新生儿鼠耳蜗外科手术技术<em>体外</em>听力研究筛选工具
Summary
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。
Abstract
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。
Introduction
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 > ， 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。
Protocol
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 准备手术台 使用70％乙醇消毒手术台。 在显微镜旁边放置两个无菌制剂垫。 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?…
Representative Results
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…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Ampicillin, Sodium Salt | Invitrogen | 11593-027 | |
| anti-CtBP2 antibody, mouse(IgG1) | BD Transduction Laboratories | 612044 | |
| anti-Myo7A antibody, rabbit | Proteus Biosciences | 25-6790 | |
| anti-NF-H antibody, chicken | EMD Millipore | AB5539 | |
| anti-PSD95 antibody, mouse(IgG2a) | Antibodies Inc. | 75-028 | |
| anti-TuJ1 antibody, mouse | BioLegend | 801202 | |
| Cell-Tak Cell and Tissue Adhesive, 5 mg | Corning | 354241 | |
| CELLSTAR 15 ml Centrifuge Tubes, Conical bottom, Graduation, Sterile | Greiner Bio-One | 188161 | |
| CELLSTAR Cell Culture Dish, 100×20 mm | Greiner Bio-One | 664160 | |
| CELLSTAR Cell Culture Dish, 35×10 mm, four inner rings | Greiner Bio-One | 627170 | |
| CELLSTAR Cell Culture Dish, 60×15 mm | Greiner Bio-One | 628160 | |
| CELLSTAR 50 ml Centrifuge Tubes, Conical bottom, Graduation, Sterile | Greiner Bio-One | 227261 | |
| Clear Nail Polish | Electron Microscopy Sciences | 72180 | |
| Clear Wall Glass Bottom Dishes (Glass 40mm), PELCO®, Sleeve/20, 50×7 mm | Ted Pella Inc. | 14027-20 | |
| Coverslips, Round, Glass, 10 mm diameter, Thickness #1, 0.13-0.16mm | Ted Pella Inc. | 260368 | |
| DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) | Thermo Fisher Scientific | D1306 | |
| Distilled water, 500 ml | Thermo Fisher Scientific | 15230-162 | |
| DMEM, high glucose, pyruvate, no glutamine, 500 ml | Thermo Fisher Scientific | 10313-039 | |
| Dumont #4 Forceps | Fine Science Tools | 11241-30 | |
| Dumont #55 Forceps (Dumostar) | Fine Science Tools | 11295-51 | |
| Ethyl alcohol, Pure, 200 proof, anhydrous, ≥99.5% | Sigma-Aldrich | 459836-1L | |
| Fetal Bovine Serum, qualified, USDA-approved regions, 500 ml | Thermo Fisher Scientific | 10437-028 | Aliquot in 50 ml tubes and store in -20°C freezer |
| Glutamate – GlutaMAX supplement, 100 ml | Thermo Fisher Scientific | 35050-061 | |
| goat anti-chicken-647 secondary antibody | Thermo Fisher Scientific | A-21469 | |
| goat anti-mouse(IgG)-568 secondary antibody | Thermo Fisher Scientific | A-11004 | |
| goat anti-mouse(IgG1)-568 secondary antibody | Thermo Fisher Scientific | A-21124 | |
| goat anti-mouse(IgG2a)-488 secondary antibody | Thermo Fisher Scientific | A-21131 | |
| goat anti-rabbit-488 secondary antibody | Thermo Fisher Scientific | R37116 | |
| H2O, sterile, EmbryoMax Ultra Pure Water, 500ml | EMD Millipore | TMS-006-B | |
| HBSS, calcium, magnesium, no phenol red, 500 ml | Thermo Fisher Scientific | 14025-092 | |
| Instrument Tray with Lid Stainless Steel | Mountainside Medical | TechMed4255 | |
| Micro (dissecting) knife – angled 30° | Fine Science Tools | 10056-12 | |
| Microscope slides, VistaVision, color-coded, 75 x 25 mm (3 x 1"), 1 mm thick, white, pack of 72 | VWR | 16004-382 | |
| N-2 Supplement (100X), 5 ml | Thermo Fisher Scientific | 17502-048 | |
| NaHCO3, Sodium Bicarbonate 7.5% solution, 100 ml | Thermo Fisher Scientific | 25080-094 | |
| NaOH, sodium hydroxide solution, 1 l | Thermo Fisher Scientific | SS266-1 | |
| Normal Horse Serum (NHS) | Invitrogen | 16050130 | |
| Operating scissors | Roboz Surgical Instruments Co. | RS-6806 | |
| Paraformaldehyde, Reagent Grade, Crystalline | Sigma-Aldrich | P6148 | Prior to use: Establish Standard Operating Procedures based on protocols available online |
| PBS, pH 7.4, 500 ml | Thermo Fisher Scientific | 10010-023 | Autoclave prior to use |
| Phalloidin, Alexa Fluor 568 | Thermo Fisher Scientific | A12380 | |
| Prep Pad, Non Sterile | Medline | 05136CS | |
| Safe-Lock Microcentrifuge Tubes, Polypropylene, 0.5 ml | Eppendorf | 022363719 | Autoclave prior to use |
| Safe-Lock Microcentrifuge Tubes, Polypropylene, 1.5 ml | Eppendorf | 022363204 | Autoclave prior to use |
| Scalpel Blades – #15 | Fine Science Tools | 10015-00 | |
| Scalpel Handle – #4 | Fine Science Tools | 10004-13 | |
| Stemi 2000-C Stereo Microscope | Zeiss | 000000-1106-133 | |
| TCS SP5 confocal microscope | Leica | N/A | |
| Triton-X (non-ionic surfactant) | Integra | T756.30.30 | |
| VectaShield antifade mounting medium for fluorescence | Vector Laboratories, Inc. | H-1000 | |
| Zipper Bag, Reclosable, 4'' x 6'' – 2 mil. thick | Zipline | 0609132541599 |
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Cite This Article
Landegger, L. D., Dilwali, S., Stankovic, K. M. Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research. J. Vis. Exp. (124), e55704, doi:10.3791/55704 (2017).