大鼠视网膜仿生化学调节的神经递质谷氨酸的体外实验方法
Summary
本议定书描述了一种新的方法, 研究一种形式的化学神经的 wholemount 大鼠视网膜体外与神经递质谷氨酸。化学神经是一种有前途的替代传统的电神经视网膜神经元治疗不可逆转性失明引起的感光变性疾病。
Abstract
感光变性疾病通过视网膜感光细胞的逐渐丧失而导致无法挽回的失明。视网膜假体是一种新兴的治疗感光退行性疾病, 寻求恢复视力的人为刺激幸存的视网膜神经元, 希望能获得可理解的视觉感知的病人。目前的视网膜假体已证明是成功的恢复有限的视力患者使用一系列电极电刺激视网膜, 但面临巨大的物理障碍, 恢复高视力, 自然视力的病人。使用天然神经递质的化学神经是一种仿生替代电刺激, 可以绕过与使用电神经的视网膜假肢相关的基本限制。具体而言, 化学神经有可能恢复更多的自然视力与可比或更好的视觉视力患者通过注射极少量的神经递质, 相同的自然药物的交流使用的视网膜化学突触, 比当前的电子假肢更精细的分辨率。然而, 作为一个相对未被探索的刺激范例, 没有建立的协议为实现视网膜的化学刺激在体外。这项工作的目的是提供一个详细的框架, 以完成化学刺激视网膜的研究者谁希望研究的潜在化学调节的视网膜或类似的神经组织体外。在这项工作中, 我们描述的实验设置和方法, 诱导视网膜神经节细胞 (王国) 穗反应类似于视觉光反应的野生型和感光 wholemount 大鼠视网膜的注射控制体积神经递质谷氨酸进入视网膜空间使用玻璃 micropipettes 和定制多端口微流控装置。这种方法和协议是通用的, 足以适应调节使用其他神经递质或甚至其他神经系统组织。
Introduction
感光性退行性疾病, 如视网膜色素变性和年龄相关的黄斑变性, 是导致视力丧失的可继承原因, 目前是无法治愈的1,2。尽管这些疾病是由各种特定的基因突变引起的, 但感光变性疾病是由视网膜感光细胞的逐渐丧失而引起的, 最终导致失明。光的丢失引发了整个视网膜的广泛重塑, 但存活的视网膜神经元, 包括双极细胞和视网膜, 仍然完好无损, 即使在感光变性的高级阶段也相对正常,3 ,4,5,6,7。
这些疾病的机制和病理特点已经很好的特征3,4,5,6,7 , 但有效的治疗仍然难以捉摸。在过去的三年中, 世界各地的研究人员已经研究了多种治疗方法, 以恢复视力的那些受影响的感光变性疾病, 包括基因治疗8, 干细胞治疗9,视网膜移植10, 人工刺激11,12存活的视网膜神经元。其中, 最有临床意义的是视网膜假体, 这是人工神经装置, 传统上使用了一系列电极, 以电刺激双极细胞或视网膜在特定模式的目标在患者中创建人工视觉感知11。当前世代电子假肢, 例如守卫 II13和阿尔法 IMS14设备, 获得了临床批准, 并且初步研究表明, 他们能改善患者的生活质量通过恢复测量视觉使用两个前 (视网膜的前面) 和视网膜 (视网膜的后面) 植入的设备15,16。世界各地的研究小组正在努力推进视网膜假肢超越这些 first-generation 设备的成功17,18,19,20 ,但已面临困难设计一个电子假肢能够恢复高视力视力低于法律失明水平的病人。最近的研究表明, 实现更高的空间分辨率比目前的代为主假肢是挑战, 因为电荷注入限制, 这就需要使用大的电极来安全地刺激视网膜神经元以空间分辨率为代价,即视觉灵敏度11,21。此外, 电刺激是进一步有限的, 因为它通常刺激所有附近的细胞, 从而引起病人的不自然和混淆的看法, 很大程度上是因为它是一个固有的非自然刺激范例21。然而, 早期的电刺激成功表明, 人工神经可以是一种有效的治疗感光变性疾病。这导致一个假设, 更有效的治疗可能是可以通过刺激视网膜与神经递质化学品, 在化学突触沟通的自然代理人。该方法的目的是探讨化学刺激的治疗可行性, 旨在模拟视网膜神经元之间的突触通信的自然系统, 作为电刺激的仿生替代品。用于视网膜假体
对化学性视网膜假体的治疗性化学刺激的概念的翻译依赖于化学活化的目标视网膜神经元, 少量的本地神经递质, 如谷氨酸, 通过微流控物释放一种装置, 包括大量的 microports 以响应视觉刺激。这样, 一个化学的视网膜假体将本质上是一个仿生的人工感光层, 把光子自然地到达视网膜的化学信号。由于这些化学信号在正常的视网膜信号中使用相同的神经递质, 并通过正常视觉通路所使用的相同突触通路刺激退化视网膜的存活的视网膜神经元, 由此产生的视觉通过一个化学的视网膜假体的知觉可以更自然和可理解相比, 通过一个电动假肢诱发。此外, 由于神经递质被释放的 microports 可以非常小, 并排列在高密度, 不像电极, 一个潜在的化学假体可能能够获得更多的焦点刺激和更高的空间分辨率比电子假肢。因此, 基于这些潜在的优势, 化学性视网膜假体提供了一个非常有前途的替代电气假肢。
然而, 直到最近, 对视网膜的化学刺激也相对较少。而电刺激的视网膜已经很好地表征了几十年的工作通过体外22,23,在体内23,24, 和临床研究13 ,14, 对化学刺激的研究仅限于少数体外工作25,26,27,28。耶齐和芬利森26和 Inayat et al.27分别使用单电极和多阵列 (前) 对视网膜的化学刺激进行了实验, 以记录视网膜神经元的谷氨酸诱发反应。最近, 朗特里et al.28显示了视网膜侧的谷氨酸和视网膜通路的差异刺激, 并记录了视网膜上多个部位的神经元反应。虽然这些工作已经初步确定了化学刺激的可行性, 但进一步的研究对于研究这种方法的许多方面是非常必要的, 除了这些方面, 到目前为止25,26,27,28, 并 fine-tune 的治疗刺激参数在两个体外和在体内动物模型之前, 把这个概念转化为化学视网膜假体如上所述。然而, 目前还没有确定的方法, 以完成化学刺激的视网膜在文献中使用的方法, 在以前的作品没有被描述的细节, 这将是必要的复制研究。因此, 本方法的基本原理是提供一个明确的框架来进行体外对视网膜的化学刺激, 对于那些有兴趣复制我们以前的研究的调查者27, 28或进一步推进这一新生的化学神经概念。
在这里, 我们演示了一种进行体外的方法, 对野生型大鼠 wholemount 视网膜神经元的化学刺激及感光变性大鼠模型的模拟疾病在人。在体外模型中开发这种刺激方法的基本原理是评估各种刺激参数的治疗范围, 并研究在中不可能或难以观察到的神经反应特性. 体内的模型, 特别是在最初的研究集中在评估这种方法的可行性。在这个过程中, 我们显示站点和同时多站点的视网膜的化学刺激通过提供少量的1毫米谷氨酸近靶视网膜神经元通过商业可用的单玻璃 micropipettes 和自定义多口微流控装置, 分别。虽然站点和多站点刺激完成了研究化学调节的治疗可行性的基本目标, 但它们都具有独特的优势。站点刺激, 这可能是完成与商业可用的 pre-pulled 玻璃 micropipettes, 可用于直接注入化学品的视网膜下在一个单一的地点, 并有助于调查, 如果可见的研资局穗率类似于视觉诱发光反应的反应可以在注射部位局部。另一方面, 多站点刺激, 需要专门制作的多端口微流控装置, 可以被用来在多个地点的视网膜表面上注入化学物质, 并有助于研究谷氨酸诱发 RCG反应模式对应于模式刺激研究中的谷氨酸注射模式。
Protocol
Representative Results
Discussion
这里介绍的方法展示了一个独特的神经刺激模式, 其中视网膜神经元的化学刺激, 注射当地神经递质化学物质进入视网膜的地下体外。这种化学刺激技术在传统的电刺激技术中提供了一些好处, 包括选择性和靶神经元的高聚焦特异性。上面的协议详细说明了如何小体积的神经递质谷氨酸注射在目标视网膜神经元附近使用单玻璃微或自定义微流控流体设备引出在生理学上重要的研资反应。虽然?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
论文中介绍的工作得到了国家科学基金会的支持, 研究和创新领域的新兴前沿 (NSF-EFRI) 方案赠款0938072。本文的内容完全是作者的责任, 不一定代表 NSF 的官方观点。作者还希望感谢 Dr. Samsoon Inayat 为他的工作设计和测试的初步实验设置的化学刺激和 Mr. 温 Raghunathan 为他的工作设计, 制作, 并评价了多端口微流控装置使用这项研究。
Materials
| Microelectrode array, perforated layout | Multi Channel Systems, GmbH | 60pMEA200/30iR-Ti-pr | http://www.multichannelsystems.com/products/microelectrode-arrays/60pmea20030ir-ti |
| MEA amplifier | Multi Channel Systems, GmbH | MEA1060-Inv | http://www.multichannelsystems.com/products/mea1060-inv |
| Bottom perfusion groundplate for pMEA | Multi Channel Systems, GmbH | MEA1060-Inv-(BC)-PGP | http://www.multichannelsystems.com/products/mea1060-inv-bc-pgp |
| 3-axis Motorized Micromanipulator | Sutter Instruments, Novato, CA | MP-285 | https://www.sutter.com/MICROMANIPULATION/mp285.html |
| Micromanipulator Control System | Sutter Instruments, Novato, CA | MPC-200 | https://www.sutter.com/MICROMANIPULATION/mpc200.html |
| Gantry style micromanipulator stand with linear slide | Sutter Instruments, Novato, CA | MT-75/LS | https://www.sutter.com/STAGES/mt75.html |
| 8-channel Programmable Multichannel Pressure Injector | OEM: MicroData Instrument, S. Plainfield, NJ Vendor: Harvard Apparatus UK |
PM-8000 or PM-8 | OEM: http://www.microdatamdi.com/pm8000.htm Vendor: https://www.harvardapparatus.co.uk/webapp/wcs/stores/servlet/product_11555_10001_39808_-1_HAUK_ProductDetail |
| Axopatch 200A Integrating Patch Clamp Amplifier | Molecular Devices, Sunnyvale, CA | Axopatch 200A | Axopatch 200A has been replaced with a newer model Axopatch 200B: https://www.moleculardevices.com/systems/axon-conventional-patch-clamp/axopatch-200b-amplifier |
| Patch clamp headstage | Molecular Devices, Sunnyvale, CA | CV 201A | http://mdc.custhelp.com/app/answers/detail/a_id/16554/~/axopatch-200a%3A-selection-cv-headstage |
| Vacuum waste kit | ALA Scientific Instruments, Farmingdale, NY | VMK | http://alascience.com/product/vacuum-waste-kit/ |
| Pipette holder | Warner Instruments, Hamden, CT | QSW-A10P | https://www.warneronline.com/product_info.cfm?id=915 |
| Pre-pulled 10 μm tip diameter glass micropipettes | World Precision Instruments, Sarasota, FL | TIP10TW1 | https://www.wpiinc.com/products/laboratory-supplies/make-selection-pre-pulled-glass-pipettes-plain/ |
| Zoom stereomicroscope | Nikon, Tokyo, Japan | SMZ-745T | https://www.nikoninstruments.com/Products/Stereomicroscopes-and-Macroscopes/Stereomicroscopes/SMZ745 |
| Microscope boom stand with dual linear ball bearing arm | Old School Industries, Inc., Dacono, CO | OS1010H-16BB | http://www.osi-incorp.com/productdisplay/dual-linear-ball-bearing-arm |
| Zoom Stereo Microscope with C-LEDS Hybrid LED Stand | Nikon, Tokyo, Japan | SMZ-445 | https://www.nikoninstruments.com/Products/Stereomicroscopes-and-Macroscopes/Stereomicroscopes/SMZ445 |
| Inverted microscope system | Nikon, Tokyo, Japan | Eclipse Ti-E | https://www.nikoninstruments.com/Products/Inverted-Microscopes/Eclipse-Ti-E |
| Ames medium | Sigma-Aldrich, St. Louis, MO | A1420 | http://www.sigmaaldrich.com/catalog/product/sigma/a1420 |
| L-Glutamic Acid (Glutamate) | Sigma-Aldrich, St. Louis, MO | G5667 | http://www.sigmaaldrich.com/catalog/product/mm/100291 |
| Sodium bicarbonate | Sigma-Aldrich, St. Louis, MO | S8761 | http://www.sigmaaldrich.com/catalog/product/sigma/s8761 |
| 60 mm Petri dish (10 mm tall) | Fischer Scientific, Waltham, MA | FB0875713A | 60 mm clear petri dish; https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-clear-lid-12/fb0875713a |
| Jewelers #5 Forceps | World Precision Instruments, Sarasota, FL | 555227F | https://www.wpiinc.com/products/laboratory-supplies/555227f-jewelers-5-forceps-11cm-straight-titanium/ |
| Standard Scalpel Blad #24 | World Precision Instruments, Sarasota, FL | 500247 | https://www.wpiinc.com/products/laboratory-supplies/500247-standard-scalpel-blade-24/ |
| Scalpel Handle #4 | World Precision Instruments, Sarasota, FL | 500237 | https://www.wpiinc.com/products/laboratory-supplies/500237-scalpel-handle-4-14cm/ |
| Vannas Tubingen Dissection Scissors | World Precision Instruments, Sarasota, FL | 503378 | https://www.wpiinc.com/products/laboratory-supplies/503378-vannas-tubingen-scissors-8cm-straight-german-steel/ |
| Nylon mesh kit | Warner Instruments, Hamden, CT | NYL/MESH | https://www.warneronline.com/product_info.cfm?id=1173 |
| Harp slice grid | ALA Scientific Instruments, Farmingdale, NY | HSG-5AD | http://alascience.com/product/standard-harp-slice-grids/ |
| Ag/AgCl reference electrode pellet | Multi Channel Systems, GmbH | P1060 | http://www.multichannelsystems.com/products/p1060 |
| 4 Channel Valve Controlled Gravity Perfusion System | ALA Scientific Instruments, Farmingdale, NY | VC3-4xG | http://alascience.com/product/4-channel-valve-controlled-gravity-perfusion-system/ |
| Zyla 5.5 sCMOS microscope camera | Andor Technology, Belfast, UK | Zyla 5.5 sCMOS | http://www.andor.com/scientific-cameras/neo-and-zyla-scmos-cameras/zyla-55-scmos |
| Silver wire (50 μm diameter) | Fischer Scientific, Waltham, MA | AA44461G5 | https://www.fishersci.com/shop/products/silver-wire-0-05mm-0-002-in-dia-annealed-99-99-metals-basis-3/aa44461g5 |
| Tygon microbore tubing (1.6 mm diameter) | Cole Parmer, Vernon Hills , IL | EW-06419-01 | https://www.coleparmer.com/i/tygon-microbore-tubing-0-020-x-0-060-od-100-ft-roll/0641901 |
| Tilting Tool Holder with Steel Cannula | ALA Scientific Instruments, Farmingdale, NY | TILTPORT | One each of these were utilized for top perfusion and suction; http://alascience.com/product/tilting-tool-holder-with-steel-cannula/ |
| Roscolux #26 Light Red Filter Sheet | Rosco Laboratories Inc., 52 Harbor View, Stamford, CT | R2611 | Manufacturer: http://us.rosco.com/en/products/catalog/roscolux Vendor: https://www.bhphotovideo.com/c/product/43957-REG/Rosco_RS2611_26_Filter_Light.html |
| Smith & Wesson Galaxy Red Flashlight | Smith & Wesson, 2100 Roosevelt Avenue, Springfield, MA | 4588 | Manufacturer: https://www.smith-wesson.com/ Vendor: http://www.mypilotstore.com/mypilotstore/sep/4588 |
| MC_Rack Software | Multi Channel Systems, GmbH | MC_Rack | http://www.multichannelsystems.com/software/mc-rack |
| Labview Software | National Instruments, Austin, TX | LabVIEW | http://www.ni.com/labview/ |
| NIS-Elements: Basic Research Software | Nikon, Tokyo, Japan | NIS-Elements BR | https://www.nikoninstruments.com/Products/Software/NIS-Elements-Basic-Research |
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