Name: neonatal murine cochlear explant technique as an in vitro screening tool in hearing research
Uploaded: 2017-06-08T15:00:00.000+00:00
Duration: 8 min 30 s
Description: Watch this Scientific Journal Video about Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research at JoVE.com

这项工作得到了国家失聪和其他传播障碍研究所资助的R01DC015824（KMS）和T32DC00038（支持SD），国防部授予W81XWH-15-1-0472（KMS），贝塔雷利基金会（KMS）， Nancy Sayles Day基金会（KMS）和Lauer耳鸣研究中心（KMS）。感谢Jessica E. Sagers，BA对稿件的深刻见解。

研究方案由马萨诸塞州眼科和耳朵的机构动物护理和使用委员会（IACUC）批准。根据世界医学协会道德守则进行实验。 1.准备解剖<ol><li> 准备手术台 <ol><li>使用70％乙醇消毒手术台。 </li><li>在显微镜旁边放置两个无菌制剂垫。 </li><li>准备仪器托盘，包括热灭菌的操作剪刀，手术刀手柄，微型刀和镊子（＃4＃＃两个）。将仪器托盘和一个50毫米，透明玻璃的玻璃培养皿放在一个非消毒垫上。 </li><li>获得无菌＃15手术刀刀片;可密封的塑料袋或容器（根据制度准则处置屠体）;一桶冰和冷的，无菌的汉克平衡盐溶液（HBSS）。将这些物品放在其他无菌垫。 </li><li>将HBSS转移到50 mL塑料实验室管中，并放在冰上。 </li></ol></li><li> 准备文化 <ol><li>准备层流罩中的所有材料。对于每四个样品，将四个高压灭菌的10毫米圆形玻璃盖玻片放入35毫米4孔培养皿的四个镶嵌中。 </li><li>对于每4个样品，将总量为300μL的微量混合物在微量离心管中混合:10μL组织粘合剂，285μLNaHCO 3和5μLNaOH。 </li><li>将45μL所得溶液放在每个盖板上，并在室温下放置至少20分钟;该解决方案可以留在盖玻片上几个小时，但不能留下O / N。 </li><li>将一个或两个35毫米4孔培养皿放在10厘米培养皿内，以产生两个防止污染的屏障。 </li><li>制备含有98％Dulbecco&#39;s Modified Eagle&#39;s Medium的培养基（DMEM），1％N-2和1％氨苄青霉素（在微量离心管中每孔65μL）。使用前将溶液温热至25°C。 </li></ol></li></ol>小鼠人工耳蜗解剖<ol><li> 斩首新生儿小鼠 <ol><li>获得一个60毫米塑料培养皿，并在其盖子上喷洒70％乙醇，使表面湿润。 </li><li>将冷的HBSS从塑料实验室管倒入60毫米塑料的底部和50毫米透明的玻璃底培养皿中。将塑料实验室管和两个陪替氏培养皿存放在冰上，以便在不解剖时保持组织冷。 </li><li>将P3-P5年龄的新生儿小鼠放在乳胶手套的切断手指上的冰上，并等到低温引起意识丧失（约1-3分钟）。 </li><li>用操作剪刀快速拆卸鼠标，将身体处理在可密封的塑料袋中。将切断的新生儿头部放在70％乙醇在60毫米塑料培养皿的盖子上。 </li></ol></li><li> 颞骨的宏观解剖 <ol><li>通过使用手术刀刀片从前端到后端（直接在矢状缝合线顶部）进行表面切割，去除头骨的皮肤。 </li><li>用手术刀切割两个外耳道并将皮肤向前折叠以暴露整个头颅。 注意:虽然此步骤通常不需要夹层显微镜，但可用于改进可视化。 </li><li>使用＃15手术刀刀片沿着矢状缝线打开颅骨。确保通过从前到后轻轻地移动刀片来切割颅骨，以避免耳蜗的压缩。 注意:颅骨在这个年龄时不应该僵硬，应该很容易切开。 </li><li>使用＃15手术刀通过在轨道的正后方进行垂直切割，去除并丢弃鼻子刀。 注意:头骨的后部应该还包含脑和耳蜗。 </li><li>用＃4镊子通过钝性解剖处理前脑，小脑和脑干。 </li></ol></li><li> 微观提取耳蜗 <ol><li>通过将镊子放在范围内并优化照明和聚焦，调整显微镜（6.5倍放大）和光源。 </li><li>将颅骨的两半放入装有冷HBSS的60 mm塑料培养皿中。 </li><li>完全暴露耳蜗/ e，可以识别为邻近周围的手腕动脉（颞骨内的曲折动脉），并使用＃4镊子将器官与颞骨平坦分离。 </li><li>将耳蜗转移到50毫米，透明的，玻璃底的陪替氏培养皿中，并将盘定位在显微镜下。 </li><li>将60毫米塑料培养皿与颅骨的另一半放在冰上。 </LI&gt; <li>从前庭系统中取出耳蜗，用＃4镊子仔细解剖耳蜗耳蜗胶囊（通常在此年龄为软骨）。使用＃55镊子进行所有进一步的步骤。 </li></ol></li><li> 组织处理的最后步骤 <ol><li>通过使用微刀或两个镊子仔细分离粘附到Corti器官的螺旋韧带与耳蜗的其余部分和组织，继续处理内耳组织。 <ol><li>切入螺旋韧带和毛细胞区域之间的缝隙处的较暗，半透明的区域，并通过在最基础的方面抓住螺旋韧带并继续移除，并在顶端移动时轻轻地展开。 </li></ol></li><li>将样品放置以获得耳蜗的纵向矢状视图，并使用微型刀将耳蜗切成两至三个部分，将这些部分分类为顶端（中间）和basal转。去除组织上下不同于毛细胞和神经突的实际层，以便实现可以水平放置在培养皿上的耳蜗外植体片。 注意:用于稳定粘附培养皿的适当尺寸大约是一个人工耳蜗的一半。 </li><li>去除胸膜，一个非常薄的半透明层，立即高于Corti的器官，用镊子轻轻剥去。 </li><li>通过用镊子触摸两侧的剥离器，将Reissner的膜立即脱离SGN，然后将其剥离。 注意:用微型刀将其切割掉，以清除任何横向位置受损的毛细胞区域。 </li></ol></li></ol> 3.将标本转移到文化板上<ol><li>从四个盖玻片中取出45μL的组织粘合剂溶液。用50μL无菌H 2 O清洗每个盖子，吸水。 </li><li>拿起1毫升移液器。用大约120μL的DMEM将吸液管吸湿，以防止样品粘附在尖端内部。 </li><li>使用1 mL移液管单独转移标本。从装有HBSS，小而透明的玻璃底的陪替氏培养皿中取出最多70μL，将样品与液体一起放在4孔培养皿中的4个镶嵌（用盖子盖子制成）之一上。 </li><li> 在显微镜下（50倍放大）检查样品处于正确的方向。确保去除胸膜的毛细胞面积向上。确保基底膜与修剪的基部部分面朝下并粘附在盖玻片上。 <ol><li>如果样品在检查时上下颠倒定位，则将残留在移液管尖端的HBSS沉积在嵌体中，并重新定位该片，直到其正确定向。 没有TE:这样可以防止毛细胞漂浮在培养基中，而不需要盖玻片的结构支撑，这可能导致退化。 </li></ol></li><li>使用1 mL移液管吸出过量的HBSS。等待15秒。 </li><li>将60μL制备的温育培养基（98％DMEM，1％N-2和1％氨苄青霉素）直接吸取到样品上。小心不要用吸液管接触标本。更换内外培养皿盖，并在37°C孵育O / N。 </li><li>将所有储备溶液放回适当位置，处理屠体和残留废物，根据制度指导（ 例如使用乙醇或次氯酸盐）去除外科手术台，清洁并高压灭菌器具，并丢弃相应容器中的尖锐物。 </li></ol> 4.添加最终文化媒体和兴趣物质注意:耳蜗外植体将在10-16小时后使用。 <ol><li>准备一个mixtu重复使用97％DMEM，1％胎牛血清（FBS），1％N-2和1％氨苄青霉素（在微量离心管中每孔65μL;在使用前将溶液温热至25℃）。 </li><li>在各个治疗组的溶液中加入感兴趣的其他物质。如果添加荧光物质（ 如绿色荧光蛋白（GFP））表达病毒;最近的出版物中，使用浓度为10 10 GC的腺相关病毒（AAV）48小时，50μL） 18 ，保护光。 </li><li>将来自培养箱的含耳蜗外植体培养皿转移到层流罩下。 </li><li>用吸液管从嵌体中吸出旧培养基（不含FBS）。 </li><li>加入60μL/孔的制备的温育培养（处理）培养基（97％DMEM，1％FBS，1％N-2,1％氨苄青霉素和目标物质）。 </li><li>将培养皿放回培养箱（37℃）。 </li><li>查看r在显微镜下显微镜观察以识别污染，细胞迁移或耳蜗外植体脱落的迹象，并在最多7天后进行染色。 </li></ol> 5.免疫荧光 - 第1天<ol><li>制备封闭溶液（94％磷酸盐缓冲盐水（PBS），5％正常山羊或马血清（NGS / NHS，取决于第二抗体）和1％非离子表面活性剂（参见材料表 ））和储备抗体溶液（98.6％PBS，1％NHS和0.4％非离子表面活性剂与相应的抗体）。 注意:抗体包括浓度为1:400+的兔抗Myo7A（肌球蛋白7A，内外毛细胞）和小鼠抗TuJ1 1:200+（β-微管蛋白，SGN）或小鼠（IgG1）抗体-CtBP2 1:200+（c-末端结合蛋白，用于突触前），小鼠（IgG2a）抗PSD95 1:50+（突触后密度，用于突触后）和鸡抗NF-H 1:1000+（神经丝，用于SGN和神经纤维）。 "+"means"或更高"。 </li><li>用移液管吸出培养基。小心不要触摸标本。 </li><li>在层流罩下，用无菌PBS冲洗耳蜗外植体两次，每次洗涤后将样品置于振荡器上5分钟。 </li><li>在4％多聚甲醛（PFA - 注意）中将组织固定在摇床上（罩下）20分钟。吸取PFA。 </li><li>将60μLPBS加入耳蜗外植体，将培养皿置于摇床上5分钟。吸出PBS。重复3次。 </li><li>将耳蜗外植体置于制备的阻塞溶液（94％PBS，5％NHS和1％非离子表面活性剂）中30分钟。 </li><li>将耳蜗外植体置于制备的储备抗体溶液（98.6％PBS，1％NHS和0.4％非离子表面活性剂）中，与RT相同的一抗（使用前漩涡）O / N。 注意:用缠绕在塑料包装中的湿纸巾（或铝箔，如果添加的溶液）包裹陪替氏培养皿离子含有诸如GFP表达病毒的荧光材料）以保持培养皿的潮湿并防止溶液O / N的蒸发。 </li></ol> 6.免疫荧光 - 第2天<ol><li>制备4&#39;，6-二脒基-2-苯基吲哚（DAPI）染色剂（300nM），并使用库存抗体溶液获得与步骤5.1中的一抗互补的正确的二抗混合物。 （ 例如 ，山羊抗兔488 1:200+和山羊抗小鼠568 1:200+或山羊抗小鼠（IgG1）568 1:1000+，山羊抗小鼠（IgG2a）488 1:500+，和山羊抗鸡647 1:200+或鬼笔环蛋白568 1:200+（用于内外毛细胞））。 </li><li>吸出一抗溶液。 </li><li>将60μLPBS加入耳蜗外植体，将培养皿置于摇床上5分钟。吸出PBS。重复3次。 </li><li>将耳蜗外植体置于制备的储备抗体溶液（98.6％PBS，1％NHS和0.4％非离子表面活性剂）中，果实二次抗体（使用前旋涡）在柜台上1.5小时，防止光照。 </li><li>吸出第二抗体溶液，并用DAPI染色剂（置于振荡器上1分钟然后抽吸）冲洗耳蜗外植体。 </li><li>将60μLPBS加入耳蜗外植体，将培养皿置于摇床上5分钟。吸出PBS。重复3次。 </li><li>使用镊子从4孔板中取出标本，将其浸入装有蒸馏水的接收器中，并垂直使其与纸巾接触，干燥盖玻片。 </li><li>将一滴抗衰老的安装介质放在显微镜载玻片上并倒置盖玻片，将向下的组织浸入介质中。 </li><li>使用透明的指甲油密封盖玻片，周向覆盖边缘（不在玻璃下破碎样品）。将幻灯片平放放置在远离光线的覆盖区域15 - 20分钟，直到干燥将它们放在盒子中或在共焦显微镜下检查。 注意:通过将载玻片存放在4或-20°C下，荧光染色最好保存。 </li></ol>共焦成像<ol><li>获取Corti地区器官的概览和放大图片，包括神经突和神经元。 注意:成像过程的标准设置:1,024 x 1,024像素的格式，速度为400 Hz，帧平均为3,20％的整体激光强度，通常为20X和63X透镜（含有2X数码变焦）。 DAPI，Myo7A和TuJ1染色方案的通道设置如下:405 5％DAPI"Smart Gain"766.8V"Smart Offset"0.0％ - 488 15％异硫氰酸荧光素（FITC），"Smart Gain"622.2V ，"智能偏移"0.0％ - 561 13％四异氰酸异硫氰酸四甲酯（TRITC），"智能增益"562.4V，"智能偏移"0.0％。 </li><li>使用z-stack合并图像软件获得z轴投影。 </li></ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+and+plasmid+electroporation+of+the+murine+organ+of+Corti.">Primary culture and plasmid electroporation of the murine organ of Corti.</a> J Vis Exp. (36), (2010).</li><li>Mulvaney, J. F., Dabdoub, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+time+lapse+imaging+of+mouse+cochlear+explants.">Long-term time lapse imaging of mouse cochlear explants.</a> J Vis Exp. (93), e52101 (2014).</li><li>Wang, Q., Green, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+role+of+neurotrophin-3+in+synapse+regeneration+by+spiral+ganglion+neurons+on+inner+hair+cells+after+excitotoxic+trauma+in+vitro.">Functional role of neurotrophin-3 in synapse regeneration by spiral ganglion neurons on inner hair cells after excitotoxic trauma in vitro.</a> J Neurosci. 31 (21), 7938-7949 (2011).</li><li>Landegger, L. D., 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=A+synthetic+AAV+vector+enables+safe+and+efficient+gene+transfer+to+the+mammalian+inner+ear.">A synthetic AAV vector enables safe and efficient gene transfer to the mammalian inner ear.</a> Nat Biotechnol. 35 (3), 280-284 (2017).</li><li>Dilwali, S., Landegger, L. D., Soares, V. Y., Deschler, D. G., Stankovic, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secreted+Factors+from+Human+Vestibular+Schwannomas+Can+Cause+Cochlear+Damage.">Secreted Factors from Human Vestibular Schwannomas Can Cause Cochlear Damage.</a> Sci Rep. 5, 18599 (2015).</li><li>Soares, V. Y., 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=Extracellular+vesicles+derived+from+human+vestibular+schwannomas+associated+with+poor+hearing+damage+cochlear+cells.">Extracellular vesicles derived from human vestibular schwannomas associated with poor hearing damage cochlear cells.</a> Neuro Oncol. , (2016).</li><li>Tong, M., Brugeaud, A., Edge, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerated+synapses+between+postnatal+hair+cells+and+auditory+neurons.">Regenerated synapses between postnatal hair cells and auditory neurons.</a> J Assoc Res Otolaryngol. 14 (3), 321-329 (2013).</li><li>Yuan, Y., 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=Ouabain-induced+cochlear+nerve+degeneration:+synaptic+loss+and+plasticity+in+a+mouse+model+of+auditory+neuropathy.">Ouabain-induced cochlear nerve degeneration: synaptic loss and plasticity in a mouse model of auditory neuropathy.</a> J Assoc Res Otolaryngol. 15 (1), 31-43 (2014).</li><li>Fernandez, K. A., Jeffers, P. W., Lall, K., Liberman, M. C., Kujawa, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+after+noise+exposure:+acceleration+of+cochlear+synaptopathy+in+&amp;#34;recovered&amp;#34;+ears.">Aging after noise exposure: acceleration of cochlear synaptopathy in &amp;#34;recovered&amp;#34; ears.</a> J Neurosci. 35 (19), 7509-7520 (2015).</li><li>Barclay, M., Constable, R., James, N. R., Thorne, P. R., Montgomery, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+sensory+stimulation+alters+the+molecular+make-up+of+glutamatergic+hair+cell+synapses+in+the+developing+cochlea.">Reduced sensory stimulation alters the molecular make-up of glutamatergic hair cell synapses in the developing cochlea.</a> Neuroscience. , 50-62 (2016).</li><li>Zinn, E., 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=In+Silico+Reconstruction+of+the+Viral+Evolutionary+Lineage+Yields+a+Potent+Gene+Therapy+Vector.">In Silico Reconstruction of the Viral Evolutionary Lineage Yields a Potent Gene Therapy Vector.</a> Cell Rep. 12 (6), 1056-1068 (2015).</li><li>Wu, Z., Yang, H., Colosi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+genome+size+on+AAV+vector+packaging.">Effect of genome size on AAV vector packaging.</a> Mol Ther. 18 (1), 80-86 (2010).</li><li>Shu, Y., 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=Identification+of+Adeno-Associated+Viral+Vectors+That+Target+Neonatal+and+Adult+Mammalian+Inner+Ear+Cell+Subtypes.">Identification of Adeno-Associated Viral Vectors That Target Neonatal and Adult Mammalian Inner Ear Cell Subtypes.</a> Hum Gene Ther. , (2016).</li><li>Kao, S. Y., Soares, V. Y., Kristiansen, A. G., Stankovic, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+TRAIL-DR5+pathway+promotes+sensorineural+degeneration+in+the+inner+ear.">Activation of TRAIL-DR5 pathway promotes sensorineural degeneration in the inner ear.</a> Aging Cell. 15 (2), 301-308 (2016).</li></ol>

作者没有什么可以披露的。

在进行耳蜗外植体实验之前，研究人员必须完善解剖技术。毛细胞通常在学习曲线早期进行的解剖过程中被破坏，并且其完整性的一个特别有问题的时刻是去除胸膜，这需要稳定的手，适当的工具和经验。为了节省时间和资源，应在夹层显微镜下进行视觉控制，并注意潜在的损伤区域。代替使用昂贵的一级和二级抗体，更便宜的试剂如鬼笔环肽更适用于初步实验。在某些实验（分析未受影响的部?...

感觉神经性听力损失（SNHL）反映内耳损伤或上升听觉通路。听力损失是人类最常见的感觉缺陷1 ，疗效尚不存在2 。虽然耳蜗或听觉脑干植入物可以恢复对严重到深刻的SNHL的患者的某种程度的听力，但是由这些装置提供的听力仍然与"自然"听觉非常不同，特别是在理解噪音或听音乐的尝试期间。 虽然毛细胞变性长期以来被认为是创伤性听觉事件（ 例如暴露于大声）的主要后果，但越来越多的证据表明，将信息从毛细胞传播到听觉神经的突触至少与声学创伤一样容易3 ， 4，5 </sup<html> &gt; ， 6 。由于人类听力阈值，目前用于评估听力功能的黄金标准，不预测内耳特异性细胞损伤，需要更精细的工具来尽快检测细胞变性并开始适当的治疗7 。 听力损失的有希望的药物治疗通常在体外在同质细胞培养物上测试，但是这样的系统不能准确地模拟人工耳蜗的微环境。已知耳蜗细胞分泌影响耳蜗内其他细胞类型的营养因子8,9 ，当Corti 10,11或螺旋神经节神经元（SGN） 12的器官被隔离培养或当时分析分子标记然而， 对 体外数据验证可能需要的体内研究可能需要大量的资源和时间，这在体外数据验证方面需要大量的资源和时间，这在考虑时尤为重要需要多少努力才能完成和进行听力测试的中耳或圆窗膜注射和随后的人工耳蜗解剖。有效筛选被称为耳蜗外植体的器官型离体培养物中有希望的化合物提供了经济和可靠的替代物14，15，16，17 。 本文详细介绍了一种用于产生，维护和评估治疗耳蜗外植体的方案。强调了该模型的具体应用，包括其在筛选中的应用的潜在治疗化合物和用于基因治疗的病毒载体的比较评估。 离体外植体方法允许研究人员将给定治疗对原位不同细胞群体的影响视觉化，促进细胞类型特异性机制的鉴定和随后的靶向治疗的改进。 总的来说，这种技术提供了一种离体研究耳蜗的模型，同时保持耳蜗内共存的巨大不同细胞类型之间的重要串扰。

<table><tbody><tr><td>Ampicillin, Sodium Salt</td><td>Invitrogen</td><td>11593-027</td><td></td></tr><tr><td>anti-CtBP2 antibody, mouse(IgG1)</td><td>BD Transduction Laboratories</td><td>612044</td><td></td></tr><tr><td>anti-Myo7A antibody, rabbit</td><td>Proteus Biosciences</td><td>25-6790</td><td></td></tr><tr><td>anti-NF-H antibody, chicken</td><td>EMD Millipore</td><td>AB5539</td><td></td></tr><tr><td>anti-PSD95 antibody, mouse(IgG2a)</td><td>Antibodies Inc.</td><td>75-028</td><td></td></tr><tr><td>anti-TuJ1 antibody, mouse</td><td>BioLegend</td><td>801202</td><td></td></tr><tr><td>Cell-Tak Cell and Tissue Adhesive, 5 mg</td><td>Corning</td><td>354241</td><td></td></tr><tr><td>CELLSTAR 15 mL Centrifuge Tubes, Conical bottom, Graduation, Sterile</td><td>Greiner Bio-One</td><td>188161</td><td></td></tr><tr><td>CELLSTAR Cell Culture Dish, 100 x 20 mm</td><td>Greiner Bio-One</td><td>664160</td><td></td></tr><tr><td>CELLSTAR Cell Culture Dish, 35 x 10 mm, 4 inner rings</td><td>Greiner Bio-One</td><td>627170</td><td></td></tr><tr><td>CELLSTAR Cell Culture Dish, 60 x 15 mm</td><td>Greiner Bio-One</td><td>628160</td><td></td></tr><tr><td>CELLSTAR 50 mL Centrifuge Tubes, Conical bottom, Graduation, Sterile</td><td>Greiner Bio-One</td><td>227261</td><td></td></tr><tr><td>Clear Nail Polish</td><td>Electron Microscopy Sciences</td><td>72180</td><td></td></tr><tr><td>Clear Wall Glass Bottom Dishes (Glass 40mm), PELCO&amp;reg;, Sleeve/20, 50 x 7 mm</td><td>Ted Pella Inc.</td><td>14027-20</td><td></td></tr><tr><td>Coverslips, Round, Glass, 10 mm diameter, Thickness #1, 0.13 - 0.16mm</td><td>Ted Pella Inc.</td><td>260368</td><td></td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Thermo Fisher Scientific</td><td>D1306</td><td></td></tr><tr><td>Distilled water, 500 mL</td><td>Thermo Fisher Scientific</td><td>15230-162&amp;nbsp;</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM), high glucose, pyruvate, no glutamine, 500 mL</td><td>Thermo Fisher Scientific</td><td>10313-039</td><td></td></tr><tr><td>Dumont #4 Forceps</td><td>Fine Science Tools</td><td>11241-30</td><td></td></tr><tr><td>Dumont #55 Forceps (Dumostar)</td><td>Fine Science Tools</td><td>11295-51</td><td></td></tr><tr><td>Ethyl alcohol (EtOH), Pure, 200 proof, anhydrous, &amp;ge;99.5%</td><td>Sigma-Aldrich</td><td>459836-1L</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS), qualified, USDA-approved regions, 500 mL</td><td>Thermo Fisher Scientific</td><td>10437-028&amp;nbsp;</td><td>Aliquot in 50 mL tubes and store in -20&amp;deg;C freezer</td></tr><tr><td>goat anti-chicken-647 secondary antibody</td><td>Thermo Fisher Scientific</td><td>A-21469</td><td></td></tr><tr><td>goat anti-mouse(IgG)-568 secondary antibody</td><td>Thermo Fisher Scientific</td><td>A-11004</td><td></td></tr><tr><td>goat anti-mouse(IgG1)-568 secondary antibody</td><td>Thermo Fisher Scientific</td><td>A-21124</td><td></td></tr><tr><td>goat anti-mouse(IgG2a)-488 secondary antibody</td><td>Thermo Fisher Scientific</td><td>A-21131</td><td></td></tr><tr><td>goat anti-rabbit-488 secondary antibody</td><td>Thermo Fisher Scientific</td><td>R37116</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O, sterile, EmbryoMax Ultra Pure Water, 500 mL</td><td>EMD Millipore</td><td>TMS-006-B</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS), calcium, magnesium, no phenol red, 500 mL</td><td>Thermo Fisher Scientific</td><td>14025-092</td><td></td></tr><tr><td>Instrument Tray with Lid Stainless Steel</td><td>Mountainside Medical</td><td>TechMed4255</td><td></td></tr><tr><td>Micro (dissecting) knife &amp;ndash; angled 30&amp;deg;</td><td>Fine Science Tools</td><td>10056-12</td><td></td></tr><tr><td>Microscope slides, VistaVision, color-coded, 75 x 25 mm (3 x 1&quot;), 1 mm thick, white, pack of 72</td><td>VWR</td><td>16004-382</td><td></td></tr><tr><td>N-2 Supplement (100X), 5 mL</td><td>Thermo Fisher Scientific</td><td>17502-048</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;, Sodium Bicarbonate 7.5% solution, 100 mL</td><td>Thermo Fisher Scientific</td><td>25080-094</td><td></td></tr><tr><td>NaOH, sodium hydroxide solution, 1 L</td><td>Thermo Fisher Scientific</td><td>SS266-1</td><td></td></tr><tr><td>Normal Horse Serum (NHS)</td><td>Invitrogen</td><td>16050130</td><td></td></tr><tr><td>Operating scissors</td><td>Roboz Surgical Instruments Co.</td><td>RS-6806</td><td></td></tr><tr><td>Paraformaldehyde (PFA), Reagent Grade, Crystalline</td><td>Sigma-Aldrich</td><td>P6148</td><td>Prior to use: Establish Standard Operating Procedures based on protocols available online</td></tr><tr><td>PBS, pH 7.4, 500 mL</td><td>Thermo Fisher Scientific</td><td>10010-023&amp;nbsp;</td><td>Autoclave prior to use</td></tr><tr><td>Phalloidin, Alexa Fluor 568&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A12380</td><td></td></tr><tr><td>Prep Pad, Non Sterile&amp;nbsp;</td><td>Medline</td><td>05136CS</td><td></td></tr><tr><td>Safe-Lock Microcentrifuge Tubes, Polypropylene, 0.5 mL</td><td>Eppendorf</td><td>022363719</td><td>Autoclave prior to use</td></tr><tr><td>Safe-Lock Microcentrifuge Tubes, Polypropylene, 1.5 mL</td><td>Eppendorf</td><td>022363204</td><td>Autoclave prior to use</td></tr><tr><td>Scalpel Blades - #15</td><td>Fine Science Tools</td><td>10015-00</td><td></td></tr><tr><td>Scalpel Handle - #4</td><td>Fine Science Tools</td><td>10004-13</td><td></td></tr><tr><td>Stemi 2000-C Stereo Microscope</td><td>Zeiss&amp;nbsp;</td><td>000000-1106-133</td><td></td></tr><tr><td>TCS SP5 confocal microscope</td><td>Leica</td><td>N/A</td><td></td></tr><tr><td>Triton-X (non-ionic surfactant)</td><td>Integra</td><td>T756.30.30</td><td></td></tr><tr><td>VectaShield antifade mounting medium for fluorescence</td><td>Vector Laboratories, Inc.</td><td>H-1000</td><td></td></tr><tr><td>Zipper Bag, Reclosable, 4 x 6&#039;&#039; - 2 mm thick</td><td>Zipline</td><td>0609132541599</td><td></td></tr></tbody></table>

neonatal murine cochlear explant technique as an in vitro screening tool in hearing research

虽然在过去几十年的听力研究方面取得了显着的进步，但仍然没有治疗感觉神经性听力损失（SNHL），这种情况通常涉及损伤或丧失内耳微妙的机械感觉结构。近年来出现了复杂的体外和离体测定，能够筛选越来越多的潜在治疗性化合物，同时最大限度地减少资源并加快努力开发SNHL的治疗。虽然某些细胞类型的同源文化在当前的研究中继续发挥重要作用，但许多科学家现在依赖于更复杂的鼠内耳的器官型培养物，也称为耳蜗外植体。内耳中有组织的细胞结构的保存促进耳蜗基础设施的各种成分的原位评估，包括内外毛细胞，螺旋神经节神经元，神经元ites和支持细胞。在这里，我们介绍新生儿鼠耳蜗外植体的制备，培养，治疗和免疫染色。仔细准备这些外植体有助于识别有助于SNHL的机制，并为听力研究界提供宝贵的工具。

虽然许多协议集中在Corti外植体的器官，但是这种技术试图保留包括SGN在内的整个耳蜗转向的解剖学。这使得研究人员有机会分析给药治疗对神经突和神经元的体细胞除了科尔蒂的器官之外。如本文所述，进行保留部分梗阻的解剖在技术上更具挑战性，而不是仅仅去除皮质的器官。然而，神经突和SGN区域是重要的，因为在应用前庭神经鞘瘤分泌物（ 图1 ）和细...

Watch this Scientific Journal Video about Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research at JoVE.com

新生儿小鼠耳蜗外植技术作为听力研究的体外筛查工具医学|视频

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

该方案的目的是证明新生儿鼠耳蜗外植体的制备，培养，治疗和免疫染色。该技术可用作听力研究中的体外筛选工具。

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 ...

neonatal-murine-cochlear-explant-technique-as-an-vitro-screening-tool

Universidad de Cartagena UDEC

Research

Education

JoVE Journal

JoVE Core

Biology

Medicine

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

Human Biology

Sensory Systems

SCIENTISTS IN ACTION

17.5K 次观看.  Massachusetts Eye and Ear. 该方案的目的是证明新生儿鼠耳蜗外植体的制备，培养，治疗和免疫染色。该技术可用作听力研究中的体外筛选工具。 

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

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

小鼠胚胎植耳蜗的文化和基因转移通过电穿孔

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

科研

发育生物学

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

听力：IMAP测试电池的决策意识

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

医学

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

一项战略，以确定在常见的疾病，如自闭症和精神分裂症的新突变

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

原代培养及质粒电穿孔尔蒂小鼠器官。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

生物学

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

老鼠耳蜗外植体的长期时间推移成像

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

神经科学

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

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

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

老鼠圆窗途径耳毒性剂递送:一个快速和可靠的技术诱导耳蜗细胞变性

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

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 ...

In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after co-culture with cochlear epithelium. The organ of Corti was immobilized on a plastic coverslip by pressing a portion of the Reissner's membrane generated during the dissection. MSCs confined by a glass cylinder migrated toward cochlear epithelium when the cylinder was removed. Their predominant localization was observed in the modiolus of the organ of Corti, aligned in a direction similarly to that of the nerve fibers. However, some MSCs were localized in the limbus area and showed a horizontally elongated shape. In addition, migration into the hair cell area was increased, and the morphology of the MSCs changed to various forms after kanamycin treatment. In conclusion, the results of this study indicate that the coculture of MSCs with cochlear epithelium will be useful for the development of therapeutics via cell transplantation and for studies of cell regeneration that can examine various conditions and factors.

In this study, we present a real-time imaging method using confocal microscopy to observe cells moving toward damaged tissue by ex vivo incubation with the cochlear epithelium containing the organ of Corti.

体外延时活细胞成像探索细胞向科尔蒂器官迁移

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after ...

Whole Neonatal Cochlear Explants as an In vitro Model

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is characterized by the cumulative and irreversible loss of sensory hair cells and auditory nerves in the cochlea. Entire and vital cochlear explants are one of the fundamental tools in hearing research to detect hair cell loss and to characterize the molecular mechanisms of the inner ear cells. Many years ago, a protocol for neonatal cochlear isolation was developed, and although it has been modified over time, it still holds potential for improvement. 
This paper presents an optimized protocol for isolating and culturing whole neonatal cochlear explants in multi-well culture chambers that enables the study of hair cells and spiral ganglion neuron cells along the entire length of the cochlea. The protocol was tested using cochlear explants from mice and rats. Healthy cochlear explants were obtained to study the interaction between hair cells, spiral ganglion neuron cells, and the surrounding supporting cells. 
One of the main advantages of this method is that it simplifies the organ culture steps without compromising the quality of the explants. All three turns of the organ of Corti are attached to the bottom of the chamber, which facilitates in vitro experiments and the comprehensive analysis of the explants. We provide some examples of cochlear images from different experiments with live and fixed explants, demonstrating that the explants retain their structure despite exposure to ototoxic drugs. This optimized protocol can be widely used for the integrative analysis of the mammalian cochlea.

Whole Neonatal Cochlear Explants as an In vitro Model

The current protocol updates previous protocols and incorporates relatively straightforward approaches for culturing high-quality cochlear explants. This provides reliable data acquisition and high-resolution imaging in live and fixed cells. This protocol supports the ongoing trend of studying inner ear cells.

整个新生儿耳蜗外植体作为 体外 模型

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is ...

小鼠耳蜗毛细胞的高效体外培养

Isolation and Culture of Primary Cochlear Hair Cells from Neonatal Mice

Cochlear hair cells are the sensory receptors of the auditory system. These cells are located in the organ of Corti, the sensory organ responsible for hearing, within the osseous labyrinth of the inner ear. Cochlear hair cells consist of two anatomically and functionally distinct types: outer and inner hair cells. Damage to either of them results in hearing loss. Notably, as inner hair cells cannot regenerate, and damage to them is permanent. Hence, in vitro cultivation of primary hair cells is indispensable for investigating the protective or regenerative effects of cochlear hair cells. This study aimed to discover a method for isolating and cultivating mouse hair cells. 
After manual removal of the cochlear lateral wall, the auditory epithelium was meticulously dissected from the cochlear modiolus under a microscope, incubated in a mixture consisting of 0.25% trypsin-EDTA for 10 min at 37 &#176;C, and gently suspended in culture medium using a 200 &#181;L pipette tip. The cell suspension was passed through a cell filter, the filtrate was centrifuged, and cells were cultured in 24-well plates. Hair cells were identified based on their capacity to express a mechanotransduction complex, myosin-VIIa, which is involved in motor tensions, and via selective labeling of F-actin using phalloidin. Cells reached &gt;90% confluence after 4 d in culture. This method can enhance our understanding of the biological characteristics of in vitro cultured hair cells and demonstrate the efficiency of cochlear hair cell cultures, establishing a solid methodological foundation for further auditory research.

作者聚焦：培养小鼠毛细胞用于听觉研究的进展 

Herein, we present a detailed protocol for isolating and culturing primary cochlear hair cells from mice. Initially, the organ of Corti was dissected from neonatal (aged 3-5 days) murine cochleae under a microscope. Subsequently, cells were enzymatically digested into a single-cell suspension and identified using immunofluorescence after several days in culture.

新生小鼠原代耳蜗毛细胞的分离和培养

Cochlear hair cells are the sensory receptors of the auditory system. These cells are located in the organ of Corti, the sensory organ responsible for ...

新生儿鼠耳蜗外科手术技术

摘要

探索更多视频

此视频中的章节

小鼠胚胎植耳蜗的文化和基因转移通过电穿孔

听力：IMAP测试电池的决策意识

一项战略，以确定在常见的疾病，如自闭症和精神分裂症的新突变

原代培养及质粒电穿孔尔蒂小鼠器官。

老鼠耳蜗外植体的长期时间推移成像

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

成年小鼠中的科利耳表面制备

体外延时活细胞成像探索细胞向科尔蒂器官迁移

整个新生儿耳蜗外植体作为体外模型

新生小鼠原代耳蜗毛细胞的分离和培养

新生儿鼠耳蜗外科手术技术

摘要

探索更多视频

此视频中的章节

小鼠胚胎植耳蜗的文化和基因转移通过电穿孔

听力：IMAP测试电池的决策意识

一项战略，以确定在常见的疾病，如自闭症和精神分裂症的新突变

原代培养及质粒电穿孔尔蒂小鼠器官。

老鼠耳蜗外植体的长期时间推移成像

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

成年小鼠中的科利耳表面制备

体外延时活细胞成像探索细胞向科尔蒂器官迁移

整个新生儿耳蜗外植体作为 体外 模型

新生小鼠原代耳蜗毛细胞的分离和培养

整个新生儿耳蜗外植体作为体外模型