Name: the mouse round-window approach for ototoxic agent delivery: a rapid and reliable technique for inducing cochlear cell degeneration
Uploaded: 2015-11-26T13:00:00.000+00:00
Duration: 12 min 21 s
Description: Watch this Scientific Journal Video about The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration at JoVE.com

This work was supported by National Institutes of Health (NIH) Grant number: NIH P50DC00422 (H.L.); NIH R01DC12058 (H.L.). This work also benefitted from the South Carolina Clinical and Translational Research Institution (SCTR) Clinical and Translational Science Award (NIH/NCRR UL1RR029882). The funders had no role in study design, data collection, and/or analysis. The authors would like to thank Lonnie E. Brown Jr. for his artistic and graphic contributions.

动物研究的所有方面均按照相应的机构动物护理和使用委员会的指导下进行。这里描述的所有脊椎动物实验方法下，南卡罗来纳州（MUSC）机构动物护理和使用委员会的医科大学（IACUC）的指导方针获得批准。 1.选型<ol><li>保持在一个低噪声动物饲养，每体制协议常规照料直到准备使用的动物模型。在动物研究设施（ARFs），保持振动稳定性，减震降噪，昼夜照明，准备空间，表面处理，密封和填缝，并通风符合NIH标准。 注:ARFs和维护的低噪声动物饲养国立卫生研究院（NIH）的指导方针可以在审查: http://www.orf.od.nih.gov/PoliciesAndGuidelines/BiomedicalandAnimalResearchFacilitiesDesignPoliciesandGuidelines/ </li><li>对于所有的实验中，用右耳为实验耳。左耳用作内动物的控制，而不是手术改变。 </li><li>检查模型动物预可操作为中间或外耳感染的迹象。潜在的症状可能包括引流液，或脓液从耳，耳廓发炎的组织，和/或嗜睡的动物。这是不寻常的，但如果说，避免进一步的手术治疗和适当处理的动物。 </li></ol> 2.术前手续<ol><li>麻醉动物30分钟在手术前和通过腹膜内（IP）注射氯胺酮（100mg / kg的IP）和赛拉嗪（20毫克/千克IP）中任围手术程序。补充麻醉必要时，作为判断的正脚趾捏反射，用氯胺酮（25毫克/千克IP）和赛拉嗪（5mg / kg的IP）的较低剂量。确定给药符合的小鼠在剂量年龄适当的调整，从制度上允许的协议。 </li><li>检查完整的Sedation模型。检查麻醉的阶段3平面为带标记的常规呼吸速率，缺乏翻正反射（在小鼠中），并且缺乏踏板及眼睑的（脚趾捏）反射所描述的协议的全部内容。维持动物在麻醉的这个水平。这是极为重要的疼痛和术中运动，出血，和间质液的泄漏的手术过程中的最小化。 </li><li>保持动物的体温在37.5℃下用闭环加热垫。 </li><li>通过皮下注射丁丙诺啡辖镇痛。交付丁丙诺啡（0.1毫克/公斤SQ一次30分钟手术前）为镇痛，以尽量减少手术的不适。基本剂量和选择适合的制度上批准的方案选择的模式。抗生素的使用是没有必要的，如果良好的无菌技术已经施行。动物接受术后镇痛，如果有疼痛和痛苦的迹象。 </li><li>执行pH值ysiologic术前检查。表演听性脑干反应（ABR）测试和/或畸变产物光声排放测试（DPOAE）这两个手术前和之前以牺牲动物作为一个客观的测量所选择的耳毒性剂对鼠标的听觉效果。 </li></ol> 3.手术准备和定位<ol><li>消毒所有仪器每次的制度规范手术前。准备手术器械和字段以一致的，无菌的和有组织的方式，以避免在操作过程中不需要的努力和运动。所需的典型手段包括锋利的解剖剪刀，几双金属镊子珠宝商的提示，一个耳科挑，一个耳科刮匙，和双极电单元（ 图1）。 </li><li>准备并通过X 1切一小三角片擦拭（〜0.7英寸×1.25消毒从labwipes事先做了纸灯芯。在75），并用一只手（〜1.25英寸）的拇指和食指之间扭紧。创建一个紧密扭曲的，非常薄的灯芯是极为重要的手术成功。共准备手术之前15-20灯芯（ 图2）。 </li><li>使用牙钻以快速穿透鼓泡的骨以受控方式。使用皮带驱动牙科手电钻的用1或2毫米的锥形尖端是优选的。一个执行显微镜要完成的协议。 （见步骤4.7） </li><li>预绘制0.2毫升含有所选的耳毒性剂的水溶液1毫升结核菌素注射器用28 G，1/2"针头。通常1滴试剂的（〜10微升）足以完全填充鼠标RWN。一种金属，钝头针头减轻药剂的递送，同时防止损坏的急剧伞尖底层结构。排出的空气注射器内的气泡可能会无意中填补RWN和/或防止正确应用代理圆窗本身。 </li><li>使用电梳理快船队在耳后皮肤取下毛皮。从auriculo-头折痕吻侧到肩胛带尾端延伸的区域删除皮毛。扩展从背，矢状中线下颌角横向脱毛。安全删除皮草是极为重要的保持清洁，术野清晰。轻轻刷，从预期的手术部位剪报。 </li><li>每次消毒机构的协议的准备区的皮肤。应用优碘（聚维酮/碘）以循环的方式，2分钟交替用乙醇局部给药。其他试剂可以每机构准则被取代。 </li><li>放置在平坦表面上的动物，以保持该过程中恒定身体定位。用一个小加热垫身下放置到平台的过程中，以控制体温保持动物的体温在36-38℃。不要奥雅纳rheat因为这可能导致轻度至重度的皮肤损伤和/或早期安乐死的动物。 <ol><li>通过咬块/头固定装置固定动物的头。利用一个0.5厘米×从黄铜加工有通过它以5毫米的间隔沿着长轴线钻出2-4毫米直径的孔2厘米咬合块。解决此夹具打开动物的嘴和适合上部中央门牙到取决于动物的大小的孔中的一个。轻轻拧紧一个小夹子在动物的口鼻部，将其固定到位 （图3）的背部。 </li><li>确认该咬合块/头支架刚性地连接到一个U形铰接臂（图3）的中心。确保了1cm宽杆到"U"形的左臂并使用杆作为左侧头枕（右侧总是术侧）。 </li><li>一旦坐稳头保持器，旋转鼠标到左侧卧位。放置身体carefuLLY在平坦操作表面，以确保它是通过在整个过程稳定，并避免对颈椎过度的扭曲应力。 </li></ol></li><li>放置在手术显微镜能够4倍，10倍，而20倍的放大倍率在外科领域。确认该显微镜可保持其位置在一个免手的方式，因为这是理想的下面讨论的两手外科协议。 </li><li>将用细尖珠宝商的手一块双极启用烧灼单元的位置上可立即在小血管和组织的解剖烧灼的援助。这也可能是必要的应重出血会遇到。 </li></ol> 4.手术方法<ol><li>在显微镜下放大，用锋利的剪刀或手术刀刀片创建1-1.5厘米耳后切口，约6-8毫米尾椎颅耳折痕。在成年小鼠中，从背中线切口侧向靠近下颌角的点就足够了。小心避免削减深深地保留基本的血管结构。 </li><li>通过它可以是可变厚度的皮下脂肪层进行仔细钝性分离。脂肪会在必要时提高曝光被安全地删除。采取谨慎的腹侧内侧方向作为解剖颈外静脉穿过这个区域和破坏这一结构可能会导致大量失血和手术野的洪水时。控制任何失血过多用吸收性明胶海绵和/或棉球。用双极电凝对出血加重。 </li><li>一旦脂肪层被正确划分，露出颈部肌肉。注意重要结构，包括cleidomastoideus的大肌肉的身体内集中暴露手术视野，颈外静脉腹侧和腮腺组织吻侧覆下颌角。一个重要标志是小神经branc小时（脑神经Ⅺ的），该缠绕在cleidomastoideus的后部/背部边缘到吻侧伸向耳廓（图4）。 </li><li>轻轻缩回cleidomastoideus肌肉体在后部/背部方向（ 图4，图5）。轻轻地划分透明筋膜包围肌体。以类似的方式，轻轻缩回在相反的（前/腹侧）方向（图5）腮腺和外部颈静脉。 </li><li>与cleidomastoideus肌肉体的良好回缩，鼓泡骨膜的光泽球机将进入视野（图6）。在泡的尾鳍方面，更深颈肌，所述sternomastoideus的插入，将进入视图 （图6）。保留面神经，成为在背部和泡圆顶喙方面可见。放置一个自固牵开器（无菌钛shaft--嵌入一次性硅氧烷粘贴）之前钻孔。 </li><li>用两手技术中，轻轻地划分大疱骨膜双极电疗以暴露下面的骨骼。使用镊子或耳科刮匙，轻轻提升和推骨膜在圆周方向广泛露出大疱圆顶。 注意:步骤4.6是关键最大化中耳空间的外科图。利用细致，温柔的软组织处理，以防止出血和/或间质液泄漏到钻孔后的肺大泡腔。 </li><li>在一个适当曝光泡的圆顶，钻透大疱骨与拱顶的尾缘和明显不透明线（表示鼓膜）之间的牙外科钻一个2毫米导孔穿过泡的喙方面延伸（图7）。照顾钻仅通过骨保留下层结构，例如镫骨动脉。钻第二个先导孔附近，以方便非屋面泡骨（&lt;STRONG&gt;图7）。 </li><li>使用一对珠宝商的尖镊子的，开盖的骨头泡在背鳍和尾鳍方向（ 图8）。在高倍镜下零敲碎打取出骨头。不要刺破的镫骨动脉，它直接位于泡盖的下方，从这个动脉出血可能会影响程序。最小化骨的删除，以防止过 ​​度的流体进入到中耳，同时仍然允许优良的可视化，并获得了圆窗龛（图8）的量。 </li></ol>耳毒性剂的5圆窗应用<ol><li>制作精细的旋转调整头支架带来的RWN正视眼帘。所述RWN通常位于中耳空间的背部和尾部方面，并显示作为听囊骨杯状缩进。在大多数情况下，镫骨动脉运行1-2毫米腹侧/延髓此。该RWN有时TUC在泡圆顶急性角度周边糟透了。在这样的情况下，仔细定位动物的头的是至关重要的。 <ol><li>采用术前准备的文件灯芯，去除所有可见的流体在中耳和RWN直至干燥骨可视化。 注意:这是整个协议中最关键的步骤，作为〜耳毒性剂的10微升（1滴）加到RWN可以容易地通过该流体稀释。 </li></ol></li><li>在最大放大倍率，请在1毫升结核菌素注射器细口径的针头直接应用耳毒性药物的一种滴（约10微升）的RWN，完全填充它。小心不要破坏镫骨动脉和密切观察的小光反射的更换在干燥的利基的底座采用了沉闷和朦胧的弯月面作为一个指标，该利基填充正确。 <ol><li>允许代理的RWN内休息约10分钟的曝光时间。在此之后，完全W¯¯ICK出剂，并将其与同一代理的一个新的应用程序替换。根据代理规范确定应用程序的重复。在此过程中总曝光时间通常是30至60分钟之间的范围内。 </li><li>在该过程结束时，完全吸走代理最后一次并应用新鲜剂与RWN。离开泡不封顶，并使用镊子关闭软组织在手术部位。 </li></ol></li><li>密封手术部位处皮肤的具有4-0，不可吸收单纤维缝合线的水平。将动物在恢复笼/站。监测动物的临床状况定期以下的操作。保持适宜的环境条件，包括外壳，柔软的被褥，并补充软的食物的动物，以减少压力。根据体制IACUC协议确定未来程序和手术后的条件。 </li><li>使用机构IACUC协议消毒仪S上的下一个动物后再使用。允许仪器的使用之间的适当的冷却时间。 </li></ol> 6.术后过程和耳蜗组织收获<ol><li>如步骤2.5中所述，在术后所需点进行听证的生理测试和以前的牺牲。根据实验的目的进行术后程序。牺牲在任何需要的术后第二天的动物。 </li><li>一旦通过IP注射全身麻醉，每个机构IACUC协议的要求，牺牲动物。使用锋利的剪刀，杀头的动物只是尾椎枕。大幅打开颅腔沿着背部和腹部中线剪刀和广泛传播。轻轻舀出脑组织暴露颞骨。 注:一些用于耳蜗后的曝光变化的调查程序，可以从这个角度分支，将讨论一节中提到。确定调查方法最relevan吨的实验目的。如果电子显微镜计划耳蜗被剖开后，可以使用心脏导管预修复组织。这超出了本讨论的范围，并已包括在深度别处。13 </li><li>切颞骨用剪刀颅底，并立即放置在固定液。直接在4％多聚甲醛浸泡骨骼1.5小时在RT。监视定影时间，因为更长的持续时间可以在后面的步骤中限制组织学分析的结果。脱钙骨解剖的时间通过浸没的可变周期中1mM的乙二胺四乙酸（EDTA）。 </li></ol>

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M., 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=Heptanol+application+to+the+mouse+round+window:+a+model+for+studying+cochlear+lateral+wall+regeneration.">Heptanol application to the mouse round window: a model for studying cochlear lateral wall regeneration.</a> Otolaryngology--head and neck surgery : official journal of American Academy of Otolaryngology-Head and Neck Surgery. 150, 659-665 (2014).</li><li>Zheng, Q. Y., Johnson, K. R., Erway, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+hearing+in+80+inbred+strains+of+mice+by+ABR+threshold+analyses.">Assessment of hearing in 80 inbred strains of mice by ABR threshold analyses.</a> Hearing research. 130, 94-107 (1999).</li><li>Hequembourg, S., Liberman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spiral+ligament+pathology:+a+major+aspect+of+age-related+cochlear+degeneration+in+C57BL/6+mice.">Spiral ligament pathology: a major aspect of age-related cochlear degeneration in C57BL/6 mice.</a> Journal of the Association for Research in Otolaryngology : JARO. 2, 118-129 (2001).</li><li>Ohlemiller, K. K., Gagnon, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apical-to-basal+gradients+in+age-related+cochlear+degeneration+and+their+relationship+to+'primary'+loss+of+cochlear+neurons.">Apical-to-basal gradients in age-related cochlear degeneration and their relationship to 'primary' loss of cochlear neurons.</a> The Journal of comparative neurology. 479, 103-116 (2004).</li><li>Tadros, S. F., D'Souza, M., Zhu, X., Frisina, R. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+approach+to+select+genes+from+RMA+normalized+microarray+data+using+functional+hearing+tests+in+aging+mice.">Novel approach to select genes from RMA normalized microarray data using functional hearing tests in aging mice.</a> Journal of neuroscience. 171, 279-287 (2008).</li><li>Tang, X., 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=Age-related+hearing+loss:+GABA,+nicotinic+acetylcholine+and+NMDA+receptor+expression+changes+in+spiral+ganglion+neurons+of+the+mouse.">Age-related hearing loss: GABA, nicotinic acetylcholine and NMDA receptor expression changes in spiral ganglion neurons of the mouse.</a> Neuroscience. 259, 184-193 (2014).</li><li>Borkholder, D. A., Zhu, X., Frisina, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Round+window+membrane+intracochlear+drug+delivery+enhanced+by+induced+advection.">Round window membrane intracochlear drug delivery enhanced by induced advection.</a> Journal of controlled release : official journal of the Controlled Release Society. 174, 171-176 (2014).</li><li>Tadros, S. F., D'Souza, M., Zhu, X., Frisina, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+changes+for+antioxidants+pathways+in+the+mouse+cochlea:+relations+to+age-related+hearing+deficits.">Gene expression changes for antioxidants pathways in the mouse cochlea: relations to age-related hearing deficits.</a> PloS one. 9, e90279 (2014).</li><li>Lang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox2+up-regulation+and+glial+cell+proliferation+following+degeneration+of+spiral+ganglion+neurons+in+the+adult+mouse+inner+ear.">Sox2 up-regulation and glial cell proliferation following degeneration of spiral ganglion neurons in the adult mouse inner ear.</a> Journal of the Association for Research in Otolaryngology : JARO. 12, 151-171 (2011).</li></ol>

没有竞争的财务权益。作者什么都没有透露。

上述协议和有代表性的结果在CBA / CAJ小鼠模型，包括男女获得。这近交系被确立为一种"听觉"的标准和"正常衰老"的模式审理研究。16-23说明使用该协议在其他哺乳动物模型的讨论已经超出了本文的范围。读者必须留意的是，RWN应用技术提供了多种优势，研究哺乳动物的内耳。这些中，最显着的是，它避免了细腻的解剖结构的直接破坏和听囊的壁中存在的生化梯度。程序如内耳开窗和输液泵植入有倾向直接违反内耳结构导致永久性阈移;必须分析的结果时，应考虑到一个事实。耳蜗侧壁结构，通过侵入其实现方法具破坏DS还可以限制使用耳毒性药物如速尿或庚，其效果特定区域仅限于该位置。15,24替代的非侵入性的方法，例如跨鼓室注射液和注射针剂也一直困扰着不可靠的成果， /或全身毒性的动物模型。这种应用方法已经证明，以避免这两个缺点，实现一致性接近于上面讨论的更侵入性的方法的水平。 这种技术的其它优点包括其被广泛的适用性的若干动物模型和可行性的掺入到现有的基础设施的实验室。关于后者，没有专门的试剂或化学品被从选择的耳毒性剂，麻醉剂，止痛剂和所需的一边。耳毒性剂通常使用在固定的浓度，并混合在足够大体积的溶液（5毫升）要持续很长一段时间考虑荷兰国际集团每一个应用程序使用约10微升（小鼠）。因此，用品和仪器的初始采购后，调查人员从耗时的溶液的制备或频繁更换材料的相对自由。该技术还提供了减少手术时间，相对于涉及外淋巴输液泵或cochleostomies植入程序，这可以是显著。一旦达到的技术熟练程度的电平，从最初的切口闭合我们的平均完成时间为通常20分钟至1.5小时，这取决于曝光所需的耳毒性剂的长度。三四个手术可以很容易地在一天内完成，从而提高效率并获得成功的结果增加了可能性。如上所述，这种技术也可以容易地应用于各种啮齿动物模型，包括小鼠，大鼠，豚鼠和沙鼠。 该方法的局限性是集中在适度陡峭的学习曲线需要掌握它，并减少预期的结果，直到技术熟练为止。如将在下面更详细讨论的，在外科手术方法或手术区域的不足可视小的错误将几乎总是导致预后较差。微妙的发现，一个新手可能无法识别，如剂的亚毫米厚的气泡阻止访问圆窗膜或间质液稀释药剂，需要时间去欣赏和发展需要纠正他们的动作技能。然而，与这些障碍物容易克服并构成那么令人畏惧的技术挑战向调查比前面提到的一些侵入性方法的步骤的重复性能。最后，该技术是与相对的限制，即耳蜗损伤只能在单个点中的手术暴露诱导时间相关联。这是可以克服的，以一定程度的，通过填充RWN与吸收性明胶海绵作为由Heydt等人所述浸泡在代理10的吸收性明胶海绵会再吸收随着时间的推移，但可以允许更长的曝光周期比是可以实现的，通过应用的单独的水溶液。 为了使研究者实现这一技术的全部优点，避免任何缺陷，重要的是认识到该技术的两个关键因素:1）始终保持中耳空间和RWN的可视化;和2）的能力，以保持手术区域自由组织液和/或血液的。在实现前者的这些，适当的头架的重要性不能被过分强调。动物的头的安全固定，确保在显微镜下稳定的图。它的重要性是十分明显的时候微妙的仪器仪表彻底的改变结构的放大倍率下的定位。一个好的^ hEAD保持器能够绕动物的延髓 - 尾轴还便于在现场研究者的线路重要动态变化。通常，旋转绕该轴线几毫米可能意味着RWN的可视化和仅听囊骨可视化之间的差别。不断地改变视图的能力也是极为重要的确保间隙液体正确地从适当位置和深处除去也是耳毒性剂充分应用之间除去在第5部分。根据我们的经验，血液，缩合，或间质液中讨论进入中耳空间具有干扰整个实验的能力。这并不奇怪，因为耳毒性剂的少量施加于圆窗（〜10微升）可以容易地通过在接触甚至少量的外来流体的稀释。出于这个原因，细致的手术剥离时，鼓泡，精心preservat零散脱帽在镫骨动脉离子都无异于一次成功的实验结果。 如果上面的关键步骤，观察和预期结果仍然没有实现，排除故障应开始。根据我们的经验，它往往是有帮助的执行两个程序要素审判的变化。第一种方法是修改与该耳毒性剂补充在圆窗的频率。根据剂被使用，总曝光时间为30分钟和1小时之间，拥有完整的排汗及随后的更换代理每10分钟的。如果暴露于较短的持续时间，提高了整体曝光可以允许代理更多时间来穿过圆窗膜扩散。附加暴露和补充也可能有助于避免不想要的稀释耳毒性剂的由血液，冷凝，或间质如以上所讨论。应使用这种方法时，可以保持谨慎，但是，因为它趋向于增加的无意的风险LY打伤镫骨动脉和/或引入间质液到RWN。 这种技术是在它所提供耳蜗生理和病理生理的调查显著。这种微创技术可以让娇嫩的生化过程的详细研究，并已经等于是在推进我们的研究旨在评估耳蜗再生潜能。12,24这种手术方式和曝光也转载在各种其他分支技术，并成功的结果使用本方法已报道耳蜗干细胞移植的研究。14还有大量未知有关耳蜗，但是，这种技术，以及提供给调查人员更广泛的医疗设备，将在缩小这一知识差距援助。

研究者已使用动物模型的各种各样的研究听觉系统的正常功能，以及听力损失的病理生理学。这些模型也可用于进行抗各种病理过程的干预研究非常有用，并作为用于人受试者平移应用程序的基础。对于大多数的研究涉及耳蜗和其相关联的听觉通路，某种程度的损伤或破坏，必须引入到系统中。通常，损伤是故意旨在创建特定病变，允许调查研究这一病变在正常功能，以及从中恢复耳蜗能力的效果。当选择一个特定的动物模型和/或技术（多个）用于引入损害，一些必须考虑的因素，以达到最佳的可能结果。各种动物模型可以有不同的反应，以干预​​，而技术的直接和间接影响可能是完全有害的所期望的结果。在大多数情况下，提供了理想内耳损伤协议将避免全身性毒性，迅速且可靠地产生损伤，建立精确的和一致的病变，并生存能力，以允许功能，细胞和分子变化进一步研究。理想情况下，这些方法也将保持耳蜗的微妙微架构和电化学梯度，以尽最大可能。 迄今为止，研究人员已经成功地建立了许多技术以诱导内耳损伤。大多数这些意味着暴露耳蜗耳毒性剂全身或通过外科手术的方法。技术包括肠胃外注射，腹膜内注射，反式鼓室注射，内淋巴囊注射和内耳开窗与外淋巴灌注。这些技术已被用于引入各种耳毒性剂，如呋塞米，庆大霉素，哇巴因，和庚1-5虽然成功地创建特定的耳蜗病变，上述技术也已经承认局限性。全身注射可以给动物剧毒，并且可以与意外的耳蜗损伤和不一致的结果相关联。后者的缺点也与反式鼓室注射相关联。技术如内耳开窗和外淋巴灌注，而能够诱导快速和高可靠的病变，是直接侵入到内耳结构和功能。被许多的手术方法也与技术难度高的关联度，并可能需要留在动物异物，如一个微型泵输液。2-4,6-8没有单一的技术是免费的缺点，和调查人员必须选择方法仔细，以满足他们的实验需要。这里，我们描述，详细地说，圆窗龛（RWN），用于在成年小鼠局部递送的耳毒性剂的应用技术。 网络连接由Husmann等人于1998年在研究在禽类模型上感觉毛细胞变性庆大霉素的效果首先描述的，这种技术被认为是能够产生显著更可靠病变不是全身庆大霉素的应用，同时避免关联的毒性。9此后，一其他一些研究人员，包括我们的实验室，已经利用该技术取得了巨大成功。 2004年，Heydt等。适应它的小鼠模型和描述的填充吸收性明胶海绵浸泡在不同庆大霉素浓度RWN控制损伤尺寸的增强能力。10 Palmgren等人，在2010年，研究了β-内银环蛇毒素的耳毒性作用，一种强效在台湾的毒液元件带状板条箱，通过应用它的一个含水形式到成年大鼠RWN 11此外，一些本实验室以前的研究已经利用圆窗方法来研究呋塞米的耳毒性作用即，哇巴因，和庚醇。这些研究的结果5,6,12-15已经证明耳蜗流体和听力正常离子平衡的重要性，发现在螺旋神经节和耳蜗侧壁细胞增殖能力，和进一步推动我们的理解与年龄相关的听力损失。 下面的方法包括通过耳后切口及骨鼓泡部分去顶术手术访问中耳。这允许RWN和膜向其中一个选择的耳毒性剂可以直接施加的优良的曝光。液剂然后在RWN的杯状中空池（或缓慢排出从包装到RWN饱和吸收性明胶海绵载体），并通过圆窗膜进入耳蜗前庭的外淋巴空间扩散。没有直接的内耳是在这种方法。这种技术的优点包括保护内耳微架构，避免全身毒性的内部动物控制耳，津贴，迅速 ​​起效，在某些耳蜗细胞类型的选择性变性（例如，I型螺旋神经节细胞哇巴因曝光和耳蜗型诱导庚治疗II纤维细胞）和可重复性/可靠的结果。这种技术可以与其他啮齿类动物，包括大鼠，豚鼠，沙鼠和之间变化很少被应用。缺点包括一个陡峭的技术学习曲线和耳毒性侮辱被限制在单个时间点的相对限制。

<table><tbody><tr><td>1-Heptanol 98%</td><td>Sigma-Aldrich</td><td>H2805&amp;nbsp;</td><td>&lt;a&gt;PubChem Substance ID 24895536&lt;/a&gt;</td></tr><tr><td>Ketaset Injectable&amp;nbsp;</td><td>Patterson Veterinary</td><td>07-803-6637&amp;nbsp;</td><td>Concentrate 100mg/ml</td></tr><tr><td>(Ketamine HCl)</td><td>10 ml</td><td>Schedule CIII controlled substance</td><td></td></tr><tr><td>Anased Injectable</td><td>Lloyd Laboratories</td><td>NADA# 139-236</td><td>Concentrate 20mg/ml</td></tr><tr><td>Buprenex Injectable&amp;nbsp;</td><td>Patterson Veterinary</td><td>07-850-2280&amp;nbsp;</td><td>Concentrate 0.3 mg/ml</td></tr><tr><td>(Buprenorphine HCl)</td><td>5 ampules per box</td><td>Schedule CIII controlled substance</td><td></td></tr><tr><td>Betadine Skin Prep Solution</td><td>Medline</td><td>&lt;a href=&quot;http://www.medline.com/sku/item/MDPMDS093941;ecomsessionid=1UZWKxhkYkuy9vK2NXBaBA__&quot;&gt;MDS093941&amp;nbsp;&lt;/a&gt;</td><td>1 Quart screw top bottle</td></tr><tr><td>0.9% Sterile Saline</td><td>Variable</td><td></td><td>For mixing solutions and injections</td></tr><tr><td>Operating Microscope</td><td>Carl Zeiss</td><td>32192</td><td></td></tr><tr><td>Controlled Acoustics Environment Sound Booth</td><td>Industrial Acoustics Company</td><td></td><td></td></tr><tr><td>Surgical Head Holder</td><td>Custom Made &amp;ndash;&amp;nbsp;</td><td>Please see Figure 3</td><td></td></tr><tr><td>Medical University of South Carolina</td><td></td><td></td><td></td></tr><tr><td>Neck Soft-Tissue Retractor (Wire Speculum, Titanium) 1.75 inch</td><td>World Precision Instruments</td><td>555801L</td><td>Maximum spread 20 mm</td></tr><tr><td>Embedded in disposable putty to affix dynamically to head holder</td><td></td><td></td><td></td></tr><tr><td>90N Dental Belt Driven Hand Drill&amp;nbsp;</td><td>Emesco</td><td>Vintage Item</td><td></td></tr><tr><td>Scalpel Handle Size 6</td><td>Bard-Parker</td><td>MEDC-011990</td><td></td></tr><tr><td>#15c Stainless Steel Surgical Scalpel Blade&amp;nbsp;</td><td>Bard-Parker</td><td>SKU: 097-7215</td><td>50 Blades/Box</td></tr><tr><td>Via ACE Surgical Supply Code</td><td></td><td></td><td></td></tr><tr><td>Straight Tip Jewelers Forceps&amp;nbsp;</td><td>Bernell</td><td>MIL17304</td><td></td></tr><tr><td>Iris Scissors Curved</td><td>Medline</td><td>&lt;a href=&quot;https://www.medline.com/sku/item/MDPDYND04026;ecomsessionid=ozXzkfIx-tj0MIi8FkGQ0A__&quot;&gt;DYND04026&amp;nbsp;&lt;/a&gt;</td><td></td></tr><tr><td>Iris Scissors Straight</td><td>Medline</td><td>&lt;a href=&quot;http://www.medline.com/sku/item/MDPDYND04025;ecomsessionid=+43JREOby+Mfz6IIgoSg5A__&quot;&gt;DYND04025&amp;nbsp;&lt;/a&gt;</td><td></td></tr><tr><td>Stevens Tenotomy Scissors Straight</td><td>Medline</td><td>&lt;a href=&quot;http://www.medline.com/sku/item/MDPMDG3222111&quot;&gt;MDG3222111&amp;nbsp;&lt;/a&gt;</td><td></td></tr><tr><td>Rosen Ear Needle Straight Shaft, Lightly Curved Tip</td><td>MytaMed</td><td>Item# 6.56.00</td><td>Figure 1 demonstrates angled shaft picks. This was later substituted for the Rosen picks</td></tr><tr><td>Rosen Ear Needle Straight Shaft, Strongly Curved Tip</td><td>MytaMed</td><td>Item# 6.56.01</td><td></td></tr><tr><td>Kimwipes Delicate Task Wipers&amp;nbsp;</td><td>Kimtech Science</td><td>CODE 34155&amp;nbsp;</td><td>White, Size 4.4x8.4 Inch. Cut to triangles and rolled into fine tip wicks.</td></tr><tr><td>House Ear Curette, 6&amp;rdquo; shaft, light angle</td><td>Medline</td><td>&lt;a href=&quot;https://www.medline.com/sku/item/MDPMDG0396486&quot;&gt;MDG0396486&amp;nbsp;&lt;/a&gt;</td><td></td></tr><tr><td>Gelfoam (absorbable gelatin sponge) Size 100</td><td>Medline</td><td>&lt;a href=&quot;https://www.medline.com/sku/item/MDPIIS34201&quot;&gt;IIS34201&amp;nbsp;&lt;/a&gt;</td><td>Substitutions may be made</td></tr><tr><td>Cotton pellets #3 4 mm</td><td>Richmond</td><td>Manufacturer Code 100108</td><td></td></tr><tr><td>ElectroSurgical Unit 100 E M/M</td><td>Elmed</td><td>List No. 52-5770</td><td>Bipolar and Monopolar Capable</td></tr><tr><td>1cc U-100 Insulin Syringe 28G, 0.5&amp;rdquo; length needle</td><td>BD</td><td>Product Number: 329410</td><td>Optional for delivery of Ototoxic agent</td></tr><tr><td>23G, blunt tip, 1&amp;rdquo; length needle</td><td>Kendall</td><td>Product Code 8881202397</td><td>For controlled delivery of Ototoxic agent with less risk of damaging stapedial artery</td></tr><tr><td>Surgical Mask</td><td>U-line</td><td>S-10478</td><td></td></tr><tr><td>Exam Grade Nitrile Surgical Gloves</td><td>U-line</td><td>&lt;a href=&quot;http://www.uline.com/Product/Detail/S-12549/Nitrile-Gloves/Nitrile-Exam-Gloves-Powder-Free&quot;&gt;S-12549&lt;/a&gt;</td><td></td></tr><tr><td>Precision Hair Clippers</td><td>Wahl</td><td></td><td>Multiple models may be substituted</td></tr><tr><td>5-0 Nylon black monofilament suture on PC-1&amp;nbsp;13 mm 3/8 circle needle</td><td>Ethilon</td><td>1855G</td><td>Substitutions may be made.&amp;nbsp;</td></tr><tr><td>Instant Sealing Sterilization Pouch</td><td>Fisher</td><td>01-812054</td><td></td></tr><tr><td>Dry Sterilizer</td><td>ROBOZ</td><td>Germinator &lt;sup&gt;TM&lt;/sup&gt; 500</td><td></td></tr></tbody></table>

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.

在利用上述协议的最新研究史蒂文斯等人，无论男女，成人CBA / CAJ小鼠经圆窗扩散暴露于庚。15庚醇是一种缝隙连接非耦合器已知会产生针对性的，对细胞可恢复伤害耳蜗侧壁。这项研究的目的是要生产有针对性的耳蜗损伤的可靠模型，允许任何损坏元件手术后再生的调查。担任官能端点手术前和手术后的听觉阈。显微镜和免疫组化染色技术被用来研究形态变化。显著增加的ABR阈值中观察到庚醇处理的小鼠（图9）。接受假手术，交付生理盐水代替庚控制动物，并没有表现出显著阈移，在任何测试的频率。 免疫染色针对内向整流钾通道（KIR）4.1担任可视化耳蜗结构的损坏/恢复的间接方法。这表明治疗和控制两耳之间的高重现性差。总体染色强度显着内血管纹（STV），并跻身螺旋韧带（SLF）庚暴露后1-3天的成纤维细胞减少，表示了大量有针对性的破坏这些地区。有在II型和IV型SLFs的区域（ 图10）和STV（图11）在基尔4.1染色强度的特定降低。的破坏核完整性和染色体缩合/泡典型细胞凋亡的证据也被看作对在这些领域耳蜗核counterstains（图10）。基尔4.1空泡区染色7天显效解决，缺席全部的14天。当基尔4.1染色强度进行定量在STV的区域，处理过的耳表现出initi人低谷之后是显著移（P &lt;0.05），走回控制强度庚曝光 （图12）后7天。 <img alt="图1" src="/files/ftp_upload/53131/53131fig1.jpg" /> 图1.仪器设置，对术前设置了仪器的描写。所有设备应是无菌的手术区域内交通便利。典型的仪器包括2锐性剥离剪刀，2-3直和/或弯曲的尖端珠宝商的镊子耳刮匙，弯轴耳科选秀权（后来与罗森代采 - 未显示），和电烙术单位有细弯的尖头珠宝商的镊子头盔。 <a href="https://www.jove.com/files/ftp_upload/53131/53131fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img aLT ="图2"SRC ="/文件/ ftp_upload / 53131 / 53131fig2.jpg"/&gt; 图2.手术耗材 。额外的供给用于保持RWN环境。灭菌labwipe切口为纸芯形成（左），紧密形成纸灯芯从灭菌实验室湿巾（中心），和4毫米棉球（右）是用来擦拭在圆窗龛过量液体和血液驱制成。 <a href="https://www.jove.com/files/ftp_upload/53131/53131fig2large.jpg" target="_blank">请这里查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53131/53131fig3.jpg" /> 图3.动物头支架 。图片描绘了头支架和牙垫。块中的孔配合并固定上中切牙。一个夹子轻轻地拧紧在背吻，以确保动物的地方。使用一个头支架是手术成功的关键OUTC青梅。理想情况下，这应该是能够围绕动物的喙，尾轴，以优化过程中的肺大泡手术的意见。 <a href="https://www.jove.com/files/ftp_upload/53131/53131fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53131/53131fig4.jpg" /> 图4. Cleidomastoideus米。示意图图示，描绘了啮齿类颈肌肉的基本解剖和其与外部颈静脉关联。该cleidomastoideus肌肉在手术方式是最容易识别的肌肉。包络筋膜其次是肌肉身体的后部/背部收缩会直接手术切除朝鼓泡（黑圈）的发布。 <a href="https://www.jove.com/files/ftp_upload/53131/53131fig4large.jpg" target="_blank">请点击此处查看人</a>arger版本这个数字。 <img alt="图5" src="/files/ftp_upload/53131/53131fig5.jpg" /> 图5.手术暴露面积 。描述了通过皮肤和皮下脂肪层剥离后曝光。值得注意的结构性景点包括脑神经十一覆cleidomastoideus肌肉（A），颈外静脉（B）的一个分支，并暴露腮腺组织（C）。脑神经XI分支通常与小血管有关，必须前程序划分。从右到左的图像对应于动物的喙-尾轴。 <a href="https://www.jove.com/files/ftp_upload/53131/53131fig5large.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/53131/53131fig6.jpg" /> 图6.揭露BULLA的cleidomastoideus的收缩和周围结构曝光后的鼓泡的。著名的地标包括cleidomastoideus肌肉的身体（A）反映后部/背部，面部神经（B），以及鼓泡骨膜（C）的闪亮的圆顶。另外，还要注意的sternomastoideus肌肉在鼓泡（星号）的左侧尾方面的插入。面神经在肺大泡的背和头端方面的存在是一个重要的里程碑，真正的鉴定泡了。从右到左的图像对应于动物的喙-尾轴。 <a href="https://www.jove.com/files/ftp_upload/53131/53131fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/53131/53131fig7.jpg" /> 图7.暴露圆窗龛我 ，图像描绘了tympaNIC泡覆骨膜剥离后充分暴露。导向孔最好放置在大疱圆顶的尾缘和一个微妙不透明线内延髓方面的大泡（表示鼓膜）可视化之间的中点处。第二，相邻的导孔可以方便容易取消屋面的肺大泡骨。深钻的避免，应采取以免损害底层的镫骨动脉。在图像底部的黑色，金属物体是钛软组织牵开器。 <a href="https://www.jove.com/files/ftp_upload/53131/53131fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图8" src="/files/ftp_upload/53131/53131fig8.jpg" /> 图8.暴露圆窗龛二 。不封顶鼓泡与圆窗龛（箭头）和镫骨动脉的曝光（红色结构1-2毫米横向利基）下放大20倍的观察。生态位往往奠定下的泡穹顶与耳囊在圆顶的尾部方面形成的锐角夹着的位置。当务之急是小生的全图表中，再使用耳毒性剂或排汗来实现。开盖过程中过度去骨也应避免，因为组织液/血液往往充斥腔创建大洞的时候。 <a href="https://www.jove.com/files/ftp_upload/53131/53131fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图9" src="/files/ftp_upload/53131/53131fig9.jpg" /> 图9.代表性的结果-庚醇性听力损失和恢复平均听性脑干反应（ABR）阈值（分贝SPL）绘制成音画中画功能频率。测量是按照预曝光（黑控制）和手术后的天（POD）的1,7，和14（红色）分组。误差棒表示SEM。图重新绘制从史蒂文斯等人。 2014年15 <a href="https://www.jove.com/files/ftp_upload/53131/53131fig9large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图10" src="/files/ftp_upload/53131/53131fig10.jpg" /> 图10.代表性的成果-目标庚曝光部分一，变化与庚治疗耳内钾整流器（基尔）通道4.1染色内血管纹（STV）和控制耳后耳蜗损伤 。 （一）正常珥4.1染色典型的控制耳朵与珥4.1（绿色）强大的血管纹细胞亲和性。 （ 二）治疗耳朵上术后第一天。 DECR大内质区缓解基尔4.1亲和力的STV（箭头）随着STV珥4.1染色强度总体下降中看出。细胞核counterstained用碘化丙啶（红色）（B）中。比例尺= 15微米。图重新绘制从史蒂文斯等人，2014年15 <a href="https://www.jove.com/files/ftp_upload/53131/53131fig10large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图11" src="/files/ftp_upload/53131/53131fig11.jpg" /> 图11.代表性的成果-目标庚曝光第二部分对耳蜗损伤的变化庚治疗耳螺旋韧带（SL）相比，控制的耳朵。 （一）正常珥4.1染色（绿色）SL典型的控制耳看似正常的II型螺旋韧带成纤维细胞（二）内。 （二）庚醇牛逼reated耳与在基尔4.1染色强度明显降低在II型螺旋韧带成纤维细胞，核的破坏和染色体缩合/凋亡（箭头）相一致泡的面积。细胞核counterstained用碘化丙啶（红色）。比例尺= 15微米。图重新绘制从史蒂文斯等人，2014年15 <a href="https://www.jove.com/files/ftp_upload/53131/53131fig11large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图12" src="/files/ftp_upload/53131/53131fig12.jpg" /> 图12.代表性的成果-恢复耳蜗染色强度以下庚曝光的平均珥的4.1染色绘制为暴露后一天的功能相对亮度。相对亮度下庚治疗共聚焦显微镜计算珥4.1反光强度编耳朵取为相同的对照耳朵的百分比。音符POD14-28数据汇集为曲线上的一个点。实心圆表示平均值，而误差棒代表平均值的标准误差。一个显著的恢复相对亮度的证明POD 7，后来时间（t检验P &lt;0.05，星号）之间。图重新绘制从史蒂文斯等人，2014年15 <a href="https://www.jove.com/files/ftp_upload/53131/53131fig12large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration at JoVE.com

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

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 ...

the-mouse-round-window-approach-for-ototoxic-agent-delivery-rapid

Universidad San Sebastian

Research

JoVE Journal

Medicine

17.2K 次观看.  Medical University of South Carolina. 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. 

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

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) has been shown to be a useful approach to local drug delivery into the inner ear. The goal of this article is to describe, in detail, the surgical techniques involved in canalostomy in both adult and neonatal mice. As indicated by fast-green dye and adeno-associated virus serotype 8 with the green fluorescent protein gene, the canalostomy facilitated broad distribution of injected reagents in the cochlea and vestibular end-organs with minimal damage to hearing and vestibular function. The surgery was successfully implemented in both adult and neonatal mice; indeed, multiple surgeries could be performed if required. In conclusion, canalostomy is an effective and safe approach to drug delivery into the inner ears of adult and neonatal mice and may be used to treat human inner ear diseases in the future.

Here we describe canalostomy procedure which allows local drug delivery into the inner ears of adult and neonatal mice through the semicircular canal with minimum damage to hearing and vestibular function. This method can be used to inoculate viral vectors, pharmaceuticals, and small molecules into the mouse inner ear.

Canalostomy 为成人和新生小鼠内耳局部药物传递的外科治疗方法

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

科研

遗传学

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

Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear. Since there is no cell line that satisfactorily represents mammalian hair cells, research on hair cells relies on primary organ cultures. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice (Figure 1) 1-6. The utricle is a vestibular organ, and the hair cells of the utricle are similar in both structure and function to the hair cells in the auditory organ, the organ of Corti. The adult mouse utricle preparation represents a mature sensory epithelium for studies of the molecular signals that regulate the survival, homeostasis, and death of these cells.
Mammalian cochlear hair cells are terminally differentiated and are not regenerated when they are lost. In non-mammalian vertebrates, auditory or vestibular hair cell death is followed by robust regeneration which restores hearing and balance functions 7, 8. Hair cell regeneration is mediated by glia-like supporting cells, which contact the basolateral surfaces of hair cells in the sensory epithelium 9, 10. Supporting cells are also important mediators of hair cell survival and death 11. We have recently developed a technique for infection of supporting cells in cultured utricles using adenovirus. Using adenovirus type 5 (dE1/E3) to deliver a transgene containing GFP under the control of the CMV promoter, we find that adenovirus specifically and efficiently infects supporting cells. Supporting cell infection efficiency is approximately 25-50%, and hair cells are not infected (Figure 2). Importantly, we find that adenoviral infection of supporting cells does not result in toxicity to hair cells or supporting cells, as cell counts in Ad-GFP infected utricles are equivalent to those in non-infected utricles (Figure 3). Thus adenovirus-mediated gene expression in supporting cells of cultured utricles provides a powerful tool to study the roles of supporting cells as mediators of hair cell survival, death, and regeneration.

Mechanosensory hair cells are the receptor cells of the inner ear. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice. We present the dissection of the adult mouse utricle, and we demonstrate adenovirus-mediated infection of supporting cells in cultured utricles.

解剖成年小鼠的椭圆囊和腺病毒介导的支撑细胞的感染

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

神经科学

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

听觉神经的光遗传学刺激

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

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

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and genetic mechanisms that contribute to hearing loss. Introduction of vectors into the inner ear must be done in a way that widely distributes the agent throughout the cochlea while minimizing injury to the existing structures. This manuscript describes a post-auricular surgical approach that can be used for mouse cochlear therapy using molecular, pharmacologic, and viral delivery to mice postnatal day 10 and older via the round window membrane (RWM). This surgical approach enables rapid and direct delivery into the scala tympani while minimizing blood loss and avoiding animal mortality. This technique involves negligible or no damage to essential structures of the inner and middle ear as well as neck muscles while wholly preserving hearing. To demonstrate the efficacy of this surgical technique, the vesicular glutamate transporter 3 knockout (VGLUT3 KO) mice will be used as an example of a mouse model of congenital deafness that recovers hearing after delivery of VGLUT3 to the inner ear using an adeno-associated virus (AAV-1).

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

通过圆窗膜病毒介导的基因传递给鼠标内耳手术方法

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...

Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. Few studies have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem, which contains second order neurons of the auditory pathway, is a potential site for gene transfer. In this protocol, a technique for direct and maximal exposure of the murine DCN via a posterior fossa approach is demonstrated. This approach allows for either acute or survival surgery. Following direct visualization of the DCN, a host of experiments are possible, including injection of opsins into the cochlear nucleus and subsequent stimulation by an optical fiber coupled to a blue light laser.&#160;Other neurophysiology experiments, such as electrical stimulation and neural injector tracings are also feasible.&#160;The level of visualization and the duration of stimulation achievable make this approach applicable to a wide range of experiments.

The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.

在鼠背耳蜗核的听觉通路的光遗传学刺激直接可视化

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. ...

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 ...

生物工程

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

新生儿鼠耳蜗外科手术技术<em&gt;体外</em&gt;听力研究筛选工具

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

医学

Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could interfere with the chemotherapeutic efficacy of the primary drugs. This is especially true for the drug cisplatin, whose anticancer actions are attenuated by antioxidants which provide adequate protection against hearing loss. Other current or potential otoprotective agents could pose a similar problem, if administered systemically. The application of various biologicals or protective agents directly to the cochlea would allow for high levels of these agents locally with limited systemic side effects. In this report, we demonstrate a trans-tympanic method of delivery of various drugs or biological reagents to the cochlea, which should enhance basic science research on the cochlea and provide a simple way of directing the use of otoprotective agents in the clinics. This report details a method of trans-tympanic drug delivery and provides examples of how this technique has been used successfully in experimental animals to treat cisplatin ototoxicity.

We present a technique for localized administration of drugs through the trans-tympanic route into the cochlea. Drug delivery through this route would not interfere with the anti-cancer efficacy of the chemotherapeutic drugs such as cisplatin.

经鼓膜药物治疗耳毒性的临床研究

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could ...

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 ...

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 ...