作者要感谢基因组学和神经功能的无创评价设施（IIBM，CSIC-UAM）提供的技术支持。这项工作是由西班牙"部:EconomiaŸCompetitividad"（FEDER-SAF2014-53979-R）和欧盟（FP7-AFHELO和FP7-人TARGEAR），以IVN的资助。

确保动物的处理程序是按照国际和国家法规。该协议遵循欧共体六十三分之二千零十/欧盟和西班牙的RD二千零十三分之五十三指引，分别。 1.一般动物处理<ol><li>小鼠饲料自由采食与标准饮食和饮水。控制健康和福祉按照联邦实验动物科学协会（FELASA）的建议。 </li></ol> 2.听力评估注意:手术后通过测试听力多次显微手术操作之前和跟踪功能的影响（在此工作2，7，14和28天postmicrosurgery）与非侵入性检查，如ABR 9。 <ol><li>对于ABR测试，麻醉持续时间短效果的方案，即小鼠腹腔注射氯胺酮（100毫克/公斤体重（BW）和甲苯噻嗪（10毫克/公斤，BW）。另外，执行吸入麻醉下听力测试。 注意:由于ABR参数可以由麻醉协议28的影响，使用同一个整个实验。 </li><li>通过测试脚趾捏反射检查麻醉深度。 注意:当撤回反射消失，动物已经达到了麻醉的合适的深度进行听觉测试。 </li><li>泪液补充，如羟丙基甲基纤维素基-凝胶局部给药防止脱水和继发性干燥性角结膜炎眼睛。 </li><li>保持在生理温度（37.5-38℃）小鼠在整个过程中。为了避免电气干扰，用温水泵和加热垫。监测体温直肠探头。务必注意不要过热动物。 注:我们建议您用老鼠之间的表面消毒剂清洁加热垫。0;对于麻醉诱导和恢复，电热垫，白炽灯或红外线灯都可以使用。 </li><li> ABR程序 注:对于ABR登记，使用计算机工作站的内侧声卡创建波形（数字到模拟，DA输出，转换）和数字化电响应波形（模拟到数字，AD输入），衰减器，示波器和低阻抗放大器。现代听觉工作站（ 即塔克戴维斯Techonologies）包括在一个紧凑的系统中的所有这些组件。 <ol><li>放置在声音衰减室中的加热垫俯卧位麻醉小鼠以避免环境噪声干扰和混响（ 图1）。 </li><li>提供声刺激到外耳道。使用预设的刺激或设计适当的软件新的信号。在DA工作站输出连接到选定的扬声器。 注:FRE插入在外耳道电子领域或封闭场扬声器可以使用。前者用，因为在封闭系统中探针插入和声音调校的困难小鼠时优先考虑。自由声场扬声器刺激两只耳朵和引起双耳响应。为了得到自由声场扬声器单声道为主的反应，对侧的活动必须由闭塞（ 即带有耳塞），或者通过屏蔽噪音被消除。 <ol><li>放置自由场扬声器在面向头或选定耳与外耳道对准的扬声器的中心的固定距离（通常为5-20厘米）。确保没有障碍是扬声器与耳朵之间和耳廓完全打开。 </li></ol></li><li>放置不锈钢皮下针状电极如下:ⅰ）在两耳之间的头皮的活性（正）电极（在颅骨的顶点），ii）所述参考值（负）电极，在腮腺区域耳廓的下方，以及iii）在后面，尾部或后肢区域的接地电极（ 图1）。 <ol><li>检查电阻抗在正电极和负电极。确保该阻抗小于3千欧（理想1千欧）。如果是较高的，重新定位，用酒精清洁或更换电极。 </li></ol></li><li>对于ABR记录，从相对声压级（SPL）在5-10分贝SPL步骤27，29，30 90至10分贝降低强度产生宽带的点击和纯音频率和现在。 <ol><li>目前短暂点击或音爆高的水平（ 即 80或90分贝SPL）开始，降低5-10分贝SPL措施力度的刺激（1-5毫秒）。登记在刺激后的第一个10毫秒的电响应（诱发ABR响应出现在6-8毫秒）。 注:由于这个原因，刺激利率不应高于50刺激/秒（正常速率20-50）。 </li></ol></li><li>放大，记录和平均每个刺激和强度诱发电反应。使用具有低噪声和良好的信噪比的放大器，并将其连接到AD输入。 注:ABR上具有非常低的振幅，通常低于1μV（峰 - 峰），并且必须使用的放大器具有非常低的噪声被记录。在听力正常小鼠，清晰ABR波平均100后出现- 200的反应，但获得高品质的录音，或听力障碍的情况下，多次重复推荐（750-1,000）27。 </li><li>直观地确定在测试过程中在ABR阈值。 注:ABR阈值的最低声音刺激强度上引发一个可靠的ABR浪I至IV清晰可见和中等峰-峰值电压2的SD以上的平均背景活动31的记录。这个数据得到证实杜环离线分析，与其他参数，包括峰值间潜伏期一起，和波振幅。 </li><li>手动或自动地进行数据分析。 <ol><li>对于人工分析，确定4-5 ABR波（Ⅰ，Ⅱ，Ⅲ...... 等 ）和标记的峰（P1，P2，P3 ...）和谷（N1，N2，N3 ...）对于每一波。一旦分析完成后，将数据导出到电子表格或文本文件。 注:电反应记录的特定软件通常会自动进行分析。额外的测量可以在该ABR记录来确定响应于一个固定的强度（ 即 70或80分贝声压级），或在相对于单个点击阈值高级 （ 即 15分贝声压级超过阈值）的强度。 </li></ol></li></ol></li><li>执行使用相应的软件ABR数据的统计分析。根据不同的实验设计，用标准的配对t检验或方差分析（ANOVA）的分析，以比较主要的ABR parameTER值在不同的组26,30。 注意:在纵向研究中，许多功能性数据被从同一动物收集在不同的时间点（ 即之前和显微之后）。在这种情况下，一般的线性模型重复测量试验提供了一个详细的方差分析。 </li></ol> 3.车辆准备<ol><li> 准备和无菌条件下使用的车辆解决方案。 注:液体溶液通常是通过耳咽管迅速清除。不同注射递送系统可用于增加药物的停留时间在中耳，包括水凝胶和纳米颗粒32。 <ol><li>为了准备CGP-水凝胶，解散75％的脱乙酰在0.2M的醋酸产生一个1.5-2％（重量/重量），壳聚糖溶液。 9％甘油（重量/重量）加入到该溶液7。制备溶液只是给药前和储存在4°C，直到使用水凝胶。 注:CGP-水凝胶是中等粘度，但仍然在此温度下注射。低于4℃时变为固相，阻止了其应用。施用后，CGP经历在37℃在约15分钟的相变到半固体凝胶。 </li><li>等分试样（0.5mL）中的商业RL缓冲器和储存在4℃直至使用。 </li></ol></li></ol> 4.显微手术操作<ol><li> 诱导全身麻醉用镇静剂和止痛剂的基于氯胺酮组合（ 即氯胺酮100毫克/千克，美托咪定0.05毫克/千克和phentanile 0.025毫克/公斤）通过腹膜内注射，接着吸入剂（ 即异氟烷）。 <ol><li>注射给药后剂，调整麻醉面罩鼠标鼻子和连接O 2供应异氟醚蒸气。维护期间的吸入麻醉显微，并监控与脚趾捏反射和呼吸模式的麻醉平面。手术开始时准备的反射是完全废除，鼠标呈现均匀的呼吸。 </li><li>保持体温加热垫在整个过程中并用羟丙基甲基纤维素基于凝胶保护眼睛角膜角膜炎。 </li></ol></li><li>通过使用无菌单准备一个干净的手术区域。消毒显微器械与手术前玻璃珠灭菌。保持整个外科手术过程中在无菌条件（无菌手套，窗帘，手术器械等 ）。 </li><li> 显微 <ol><li> Bullostomy 注:Bullostomy是单方面的过程。操作鼠标的一只耳朵和使用对侧耳朵作为对照。 <ol><li>鼠标放置在一个褥疮仰卧位。在使用指甲刀到颈部腹面准备手术区除去皮毛。清洁用聚维酮碘基于消毒液的皮肤上，并用无菌窗帘覆盖。 </li><li>使用手术刀，从下颌骨到锁骨做2厘米纵行切口。 </li><li>根据与手术显微镜的放大倍率，确定颌下腺和钳分开两者。缩回颌下腺和定位二腹肌的起源和面神经。 </li><li>使用剪刀二腹肌的起源切口，腹面收回它，露出了鼓泡的基本劣内侧面。 </li><li>通过钻入其与27克的针（ 图2A）使得在泡的开口。本地化镫骨动脉和RW膜尾椎它（ 图2B）。从可吸收性明胶海绵钻孔区域清洁血液。 </li><li>使用34克导管和玻璃微量注射器，缓缓注入3-5μ升通过直接bullostomy到RW利基车辆溶液（CGP-水凝胶或RL），填充它（ 图2C）。轴封采用组织粘合剂1-2滴的bullostomy。 </li><li>返回颌下腺其初始位置，然后关闭皮肤切口用5-0丝线缝合手术。在一台基于洗必泰切口周围防腐剂，以避免伤口感染。注意:可以使用可吸收性和非吸收性缝线。不可吸收的缝合线，必须在2周内被移除。不推荐用于皮肤缝合丝，因为它的使用与切口感染和局部组织反应有关。 </li></ol></li><li>双侧鼓室注射<ol><li>鼠标放置在侧卧位并如4.3.1.1中所述制备外耳道下面无菌手术区。 </li><li>使将0.5cm纵向切口在外耳道的靠近耳屏和s的垂直部挠度耳廓（可选）的内部皮肤褶皱。 </li><li>定位在使用手术显微镜（ 图2E）的外耳道的端部的鼓膜，并确定上部鼓膜松弛部和下收杆tensa，这是由锤骨的柄分为前部和后部部分（ 图2F） 。 </li><li>使在鼓膜松弛部的尾侧的小myringostomy。进行额外的切口在鼓膜的收杆tensa，以允许注射33中的空气排空。轻轻注入10-15微升车辆（CGP-水凝胶或RL）通过所述鼓膜松弛部 ，靠近RW利基直到中耳显然充分连接到34克的导管的玻璃微量注射器的解决方案。 </li><li>用5-0丝线缝合手术闭合皮肤切口和清洁如4.3.1.7所述。 </li><li>把鼠标放在它的另一边，OPER吃了对侧耳朵（步骤4.3.2.1至4.3.2.5）。 </li></ol></li></ol></li><li>按住鼠标上一个加热垫，直到它已经恢复了足够的意识，以保持胸骨斜卧。不要返回已动过手术，以公司的其他动物，直到完全康复的动物。 </li><li>监视身体状况，活性和疼痛或压力的迹象的存在。如果需要的话提供止痛剂（ 即丁丙诺啡0.05毫克/公斤，卡洛芬5-10毫克/千克）。日常检查手术伤口和皮肤去除关闭验证的伤口已经愈合后术后7-14天。 </li></ol> 5.耳蜗细胞构筑的形态学评估<ol><li>安乐死鼠标在实验（在该工作28天postmicrosurgery）的端部，用腹膜内戊巴比妥过量（100毫克/千克）以研究在手术的长期影响。 </li><li>执行冷0.1M的磷酸盐缓冲一个穿心灌注盐水（PBS），pH为7.5，在0.1M PBS中，随后4％（重量/体积）低聚甲醛（PFA），pH值7.5所描述的26。 注意:多聚甲醛是高毒;避免与皮肤，眼睛或粘膜接触。避免测量和准备过程中呼吸的粉末。 </li><li>使用立体显微镜如所述34,35不分离前庭和内耳的耳蜗部件从颞骨解剖出来的内耳。 </li><li>用4％（重量/体积）的PFA固定的分离的内耳在0.1M PBS中，pH为7.5，在4℃下轻轻摇动12小时。洗涤3×5分钟，用0.1M的PBS，pH为7.5。 </li><li>脱钙用在0.1M PBS中，pH值6.5，在4℃下进行10天持续振荡制备的10％乙二胺四乙酸（EDTA）的样品，改变EDTA溶液每3天。 </li><li>当耳蜗获得柔软一致性，除去EDTA和用0.1M PBS中，pH值7.5，无线洗3次5分钟日在室温下晃动。 </li><li>嵌入到石蜡样品描述34并平行于蜗轴7微米厚的耳蜗部分。 </li><li>为了评估耳蜗细胞结构，苏木和伊红（H＆E）染色30节和使用连接到数码相机的光学显微镜与4X和20X的镜头捕捉到的图像。 </li></ol> 6.耳蜗基因表达<ol><li>清洁工作表面和手术器械用核糖核酸酶净化解决方案。 </li><li>如5.1中所述，并迅速从使用显微镜的颞骨解剖出内耳安乐死鼠标。浸泡在含有核糖核酸（RNA）的保护和稳定剂的试剂的玻璃皿内耳。 </li><li>删除与珠宝商镊子剩下的岩骨，轻轻耳蜗从前庭使用万纳的眼科剪35分开。 </li><li>立即TRANSF呃耳蜗与80微升的RNA保护和稳定剂溶液2毫升的离心管，并通过将所述管在干冰冷冻组织。在-70℃下直到使用保存耳蜗样品。 </li><li>隔离耳蜗RNA为描述35，并确定其质量和数量分光光度计。 </li><li>产生从等量的总小鼠的RNA利用逆转录商业试剂盒耳蜗的cDNA。 </li><li>执行定量RT-PCR一式三份扩增互补脱氧核糖核酸（基因）来衡量基因转录35,36。 注:在此工作和亲IL1B，IL6，TGFB1，肿瘤坏死因子，IL10和DUSP1抗炎基因转录进行了测量。 </li><li>通过归一靶转录循环阈值（CT）的水平与参考基因水平的算术平均值和由规格化相对定量计算相对表达比问题组的转录水平的校准器组37的算术平均值。 </li></ol>

The authors declare that they have no competing financial interests.

局部药物递送至内耳可通过鼓室内施用直接由耳蜗内注射或间接完成，将药物在中耳4，19，39。耳蜗内给药提供受控和精确的药物递送至耳蜗，避免扩散通过窗膜，基底到心尖浓度梯度和清除通过咽鼓管。然而，这通常是需要复杂的和精致的显微7，39高度侵入性手术。在此背景下，业界正在开发新的，包衣，可植入的持续释放药物40,41装置。另一方面，鼓室内施用是一种微创且易于操作过程，它允许更大的对d的卷的注射地毯进入中耳，虽然药物动力学是不容易控制。大多数药物通过咽鼓管清零，剩余部分具有穿过RW膜扩散到达耳蜗18。 RW是从中耳到耳蜗7的外淋巴填充鼓膜导管的物质的最大吸收的位点。它是半透的三层结构，虽然它的渗透性取决于药物特性（大小，浓度，溶解度和电荷）和跨膜运输系统（扩散，主动转运或吞噬作用）42。卵圆窗和耳胶囊的替代，但效果较差入口耳蜗43，44。 这里，我们证明并比较靶向给药到鼠标中穗两显微方法:bullostomy和transtympaNIC注入程序。这些程序共同关键步骤包括:i）听力的评估之前和显微后，ⅱ）制备在无菌条件下均相车辆的溶液，iii）所述麻醉过程和动物体温和常数的监测的仔细监督，四）缓慢车辆靶向RW的适当体积的位置，以及iv）采取耳蜗样品来完成分子和形态学分析。 为bullostomy耳和腹侧的方法已被描述7，45。我们所使用的腹侧近似，因为在我们的经验，已经导致更少的发病率，并提供给RW 46更好的访问。经鼓室注射通常通过睫状tensa鼓膜进行，前或后，以锤骨柄12。在这项工作我们进行了技术，注入的修改，通过以后的标准杆tensa的前一个额外的穿刺锤骨的鼓膜松弛部允许在注射过程中的空气排出。 该鼓室注射比bullostomy创伤更小，虽然两者microsurgeries是快速（每穗20和5分钟的bullostomy分别鼓室办法），有短，术后康复时间，并没有发病。最重要的是，这两个过程保持听觉和该ABR参数的那些相同的显微之前确定的。在经鼓室途径花费较少的时间比bullostomy并且可以在相同的动物的两只耳朵相同介入期间进行。所述经鼓室注射的优点是由此，如果需要，可以双边进行和重复。另一方面，bullostomy提供给RW膜直接视觉获取并且允许Filli酒店该RW小生纳克。与此相反，经鼓室注射不允许在RW利基车辆放置的控制。 在这项工作报告的过程描述如何执行局部药物交车中耳临床前应用，如听力损失耳毒性和疗效评价的评价。两个显微过程描述了提供具有特定的优点和缺点的替代方法。这两种保护听力，并且不会导致形态学改变。局部炎症被描述为bullostomy的潜在并发症。一系列的补充技术也为术后程序，包括听证会，形态和炎症标记物表达的评估说明。对这些技术的未来的应用包括新疗法的临床前评价听力损失，包括遗传，细胞和药理学方法，在动物模型中。鼓室内管理的程序离子保证治疗的中耳的传递，在与圆窗膜接触，促进通路进入淋巴而不明显耳蜗损伤。

听力损伤是最常见的人类感觉缺陷并影响全世界人口的5.3％，和65岁以上的个体的30％（ <a href="http://www.who.int/topics/deafness/en">http://www.who.int/topics/deafness/en</a> ，更新2016）。听力损失会影响儿童语言习得和加速老年人认知能力下降。因此，它是一个显著保健问题具有巨大的社会和经济影响。它可以通过遗传缺陷，环境因素或两者1，这在端诱导的耳蜗损伤和毛细胞和神经元的死亡的组合引起的。这些细胞并不在哺乳动物中再生，因此细胞损失和随之而来的听力损失不得转回。临床选项是基于假体装置，包括助听器人工耳蜗，中耳骨导植入体2。不幸的是，没有任何具体的医疗恢复TREA为听力障碍tments，因此一些研究线路主要集中在预防和修复疗法的发展。新的治疗选择包括基因和细胞疗法以及小分子的发展为药物治疗2。 一个人工耳蜗药物治疗最重要的挑战是给药。全身性治疗方法已在耳蜗功效由于血-迷路屏障3的限制，连续内皮与耳蜗的血管，其作为一个物理和生化屏障保持内耳流体稳态，因此限制了药物通道到内耳接触。它是可渗透只有小的脂溶性的分子，尽管渗透性可以耳蜗炎症过程中与使用利尿剂或渗透剂的增加，并且也。药物，最终达到全身给药降低后耳蜗的体积;因此，需要高剂量，可能导致有机毒性。此外，药物的肝代谢可产生有毒的或无活性的代谢物4，5，6，7。相比之下，局部干预允许药物的已知数量有限到中耳或内耳的无不良副作用4，7，8，9的位置。在目前的临床实践中，鼓室内施用限于某些耳蜗病状，如在美尼尔氏病10庆大霉素，突发性耳聋的皮质类固醇，美尼尔氏病，免疫介导的和噪声引起的听力损失，11，12，13，1突发性耳聋4，16，17 4，15和胰岛素样生长因子1（IGF1）。 局部给药的制剂应保留耳蜗流体的微妙平衡（pH值和渗透压）。此外，这是非常重要的，以保持在整个过程中不育，以避免脑脊液细菌污染。用于药物递送的赋形剂应该是生物相容的，nonototoxic和适当的一致性。液体溶液被推荐用于耳蜗内注射，但不适合于在鼓室内路线由于通过咽鼓管的间隙。在这种情况下，药物通常通过半固体凝胶进行以提高其耐久性在中耳4，18，19。替代交货SYSTE用作载体，以增加药物的通路到内耳毫秒是纳米颗粒20和腺病毒21在这里，我们比较了两种车辆:CGP和RL溶液。 CGP是由脱乙酰壳多糖形成的凝胶，D-葡糖胺和从甲壳动物贝壳得到的N-乙酰基-D-葡糖胺，和β-甘油磷酸盐，形成围绕壳聚糖链的水的屏蔽，并保持它在多元醇组成的直链多糖液体形式。 CGP是热敏并且可以由溶菌酶降解，允许在中耳22，23，24，25一个持续的药物释放。壳聚糖基水凝胶进行临床应用，例如药物递送合适的车辆，因为它们缺乏免疫原性，并缺乏局部炎症反应23,24的激活的。在OTH呃手，RL缓冲器是用于静脉内给药在人类中无热原的等渗溶液（273毫渗透摩尔浓度/ L和pH为6.5），为水和电解质源，尤其是在失血，创伤或烧伤，因为乳酸代谢的副产物在肝脏抵消酸中毒。 在这里，我们描述和比较已细化为当地的药物输送到小鼠内耳两种手术方法。这两种技术的安全性，通过使用功能性的，形态学和分子测试评价。采用听性脑干反应（ABR）26，之前在不同的时间后，显微进行27日讯评价。终点程序被用来解剖耳蜗并比较这两个显微程序的解剖，细胞和分子的影响。

<table border="0" cellpadding="0" cellspacing="0" width="1375">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:455px;">Ketamine (Imalgene)</td>
			<td style="width:251px;">Merial</td>
			<td style="width:207px;"># 2529</td>
			<td style="width:464px;">CAUTION: avoid contact of the drug with skin or eyes or accidental self-inflicted injections</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Xylacine (Xilagesic)&#160;</td>
			<td style="width:251px;">Calier</td>
			<td style="width:207px;"># 6200025225</td>
			<td style="width:464px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Lubricant eye gel (Artific)</td>
			<td style="width:251px;">Angelini</td>
			<td style="width:207px;"># 784710</td>
			<td style="width:464px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Water pump&#160;</td>
			<td style="width:251px;">Gaymar</td>
			<td style="width:207px;"># TP472</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Subdermal needle electrodes&#160;</td>
			<td style="width:251px;">Spes Medica</td>
			<td style="width:207px;"># MN4013D10SM</td>
			<td style="width:464px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Low Impedance Headstage&#160; (RA4LI)</td>
			<td style="width:251px;">Tucker-Davis Technologies</td>
			<td style="width:207px;"></td>
			<td style="width:464px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Speakers (MF1 Multi-Field Magnetic Speaker)</td>
			<td style="width:251px;">Tucker-Davis Technologies</td>
			<td style="width:207px;"></td>
			<td style="width:464px;"></td>
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		<tr height="64">
			<td height="64" style="height:64px;width:455px;">System 3 Evoked Potential Workstation</td>
			<td style="width:251px;">Tucker-Davis Technologies</td>
			<td style="width:207px;"></td>
			<td style="width:464px;">The System is composed of: RP2 processor, RA16 base station, PA5 attenuator, SA1 amplifier, MA3 microphone amplifier, RA4LI impedance headstage and RA4A medusa pre-amplifier&#160;</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:455px;">SigGenRP software</td>
			<td style="width:251px;">Tucker-Davis Technologies</td>
			<td style="width:207px;"></td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Warming pads (TP pads)</td>
			<td style="width:251px;">Gaymar</td>
			<td style="width:207px;"># TP3E</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Statistics software (SPSS)</td>
			<td style="width:251px;">IBM</td>
			<td style="width:207px;"></td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Chitosan (deacetylated)</td>
			<td style="width:251px;">Sigma-Aldrich</td>
			<td style="width:207px;"># C3646</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:455px;">Acetic acid (glacial)</td>
			<td style="width:251px;">VWR</td>
			<td style="width:207px;"># 20103.295</td>
			<td style="width:464px;">CAUTION: flammable liquid, skin corrosion and respiratory and skin sensitizer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Glycerophosphate</td>
			<td style="width:251px;">Sigma</td>
			<td style="width:207px;"># SLBG3671V</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Ringer&#180;s lactate buffer</td>
			<td style="width:251px;">Braun</td>
			<td style="width:207px;"># 1520-ESP</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Medetomidine (Domtor)</td>
			<td style="width:251px;">Esteve</td>
			<td style="width:207px;"># 02400190</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:455px;">Phentanile (Fentanest)</td>
			<td style="width:251px;">Kern Pharma</td>
			<td style="width:207px;"># 756650.2</td>
			<td style="width:464px;">CAUTION: &#160;&#160;avoid contact of the drug with open wounds or accidental self-inflicted injections</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:455px;">Isoflurane (IsoVet)</td>
			<td style="width:251px;">Braun</td>
			<td style="width:207px;"># 469860</td>
			<td style="width:464px;">CAUTION: Avoid exposures at ceiling concentrations greater than 2ppm of any halogenated anesthetic agent over a sampling period not to exceed one hour.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Surgical microscope (OPMI pico)</td>
			<td style="width:251px;">Zeiss</td>
			<td style="width:207px;"></td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Sterile drape (Foliodrape)</td>
			<td style="width:251px;">Hartmann</td>
			<td style="width:207px;"># 277546</td>
			<td style="width:464px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Sterilizer&#160;</td>
			<td style="width:251px;">Fine Science Tools</td>
			<td style="width:207px;"># 18000-45</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Scalpel blade</td>
			<td style="width:251px;">Swann Morton</td>
			<td style="width:207px;"># 0205</td>
			<td style="width:464px;">CAUTION</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Scalpel handle</td>
			<td style="width:251px;">Fine Science Tools</td>
			<td style="width:207px;"># 91003-12</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Pividone iodine based antiseptic (Betadine)</td>
			<td style="width:251px;">Meda Pharma SAU</td>
			<td style="width:207px;"># M-12207</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Adventitia scissors (SAS18-R8)</td>
			<td style="width:251px;">S&#38;T</td>
			<td style="width:207px;"># 12075-12</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Curved scissors</td>
			<td style="width:251px;">CM Instrumente</td>
			<td style="width:207px;"># AJ023-18</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Forceps</td>
			<td style="width:251px;">CM Instrumente</td>
			<td style="width:207px;"># BB019-18</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Gelatine sponge (Spongostan)</td>
			<td style="width:251px;">ProNaMAc</td>
			<td style="width:207px;"># MS0001</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Microlance 27G</td>
			<td style="width:251px;">Becton Dickinson</td>
			<td style="width:207px;"># 302200</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Microliter syringe (701 RN SYR)</td>
			<td style="width:251px;">Hamilton</td>
			<td style="width:207px;"># 80330</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Catheter (Microfil 34G)</td>
			<td style="width:251px;">World Precision Instruments&#160;</td>
			<td style="width:207px;"># MF34G-5</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Tissue Adhesive (Vetbond)</td>
			<td style="width:251px;">3M</td>
			<td style="width:207px;"># 1469SB</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Needle holder (Round handled needle holder)&#160;</td>
			<td style="width:251px;">Fine Science Tools</td>
			<td style="width:207px;"># 12075-12</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Silk surgical suture (Braided Silk 5/0)</td>
			<td style="width:251px;">Arago</td>
			<td style="width:207px;"># 990011</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Chlorhexidine (Cristalmina)</td>
			<td style="width:251px;">Salvat</td>
			<td style="width:207px;"># 787341</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:455px;">Pentobarbital&#160; (Dolethal)</td>
			<td style="width:251px;">Ventoquinol</td>
			<td style="width:207px;"># VET00040</td>
			<td style="width:464px;">CAUTION: &#160;&#160;avoid contact of the drug with open wounds or accidental self-inflicted injections</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Stereomicroscope (Leica)</td>
			<td style="width:251px;">Meyer Instruments</td>
			<td style="width:207px;"># MZ75</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Vannas Micro-dissecting (Eye) Scissors Spring Action</td>
			<td style="width:251px;">Harvard Apparatus</td>
			<td style="width:207px;"># 28483</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Jeweller&#8217;s forceps (Dumont)</td>
			<td style="width:251px;">Fine Science Tools</td>
			<td style="width:207px;"># 11252-00</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">RNase Decontamination Solution&#160; (RNaseZap)</td>
			<td style="width:251px;">Sigma-Aldrich</td>
			<td style="width:207px;"># R2020</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">RNA Stabilization Solution&#160; (RNAlater)</td>
			<td style="width:251px;">Thermo Fisher Scientific</td>
			<td style="width:207px;"># R0901</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Purification RNA kit (RNeasy)</td>
			<td style="width:251px;">Qiagen</td>
			<td style="width:207px;"># 74104</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">cDNA Reverse Transcription Kit</td>
			<td style="width:251px;">Thermo Fisher Scientific</td>
			<td style="width:207px;"># 4368814</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="22">
			<td height="154" rowspan="1" style="height:154px;width:455px;">Gene expression assay (TaqMan probes)</td>
			<td rowspan="1" style="width:251px;">Thermo Fisher Scientific</td>
			<td height="22" style="height:22px;width:207px;">Il1b: Mm00446190_m1 
			Il6: Mm00446190_m1 
			Tgfb1: Mm01178820_m1 
			Tnfa: Mm99999068_m1 
			Il10: Mm00439614_m1 
			Dusp1: Mm00457274_g1 
			Hprt1: Mm00446968_m1</td>
			<td rowspan="1" style="width:464px;"></td>
		</tr>
		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:455px;">Real-time PCR System (7900HT)</td>
			<td rowspan="2" style="width:251px;">Applied Biosystems</td>
			<td rowspan="2" style="width:207px;"># 4329001</td>
			<td rowspan="2" style="width:464px;"></td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Paraformaldehyde (PFA)</td>
			<td style="width:251px;">Merck</td>
			<td style="width:207px;"># 1040051000</td>
			<td style="width:464px;">TOXIC: PFA is a potential&#160;carcinogen</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:455px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:251px;">Merck</td>
			<td style="width:207px;"># 405491</td>
			<td style="width:464px;">CAUTION:&#160; harmful if inhaled, may cause damage to respiratory tract through prolonged or repeated exposure if 
			inhaled.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Hematoxylin solution</td>
			<td style="width:251px;">Sigma-Aldrich</td>
			<td style="width:207px;"># HHS16</td>
			<td style="width:464px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:455px;">Eosin Y</td>
			<td style="width:251px;">Sigma-Aldrich</td>
			<td style="width:207px;"># E4382</td>
			<td style="width:464px;">Hazards: causes serious eye irritation</td>
		</tr>
	</tbody>
</table>

a comparative study of drug delivery methods targeted to the mouse inner ear: bullostomy versus transtympanic injection

我们提出在啮齿类动物中对特定药物递送进入中耳2微创显微外科技术，使得它有可能达到内耳。第一个过程由鼓泡，被称为bullostomy穿孔;第二个是一个经鼓室注射。这两种模拟人体临床鼓室内手续。 壳聚糖 - 甘油磷酸盐（CGP）和Ringer&#39;s乳酸缓冲液（RL）被用作局部药物递送的生物相容的车辆。 CGP是无毒的可生物降解的聚合物广泛用于药物应用。它是在RT粘性液体，但它在体温下凝结成半固体相。 RL是用于人类静脉内给药的等渗溶液。这款车的一小容量由bullostomy的手段恰恰放在圆窗（RW）的利基。一经鼓膜注射填充中耳和允许控制少，但内耳更广泛的访问。 这两个程序都适合作为给药方式到小鼠中耳，虽然经鼓膜注射证明相比bullostomy是微创。

听力由ABR之前和在几次显微后进行测试，以评估对听觉功能（ 图1A）产生的影响。 ABR寄存器麻醉下进行，以避免动物的运动和电压文物，从而提高其可重复性27。腹腔注射氯胺酮基于组合或inhalatory异氟烷通常采用在ABR测试麻醉动物。氯胺酮/甲苯噻嗪的组合提供了一个短效（2-3分钟）诱导和稳定的，安全的维护阶段，同时执行ABR寄存器。应当指出的是异氟醚可以影响的ABR测量灵敏度38。对ABR寄存器，真皮下电极被放置在特定的位置（ 图1B）和电阻抗测量。如果阻抗是3欧姆或更高，电极定位必须检查，以避免中高音操作在ABR波幅度。 鼓室内递送在小鼠中由两个显微程序（ 图2）执行。 bullostomy在泡的曝光涉及颌下腺和二腹肌回缩。此过程是非常小心进行，因为在颈动脉和迷走神经是非常接近（ 图2A）。接着，将泡钻出本地化镫骨动脉和RW膜（ 图2B）。为了避免开裂骨，小0.5毫米孔钻井前的27克针的。在34克导管通过朝向RW膜和体积小的车辆的bullostomy定向被输送到所述窗龛（ 图2C）。该经鼓室注射是通过在标准杆的切口进行松弛部与27克针鼓膜;一个较大的一个可引发耳的膜。在注射前，我们建议在睫状tensa作出额外的切口，以使车辆（ 图2F）的注射期间空气的流出。关键是要避免的镫骨动脉，颈内动脉的分支，这将导致危及生命的出血的损伤。 小鼠保留在整个实验中，类似于非手术对照（ 图3）听力bullostomy或鼓室手术。响应于点击次数和音调脉冲串ABR阈值没有显著显微后相比基线值的变化。 bullostomy和鼓室方法之间未见显著差异。形态学研究进行了确认正确车辆输送到中耳，并评估所引起的耳蜗细胞构筑程序的电位变化。主要耳蜗区域S的无从两个程序howed形态上的变化和动物提供的所有耳蜗结构（ 图4A）的形态相似。此外，对于亲和抗炎细胞因子的基因表达的耳蜗型材进行了研究。尽管缺乏两个程序之间的ABR数据的功能差异，bullostomy引起比经鼓室途径（ 图4B）更强的炎症反应。 <img alt="图1" src="/files/ftp_upload/54951/54951fig1.jpg" /> 图1.实验设计和听力评估。实验过程中（A）图。听证会是之前和显微后ABR评估。显微28天后获得人工耳蜗样品。 （B） 在超过一个声音衰减室内部的加热垫俯卧麻醉小鼠，真皮下electrodES在头骨（主动，积极）的顶点两耳之间放置在头皮上;在下面的耳廓（参考，负）腮腺区域，并在背面（地）。自由场扬声器被放置在面对右耳一个固定的距离（5厘米）。 <a href="http://ecsource.jove.com/files/ftp_upload/54951/54951fig1large.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/54951/54951fig2.jpg" /> 图2.显微车辆的应用。 （ 一 ） 鼓泡腹面。该bullostomy执行尾面神经与27克针。 （B）的RWN和镫骨动脉可通过穿孔被观察到。 （C）的34克导管通过朝向RW利基bullostomy定向。 （ 四 ）bullostomy一个月后，小骨瘢痕中存在的开口部位（箭头）。 （E）的耳侧视图，示出了在外耳道和切口鼓膜（正方形）。 （F）详细鼓膜。一种穿刺在使用27克针鼓膜尾上腹发（黑色星号，在鼓膜松弛部 ）;注射是使用34克导管通过该穿孔制成。一个附加的孔是在该膜的颅下象限到喷射制成（白星号，在睫状tensa）之前平衡鼓室压力。通过鼓膜穿刺的34克导管（G）查看。显微外科手术后耳蜗区域24小时的（H）查看。 RWN填充有载体溶液（星号）。纬度，侧;罗，喙;这样做，背;马，锤骨;有限公司耳蜗; OW，椭圆形窗口; RWN，圆窗龛。比例尺= 200微米，A，D，F;比例尺=<html> 100微米的B，C，H;比例尺= 1000微米，E，G。 <a href="http://ecsource.jove.com/files/ftp_upload/54951/54951fig2large.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/54951/54951fig3.jpg" /> 图3.听力评估。 ABR阈值（平均值±SEM，以dB SPL）响应的进化点击（A）和音调脉冲串（B）的刺激，前和7，显微外科手术中雄性八周龄C57BL / 6J后14天和28天老鼠。 Bullostomy（橙色; N = 11）;经鼓室注射（蓝色; N = 6）;非操作（灰色; N = 11）， <a href="http://ecsource.jove.com/files/ftp_upload/54951/54951fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> JPG"/&gt; 图4.耳蜗形态和基因表达分析。在耳蜗底部的主耳蜗结构（A）形态。从非操作小鼠中期蜗轴石蜡切片（7微米），耳的，和小鼠一个月后由bullostomy或经鼓室注射显微干涉Haematoxilin精 - 曙红染色。 中阶仓（A，B，C）介绍所有的主要组件。每个这些结构（编号箱）的细节示于随后的图片:螺旋神经节（1）中，科尔蒂的器官（2），螺旋韧带（3）和血管纹（4）。内毛细胞（星号）;外毛细胞（箭头的头）。比例尺= 100微米的，B，C;比例尺=在一个-1,2,3,4- 50微米。 （ 二 ）炎症标志物显微外科手术后28天耳蜗的表达。 bullostomy（橙色）和经鼓室注射（蓝色）之间和非手术小鼠（白色）的比较。 *:非操作与手术组; ^:工作组间比较。基因表达水平被表示为2 - ΔΔCT，或n倍相对于非操作group.Values的差表示为平均一式三份的平均值±SEM从每种条件的3只小鼠的池样品。统计意义:** P≤0.01; *** P≤0.001; ^^ p≤0.01; ^ ^ ^ p≤0.001。 <a href="http://ecsource.jove.com/files/ftp_upload/54951/54951fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection at JoVE.com

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. ...

a-comparative-study-drug-delivery-methods-targeted-to-mouse-inner-ear

Research

JoVE Journal

Biology

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

13.1K 次观看.  Instituto de Investigaciones Biomédicas (IIBm) Alberto Sols CSIC-UAM. 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.

视频: A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

格罗斯和精细解剖内耳感觉上皮细胞在成年斑马鱼（斑马鱼）

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

Methods for the Study of the Zebrafish Maxillary Barbel

Barbels are skin sensory appendages found in fishes, reptiles and amphibians. The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal pair and a longer maxillary pair. Barbel tissue contains cells of ectodermal, mesodermal and neural crest origin, including skin cells, glands, taste buds, melanocytes, circulatory vessels and sensory nerves. Unlike most adult tissue, the maxillary barbel is optically clear, allowing us to visualize the development and maintenance of these tissue types throughout the life cycle. 
This video shows early development of the maxillary barbel (beginning approximately one month post-fertilization) and demonstrates a surgical protocol to induce regeneration in the adult appendage (&gt;3 months post-fertilization). Briefly, the left maxillary barbel of an anesthetized fish is elevated with sterile forceps just distal to the caudal edge of the maxilla. A fine, sterile spring scissors is positioned against the forceps to cut the barbel shaft at this level, establishing an anatomical landmark for the amputation plane. Regenerative growth can be measured with respect to this plane, and in comparison to the contralateral barbel. Barbel tissue regenerates rapidly, reaching maximal regrowth within 2 weeks of injury.
Techniques for analyzing the regenerated barbel include dissecting and embedding matched pairs of barbels (regenerate and control) in the wells of a standard DNA electrophoresis gel. Embedded specimens are conveniently photographed under a stereomicroscope for gross morphology and morphometry, and can be stored for weeks prior to downstream applications such as paraffin histology, cryosectioning, and/or whole mount immunohistochemistry. These methods establish the maxillary barbel as a novel in vivo tissue system for studying the regenerative capacity of multiple cell types within the genetic context of zebrafish.

The zebrafish maxillary barbel is an integumentary sense organ containing ectodermal, mesodermal and neural crest derivatives. Importantly, the adult barbel can regenerate after proximal amputation. This video introduces maxillary barbel development and demonstrates a surgical protocol to induce regeneration, followed by collection, embedding and downstream imaging of barbel specimens.

上颌Barbel斑马鱼的研究方法

Barbels are skin sensory appendages found in fishes, reptiles and amphibians.  The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal ...

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.

A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear.

通过注射和电穿孔的斑马鱼Otocyst的MIF Morpholinos直接运抵影响内耳发展

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the ...

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions1,2. Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer.
The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma3. This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals4. The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators.
Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

高通量原位杂交方法表征在胎鼠下泌尿生殖道的基因表达模式

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

导管内注射局部给药的小鼠乳腺

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once the pre-cursor round cell begins to elongate and differentiate into what is morphologically recognized as a spermatozoon. However, the spermatozoon is very immature, having no ability for motility or egg recognition. Both of these events occur once the spermatozoa transit a secondary organ known as the epididymis. During the ~12 day passage that it takes for a sperm cell to pass through the epididymis, post-translational modifications of existing proteins play a pivotal role in the maturation of the cell. One major facet of such is protein phosphorylation. In order to characterize phosphorylation events taking place during sperm maturation, both pure sperm cell populations and pre-fractionation of phosphopeptides must be established. Using back flushing techniques, a method for the isolation of pure spermatozoa of high quality and yield from the distal or caudal epididymides is outlined. The steps for solubilization, digestion, and pre-fractionation of sperm phosphopeptides through TiO2 affinity chromatography are explained. Once isolated, phosphopeptides can be injected into MS to identify both protein phosphorylation events on specific amino acid residues and quantify the levels of phosphorylation taking place during the sperm maturation processes.

Proteomic analysis of any cell type is highly dependent on both purity and pre-fractionation of the starting material in order to de-complexify the sample prior to liquid chromatography mass spectrometry (MS). By using back-flushing techniques, pure spermatozoa can be obtained from rodents. Following digestion, phosphopeptides can be enriched using TiO2.

啮齿动物附睾精子磷酸分析

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

胚胎干细胞的跨内细胞质量注射导致嵌合体率增高

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the host chromosome and is passed on to progeny. Thus, they are ideal vectors for creation of stable cell lines, in vivo delivery of indicators, and transduction of single cell fertilized eggs to create transgenic animals. However, mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against lentiviral gene delivery. Lentiviruses are too large to penetrate the zona and are typically delivered by microinjection of viral particles into the perivitelline cavity, the space between the zona and the embryonic cells. The requirement for highly skilled technologists and specialized equipment has minimized the use of lentiviruses for gene delivery to mouse embryos. This article describes a protocol for permeabilizing the mouse fertilized eggs by perforating the zona with a laser. Laser-perforation does not result in any damage to embryos and allows lentiviruses to gain access to embryonic cells for gene delivery. Transduced embryos can develop into blastocyst in vitro, and if implanted in pseudopregnant mice, develop into transgenic pups. The laser used in this protocol is effective and easy to use. Genes delivered by lentiviruses stably incorporate into mouse embryonic cells and are germline transmittable. This is an alternative method for creation of transgenic mice that requires no micromanipulation and microinjection of fertilized eggs.

Mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against gene delivery. This article describes a protocol for perforating the zona with a laser to transduce embryonic cells with lentiviral vectors and to create transgenic mice.

激光辅助慢病毒基因传递到小鼠受精卵

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

原发性小鼠视网膜色素上皮细胞的有效解剖与培养

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...