比例尺，1毫米。 （A - E），P31鼠标。 （F），P33鼠标。 <a href="http://ecsource.jove.com/files/ftp_upload/55054/55054fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55054/55054fig2.jpg" /> 图2.解剖锤骨。 （A）右侧听泡和胶囊的腹外侧视图。沟tympanicus（ST，虚线箭头）是鼓膜的附着位点。骨横向于ST是外耳的一部分，和骨内侧到ST形成中耳腔的楼层。 A，前壁; P，后路; D，背;五，腹。清除外耳道后（B）查看揭示鼓膜（TM），包括鼓膜松弛部（PF）和收杆tensa（PT）。 （C）去除鼓室骨（虚线和＃）附近的malleal短突（MPB）的部件。男，锤骨; MM，malleal柄。箭头，气泡在通过鼓膜看出中耳空腔。 （D）裸露锤。马力士头表示。带点线表示砧骨的关节面。 （E）的张量鼓肌肌腱（TT）连接到锤骨。当锤骨被抬起（F）的张鼓被拉动。 *，肌肉发达的过程。 （G）的张量鼓被使用针切断。 （H）去除鼓膜后3听小骨。该incudo，踝关节脱臼。男，锤骨;我，砧骨; S，镫骨;去吧，goniale（融合到锤骨和鼓膜环，TR）。所有比例尺，0.5毫米。 （A，H），P33鼠标。 （B - G），P31鼠标。 <a href="http://ecsource.jove.com/files/ftp_upload/55054/55054fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55054/55054fig3.jpg" /> 图3.砧骨和镫骨解剖。 （A）和砧骨除去锤骨的后镫骨。的镫骨动脉（SA）穿过镫骨（多个）。虚线表示砧骨的关节面。注意，该短脚砧骨（ⅰ）的（ICB，小腿杆菌）由后韧带固定（未示出）。星号，镫骨肌过程。 （B）去除砧骨镫骨后。针尖用于切割镫骨动脉（SA）。箭头，血流方向。虚线表示镫骨的关节面。 （C）中镫骨动脉从镫骨除去。 X表示镫骨动脉（SA）的切口处。 （D）卵圆窗（嗷， 卵圆窗或前庭膜孔 ）的去除镫骨后可见。 RW，圆窗（ 窗孔圆形或窗孔耳蜗 ）。比例尺，0.5毫米。 <a href="http://ecsource.jove.com/files/ftp_upload/55054/55054fig3large.jpg" target="_blank">请点击此处查看这个大版本</a>数字。 <img alt="图4" src="/files/ftp_upload/55054/55054fig4.jpg" /> 图4 为嵌入在纵向定向听泡和胶囊（矢状窦旁，A - C）和水平（D - E） 切片 锤骨。 （A - C）的颈部和横向锤骨层被平行放置嵌入盘的底部。 （A）侧视图:显微CT图像显示在泡（伪彩色蓝色）右侧锤骨嵌入。锤骨和砧骨的伪彩色绿色。虚线，所希望的切割平面。实线，嵌入菜底部。男，锤骨;箭头，背嵴。男，内侧; L，横向; D，背;五，腹。 （B）顶视图:显微CT图像。需要注意的是，除去泡（styliform过程）的前端。我，砧骨。 （C）俯视图:显微照片（与拍摄滤色器）。 AC，前（上级）半规管; SF，subarcuate窝; SP，styliform过程。 A，前壁; P，后路; D，背;五，腹。 （D - F）的锤骨的突短置于垂直于嵌入盘的底部。 （D）侧视图:显微CT图像显示正确的锤骨嵌入。虚线，所希望的切割平面。实线，嵌入菜底部。 （E）顶视图:显微CT图像。 MM，malleal柄。 （F）顶视图:显微照片（用彩色滤光片拍摄）。比例尺1毫米。在5微米的体素分辨率获得显微CT图像，如前所述7。 <a href="http://ecsource.jove.com/files/ftp_upload/55054/55054fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/55054/55054fig5.jpg" /> Figure 5.组织学。 （一）H＆E染色。在P14听觉泡（虚线）的石蜡包埋右锤（M），纵向（矢状窦旁）一节。 TM，鼓膜。 （二）骨骼钙黄绿标志。在P21听觉泡冷冻，脱钙左锤（米）的纵切面。染液，DAPI。 （C）H＆E染色。在听泡和胶囊（5周龄鼠）冷冻，脱钙离开malleal短突（MPB）的水平段。有限公司，耳蜗。比例尺1毫米。 <a href="http://ecsource.jove.com/files/ftp_upload/55054/55054fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/55054/55054fig6.jpg" /> 图6. 听骨内侧观。 （a）芘的右听小骨31鼠标。 A，前壁; P，后路; D，背;五，腹。比例尺，1毫米。锤骨头（卡普特鼻疽，头状花序鼻疽）;颈部（科勒姆鼻疽）;叶片（横向叶片）;毫米（柄鼻疽）;黑色星号（锤骨肌工艺）; MPA（前突，突纤细）; MPB（短突）;砧骨体（科珀斯克里incudis）; ICB（小腿短双歧杆菌，短脚，短流程）; ICL（小腿茇，小腿长，漫长的过程）; IPL（荚状突，透镜状过程中，西尔隆起）;镫头（卡普特stapedis）;白星号（镫肌肉工艺）; SCA（小腿前段，前小腿）; SCP（小腿后韧带，后小腿）;基地（基础stapedis，踏板）; SOF（闭孔，intercrural孔）。 （B）一个76岁的人类女性的权利听骨（解剖学教研室，庆应义塾大学医学部提供）。 P31小鼠的听小骨（右下）在相同的放大倍率成像作为用于人类听小骨。 CURV编箭头指示的解剖轴线和柄（虚线）之间的角度。比例尺，2毫米。 <a href="http://ecsource.jove.com/files/ftp_upload/55054/55054fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis at JoVE.com

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis.

在出生后的小鼠听泡解剖:中耳骨骼和组织学分析的分离

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...

dissection-auditory-bulla-postnatal-mice-isolation-middle-ear-bones

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

29.9K 次观看.  Keio University School of Medicine. We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis. 

视频: Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton&#39;s jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

The goal of this protocol is to demonstrate an effective method to decellularize and decalcify mouse cochleae for utilization as scaffolds for tissue engineering applications.

用于内耳组织工程的 Decellularizing 小鼠耳蜗协议

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...

科研

生物工程

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

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 spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the 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 ...

神经科学

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research involving the cochlea and its associated neural pathways entails exposure of model cochleae to a variety of ototoxic agents. This allows investigators to study the effects of targeted damage to cochlear structures, and in some cases, the self-repair or regeneration of those structures. Various techniques exist for delivery of ototoxic agents to the cochlea. When selecting a particular technique, investigators must consider a number of factors, including the induction of inadvertent systemic toxicity, the amount of cochlear damage produced by the surgical procedure itself, the type of lesion desired, animal survivability, and reproducibility/reliability of results. Currently established techniques include parenteral injection, intra-peritoneal injection, trans-tympanic injection, endolymphatic sac injection, and cochleostomy with perilymphatic perfusion. Each of these methods has been successfully utilized and is well described in the literature; yet, each has various shortcomings. Here, we present a technique for topical application of ototoxic agents directly to the round window niche. This technique is non-invasive to inner ear structures, produces rapid onset of reliably targeted lesions, avoids systemic toxicity, and allows for an intra-animal control (the contra-lateral ear). Results stemming from this approach have helped deeper understanding of auditory pathophysiology, cochlear cell degeneration, and regenerative capacity in response to an acute injury. Future investigations may use this method to conduct interventional studies involving gene therapy and stem cell transplantation to combat hearing loss.

Various methods exist for introducing ototoxic agents to the cochleae of animal models. Presented is a surgical protocol for delivery of ototoxic agents to the round window niche. The procedure is reliable, creates targeted intra-cochlear lesions, and avoids mechanical damage to the microarchitecture. Examination of cochlear self-repair/regeneration is possible.

老鼠圆窗途径耳毒性剂递送:一个快速和可靠的技术诱导耳蜗细胞变性

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research ...

医学

Evaluation of Planar-Cell-Polarity Phenotypes in Ciliopathy Mouse Mutant Cochlea

In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest signaling pathways shown to be associated with ciliary function was the non-canonical Wnt signaling pathway, also referred to as planar cell polarity (PCP) signaling. One of the best places in which to study the effects of planar cell polarity (PCP) signaling during vertebrate development is the mammalian cochlea. PCP signaling disruption in the mouse cochlea disrupts cochlear outgrowth, cellular patterning and hair cell orientation, all of which are affected by cilia dysfunction. The goal of this protocol is to describe the analysis of PCP signaling in the developing mammalian cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy. Defects in convergence and extension are manifested as a shortening of the cochlear duct and/or changes in cellular patterning, which can be quantified following dissection from developing mouse mutants. Changes in stereociliary bundle orientation and kinocilia length or positioning can be observed and quantitated using either immunofluorescence or scanning electron microscopy (SEM). A deeper insight into the role of ciliary proteins in cellular signaling pathways and other biological phenomena is crucial for our understanding of cellular and developmental biology, as well as for the development of targeted treatment strategies.

Primary cilia influence various signaling pathways. The mammalian cochlea is ideal for examining planar cell polarity (PCP) signaling. Cilia dysfunction affects cochlear outgrowth, cellular patterning and hair cell orientation, readouts of PCP. Our goal is to analyze PCP signaling in mouse cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy.

在Ciliopathy鼠标突变耳蜗平面 - 细胞极性表型的评价

In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest ...

Cryosectioning and Immunostaining Mouse Inner Ear Tissue: From Embryonic to Adult Stages

This protocol details general histology methods for preparing inner ear samples from embryonic, neonatal, and adult mice. The purpose of this protocol is to provide a straightforward and standardized method for inner ear tissue processing for researchers, possibly new to the field, interested in working with the mouse cochlea. Included here are protocols for dissection, fixation, embedding, cryosectioning, and immunostaining. Section immunostaining is one of the best methods for examining individual cells within the context of the entire cochlea.
Key steps include dissecting the inner ear away from the skull, fixing the tissue using 4% paraformaldehyde, embedding with Optimal Cutting Temperature compound, cryosectioning, and immunostaining with antibodies targeting specific proteins expressed by the cochlea.
Included in our methods are special considerations made for the cochlea given its 
shape and structure.&#160;Reasonably good focus and dissection skills are needed, as tissue damage can affect the quality of immunostaining and consistency among samples. However, with the procedures we outline, most users can be trained in a few weeks to make beautiful preparations. Overall, this protocol offers a valuable way to promote research to understand auditory development, function, and disease in mouse models.

This protocol, intended for novice users of mouse inner ear tissue, comprehensively details steps for processing mouse inner ears at different developmental stages for section immunostaining.

小鼠内耳组织的冷冻切片和免疫染色：从胚胎阶段到成人阶段

This protocol details general histology methods for preparing inner ear samples from embryonic, neonatal, and adult mice. The purpose of this protocol is ...

发育生物学

Cochlear Implant Surgery and Electrically-evoked Auditory Brainstem Response Recordings in C57BL/6 Mice

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of hearing. Improvement of the implant technology is needed if CI users are to perceive music and perform in more natural environments, such as hearing out a voice with competing talkers, reflections, and other sounds. Such improvement requires experimental animals to better understand the mechanisms of electric stimulation in the cochlea and its responses in the whole auditory system. The mouse is an increasingly attractive model due to the many genetic models available. However, the limited use of this species as a CI model is mainly due to the difficulty of implanting small electrode arrays. More details about the surgical procedure are therefore of great interest to expand the use of mice in CI research.
In this report, we describe in detail the protocol for acute deafening and cochlear implantation of an electrode array in the C57BL/6 mouse strain. We demonstrate the functional efficacy of this procedure with electrically-evoked auditory brainstem response (eABR) and show examples of facial nerve stimulation. Finally, we also discuss the importance of including a deafening procedure when using a normally hearing animal. This mouse model provides a powerful opportunity to study genetic and neurobiological mechanisms that would be of relevance for CI users.

Animal models of cochlear implants can advance knowledge of the technological bases of treating permanent sensorineural hearing loss with electrical stimulation. This study presents a surgical protocol for acute deafening and cochlear implantation of an electrode array in mice as well as the functional assessment with auditory brainstem response.

c57bl6 小鼠耳蜗植入术与电唤起脑干反应记录

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of ...

Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have indicated that hair cell synapses may be the initial targets in sensorineural hearing loss (SNHL). Such studies have proposed the concept of cochlear &#34;synaptopathy&#34;, which refers to alterations in ribbon synapse number, structure, or function that result in abnormal synaptic transmission between IHCs and SGNs. While cochlear synaptopathy is irreversible, it does not affect the hearing threshold. In noise-induced experimental models, restricted damage to IHC synapses in select frequency regions is employed to identify the environmental factors that specifically cause synaptopathy, as well as the physiological consequences of disturbing this inner ear circuit. Here, we present a protocol for analyzing cochlear synaptic morphology and function at a specific frequency region in adult mice. In this protocol, cochlear localization of specific frequency regions is performed using place-frequency maps in conjunction with cochleogram data, following which the morphological characteristics of ribbon synapses are evaluated via synaptic immunostaining. The functional status of ribbon synapses is then determined based on the amplitudes of auditory brainstem response (ABR) wave I. The present report demonstrates that this approach can be used to deepen our understanding of the pathogenesis and mechanisms of synaptic dysfunction in the cochlea, which may aid in the development of novel therapeutic interventions.

This manuscript describes an experimental protocol for evaluating the morphological characteristics and functional status of ribbon synapses in normal mice. The present model is also suitable for noise-induced and age-related cochlear synaptopathy-restricted models. The correlative results of previous mouse studies are also discussed.

小鼠耳蜗特定频率区域带状带突起的形态和功能评估

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have ...

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

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 ...

Dissection of the Endolymphatic Sac from Mice

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the human phenotypes. Although important progress has been done in the understanding of the development and function of the neurosensory epithelia of the cochlea and vestibula, limited knowledge is available regarding the development, cellular composition, molecular pathways and functional characteristics of the endolymphatic sac. This is, in large part, due to the difficulty of visualizing and microdissecting this tissue, which is an epithelium comprised of only one cell layer. The study presented here describes an approach to access and microdissect the endolymphatic sac from the wild-type mouse inner ear at different ages. The result of a similar dissection is shown in a pendrin-deficient mouse model of enlargement of the vestibular aqueduct. A transgenic mouse with a fluorescent endolymphatic sac is presented. This reporter mouse can be used to readily visualize the endolymphatic sac with limited dissection and determine its size. It can also be used as an educational tool to teach how to dissect the endolymphatic sac. These dissection procedures should facilitate further characterization of this understudied part of the inner ear.

<meta charset="utf8"></head><body>小鼠内淋巴囊解剖

This study describes how to dissect the endolymphatic sac from the inner ear of the mouse at different ages. The result of a similar dissection is shown in an Slc26a4-null mouse model of enlargement of the endolymphatic sac. A transgenic mouse with a fluorescent reporter expressed in the endolymphatic sac is presented as a model to readily visualize the endolymphatic sac, and educational tool.

小鼠内淋巴囊解剖

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the ...

在出生后的小鼠听泡解剖:中耳骨骼和组织学分析的分离

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用于内耳组织工程的 Decellularizing 小鼠耳蜗协议

全山解剖和成年小鼠耳蜗的免疫

老鼠圆窗途径耳毒性剂递送:一个快速和可靠的技术诱导耳蜗细胞变性

在Ciliopathy鼠标突变耳蜗平面 - 细胞极性表型的评价

小鼠内耳组织的冷冻切片和免疫染色：从胚胎阶段到成人阶段

c57bl6 小鼠耳蜗植入术与电唤起脑干反应记录

小鼠耳蜗特定频率区域带状带突起的形态和功能评估

成年小鼠中的科利耳表面制备

小鼠内淋巴囊解剖

小鼠内淋巴囊解剖