同时<em>体外</em两个由视网膜>功能测试<em>在体内</em>电图系统
Summary
Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.
Abstract
An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.
Introduction
电图（ERG）是一种公认的技术，可以用来记录由光引发的视网膜的电活动。 ERG的信号由引起径向电流的电压变化（沿感光细胞和双极细胞的轴）中流动的视网膜的电阻外空间，主要产生的。第一ERG信号是从的鱼眼1表面记录在1865年霍姆格伦。艾因特霍芬和乔利1908除以2的ERG响应于光的发作分为三个不同的波，称为A，B，和c波，现在已知以反映感光体的主要的活性，ON双极细胞，和色素上皮细胞，分别3-8。 ERG可以从麻醉动物或人类（ 体内 ）的眼睛被记录时，从分离的眼制剂9，穿过分离完整视网膜（ 离体 ）3,10-15或跨越特定视网膜层与微电极（本地ERG）4,16。在这些中， 在体内 ERG是目前最广泛使用的方法，以评估视网膜的功能。它是一种非侵入性的技术，可用于诊断目的或跟随在动物或患者的视网膜疾病的进展。然而， 在体内 ERG录音制作一个复杂的信号，层峦叠成分，往往是由生理性眼外噪音（ 如呼吸和心脏活动）污染。
本地ERG可用于记录整个视网膜的特定层的信号，但它是最侵入性和具有最低信噪比（SNR），为相对于其他的ERG记录的配置。本地ERG也是技术要求高，需要昂贵的设备（ 如，显微镜和显微操作）。 Transretinal ERG从完整的，孤立的视网膜（ 体外 ERG）提供了在体内和局部ERG方法之间的妥协，允许稳定HIG^ h SNR录音来自动物或人17完整的视网膜。最近，这种方法已成功地用于研究哺乳动物，灵长类动物和人类视网膜18-20杆和视锥感光功能。另外，由于不存在在体外视网膜色素上皮，视网膜电图信号的正C波分量被除去，一个突出的负慢PIII成分显露在体外的录音。缓慢PIII组件已经显示从米勒胶质细胞的活性，以发起在视网膜21-23。因此，也可以采用离体的ERG的方法来研究Müller细胞在完整视网膜。几项研究也表明， 体外 ERG记录可以用来测量药剂浓度周围视网膜24和 测试药物25-27的安全性和有效性。
在体内系统中的多个商业可用和许多实验室不一定具有广泛的背景电使用。相比之下， 体外设备还没有着落直到最近17并因此只有极少数的实验室目前正在这个强大的技术优势。这将是有益的，使提供给更多的实验室体外 ERG录音，以推进我们对视网膜的生理和病理知识，并开发新的疗法致盲疾病。我们在这里展示了一个简单而经济实惠的体外 ERG装置17，并显示如何可以组合使用几种市售体内的ERG系统来记录棒状和锥形介导的信号（a-和b-波）和功能Müller细胞（慢PIII）从完整的野生型小鼠视网膜。
Protocol
Representative Results
Discussion
我们在这里表明，用于通过使用在体内的ERG系统组件与离体的ERG适配器一起同时获得高品质的离体的ERG记录从两个分离的小鼠视网膜的关键步骤。在此研究中，我们灌注既视网膜从具有相同的溶液（或者艾姆斯，洛克的或林格氏）的动物，但它也可以将灌注每个视网膜与不同的溶液如用于药物测试的目的。为了获得高质量的数据，最重要的步骤是从电磁噪声，小心?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由美国国立卫生研究院资助EY019312和EY021126（VJK），EY002687到眼科及视觉科学系在华盛顿大学的支持，以及防盲研究。
Materials
| In vivo ERG system | OcuScience | HMsERG | www.ocuscience.us/id77.html |
| In vivo ERG system | LKC Technologies | UTAS-E 3000 | www.lkc.com/products/UTAS/bigshot.html |
| Ex vivo adapter | OcuScience | Ex VIVO ERG adapter | www.ocuscience.us/id107.html |
| Dissection microscope | North Central Instruments | Leica M80 | May use any brand |
| IR emitter | Opto Diode Corp. | OD-50L | www.optodiode.com |
| Prowler Night Vision Scopes | B.E. Meyers Electro Optics | D4300-I | Military grade product. |
| Red filter | Rosco Laboratories | Roscolux #27 Medium Red | May be used instead of IR system |
| Red head light | OcuScience | ERGX011 | www.ocuscience.us/catalog/i29.html |
| Microscissors | WPI, Inc. | 500086 | www.wpiinc.com/ |
| Dumont tweezers #5 | WPI, Inc. | 14101 | |
| Razor blades | Electron Microscopy Sciences | 72000 | www.emsdiasum.com |
| Scale | Metler Toledo | AB54-S/FACT | May use any brand |
| pH meter and electrode | Beckman Coulter | pHI 350 | May use any brand |
| NaCl | Sigma-Aldrich | S7653 | May use any brand |
| KCl | Sigma-Aldrich | 60129 | May use any brand |
| MgCl2 | Sigma-Aldrich | 63020 | 1.0 M solution |
| CaCl2 | Sigma-Aldrich | 21114 | 1.0 M solution |
| EDTA | Sigma-Aldrich | 431788 | May use any brand |
| HEPES | Sigma-Aldrich | H3375 | May use any brand |
| Sodium Bicarbonate | Sigma-Aldrich | S6297 | May use any brand |
| Ames medium | Sigma-Aldrich | A1420 | May use any brand |
| BaCl2 | Sigma-Aldrich | B0750 | May use any brand |
| DL-AP4 | Tocris Bioscience | 101 | May use any brand |
| Succinic acid disodium salt | Sigma-Aldrich | 224731 | May use any brand |
| L-Glutamic acid | Sigma-Aldrich | G2834 | May use any brand |
| D-(+)-Glucose | Sigma-Aldrich | G7528 | May use any brand |
| Leibovitz culture medium L-15 | Sigma-Aldrich | L4386 | May use any brand |
| MEM vitamins | Sigma-Aldrich | M6895 | |
| MEM amino acids | Sigma-Aldrich | M5550 | |
| Carbogen | Airgas | UN3156 | 5% CO2 |
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