电图同时记录和麻醉大鼠视觉诱发电位
Summary
This protocol describes simultaneous measurement of electroretinogram and visual evoked potentials in anesthetized rats.
Abstract
The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the electrical responses of the retina to light stimulation, while the VEP measures the corresponding functional integrity of the visual pathways from the retina to the primary visual cortex following the same light event. The ERG waveform can be broken down into components that reflect responses from different retinal neuronal and glial cell classes. The early components of the VEP waveform represent the integrity of the optic nerve and higher cortical centers. These recordings can be conducted in isolation or together, depending on the application. The methodology described in this paper allows simultaneous assessment of retinal and cortical visual evoked electrophysiology from both eyes and both hemispheres. This is a useful way to more comprehensively assess retinal function and the upstream effects that changes in retinal function can have on visual evoked cortical function.
Introduction
电图（ERG）和视觉诱发电位（VEP）的测量提供视觉通路的完整性有用的定量评估。该ERG测量视网膜对光刺激的电反应，而VEP措施而相同的光从事件视网膜初级视觉皮层的视觉通路的相应功能的完整性。该原稿描述了在一个通常使用的实验室模型，大鼠的记录和ERG和VEP响应分析的协议。
该ERG通过量化来一道闪光视网膜的总电反应提供了许多关键的视网膜细胞类的功能完整性的指标。一个协调的一系列离子通量的光引发发病和偏移，产生可使用放置在眼外表面的电极来测量电压检测的变化。所得波形表示的本身在组合明确定义的部件的里斯，在幅度，定时和频率不同。的研究有相当体已经表明这些分量在许多脊椎动物视网膜并且该组件可从彼此分离相对保守。通过审慎地选择刺激（闪光刺激，背景，刺激间隔）的条件下和在选择合成波形的特定特性来分析一个可以确信返回视网膜细胞1,2的一组特定的量度。这些特征背后的效用，因此ERG的广泛应用，视网膜功能的非侵入性的措施。此手稿着重于方法用于测量ERG和分析其功能返回有关一些在视网膜的主要细胞类，即光感受器（PIII的成分），双极细胞（PII中成分）和视网膜神经节细胞的信息（正暗视阈值反应或PSTR）。
<p class="“jove_content”">的VEP提供光皮质反应的检测;首先从视网膜始发并且此后经由视神经，视束，丘脑（外侧膝状体，LGN）和皮层3的区域V1的视辐射串行通信。在啮齿类动物中，多数-从每只眼睛交互对4视神经纤维的（90 95％）和支配对侧中脑。不像ERG，它是作为还不可能将VEP的不同部件归因于特定的细胞类，从而5改变任意位置沿视觉通路可能影响VEP波形。尽管如此，VEP是视觉性能和视觉通路的完整性的有用的非侵入性的措施。的VEP，与ERG一起使用时，可以提供视觉系统（ 即，视网膜/视觉通路）的更完整的评价。ERG和VEP录音可以在隔离或组合来进行，这取决于APPLI阳离子。本文介绍的方法可以从两个眼睛视网膜和大脑皮质视觉诱发电生理和麻醉大鼠两个半球同时评估。这是为了更全面地评估视网膜功能和上游的效果，在视网膜功能变化可能对视觉诱发皮质功能的有效方法。
Protocol
所有的实验过程是根据实践的照顾和动物用于科学目的的使用澳大利亚代码，设置了由国家健康与医学研究委员会在澳大利亚进行。来自墨尔本大学，理学院，动物伦理委员会（批准文号0911322.1）获得伦理关。 慢性VEP电极1.植入前注：如果同时ERG和VEP信号要收集的动物必须VEP通过手术植入电极至少1周的信号收集前。 通过用氯己定（在70％乙醇0.5％）清洗消毒前实验手术台上。使…
Representative Results
ERG的a波（> -1.38日志cd.sm -2），B波（> – 4.99日志cd.sm -2）个STR（< – 4.99日志cd.sm -2）和VEPs（> – 0.52登录cd.sm -2）同时（ 图1和记录3）。在非常暗淡闪烁，一个正STR（PSTR）被认为在约闪光后110毫秒，并且在大约220毫秒的负STR（NSTR）（ 图1和2）。具有大b波的ERG，50到100毫秒的中等闪光可以以其PII响?…
Discussion
在ERG和VEP分别从视网膜和皮质，视觉功能的目标的措施。同时记录的优点是，整个视觉通路的更全面的观点得到。具体地，从它们的并发评估中的补充信息可在视觉通路提供损伤部位的一个更清楚的划分（ 例如，对于具有重叠ERG但不同VEP表现18，病症当视神经病变可以与初级脑萎缩19共存， 20时，或VEP的损失可能会受到伤病的表现在多个地点在视觉通路21,22?…
Declarações
The authors have nothing to disclose.
Acknowledgements
Funding for this project was provided by the National Health and Medical Research Council (NHMRC) 1046203 (BVB, AJV) and Melbourne Neuroscience Institute Fellowship (CTN).
Materials
| Alligator clip | generic brand | HM3022 | Stainless steel 26 mm clip for connecting VEP screw electrodes to cables |
| Bioamplifier | ADInstruments | ML 135 | For amplifying ERG and VEP signals |
| Carboxymethylcellulose sodium 1.0% | Allergan | CAS 0009000-11-7 | Viscous fluid for improving signal quality of the active ERG electrode |
| Carprofen 0.5% | Pfizer Animal Health Group | CAS 53716-49-7 | Proprietary name: Rimadyl injectable (50 mg/mL). For post-surgery analgesia, diluted to 0.5% (5 mg/mL) in normal saline |
| Chlorhexadine 0.5% | Orion Laboratories | 27411, 80085 | For disinfecting surgical instruments |
| Circulating water bath | Lauda-Königshoffen | MGW Lauda | For maintaining body temperature of the anesthetized animal during surgery and electrophysiological recordings |
| Dental amalgam | DeguDent GmbH | 64020024 | For encasing the electrode-skull assembly to make it more robust |
| Dental burr | Storz Instruments, Bausch and Lomb | #E0824A | A miniature drill head of ~0.7mm diameter for making a small hole in the skull over each hemisphere to implant VEP screws |
| Drill | Bosch | Dremel 300 series | An automatic drill for trepanning |
| Electrode lead | Grass Telefactor | F-E2-30 | Platinum cables for connecting silver wire electrodes to the amplifier |
| Faraday Cage | custom-made | Ensures light proof to maintain dark adaptation. Encloses the Ganzfeld setup to improve signal to noise ratio | |
| Gauze swabs | Multigate Medical Products Pty Ltd | 57-100B | For drying the surgical incision and exposed skull surface during surgery |
| Ganzfeld integrating sphere | Photometric Solutions International | Custom designed light stimulator: 36 mm diameter, 13 cm aperture size | |
| Velcro | VELCRO Australia Pty Ltd | VELCRO Brand Reusable Wrap | Hook-and-loop fastener to secure the electrodes and the animal on the recording platform |
| Isoflurane 99.9% | Abbott Australasia Pty Ltd | CAS 26675-46-7 | Proprietary Name: Isoflo(TM) Inhalation anaaesthetic. Pharmaceutical-grade inhalation anesthetic mixed with oxygen gas for VEP electrode implant surgery |
| Ketamine | Troy Laboratories | Ilium Ketamil | Proprietary name: Ketamil Injection, Brand: Ilium. Pharmaceutical-grade anesthetic for electrophysiological recording |
| Luxeon LEDs | Phillips Lighting Co. | For light stimulation twenty 5 watt and one 1 watt LEDs. | |
| Micromanipulator | Harvard Apparatus | BS4 50-2625 | Holds the ERG active electrode during recordings |
| Needle electrode | Grass Telefactor | F-E2-30 | Subcutaneously inserted in the tail to serve as the ground electrode for both the ERG and VEP |
| Phenylephrine 2.5% minims | Bausch and Lomb | CAS 61-76-7 | Instilled with Tropicamide to achieve maximal dilation for ERG recording |
| Povidone iodine 10% | Sanofi-Aventis | CAS 25655-41-8 | Proprietory name: Betadine, Antiseptic to prepare the shaved skin for surgery 10%, 500 mL |
| Powerlab data acquisition system | ADInstruments | ML 785 | Controls the LEDs |
| Proxymetacaine 0.5% | Alcon Laboratories | CAS 5875-06-9 | For corneal anaesthesia during ERG recordings |
| Saline solution | Gelflex | Non-injectable, for electroplating silver wire electrodes | |
| Scope Software | ADInstruments | version 3.7.6 | Simultaneously triggers the stimulus via the Powerlab system and collects data |
| Silver (fine round wire) | A&E metal | 0.3 mm | Used to make active and inactive ERG electrodes, and the inactive VEP electrode |
| Stainless streel screws | MicroFasterners | 0.7 mm shaft diameter, 3 mm in length to be implanted over the primary visual cortex and serve as the active VEP electrodes | |
| Stereotaxic frame | David Kopf | Model 900 | A small animal stereotaxic instrument for locating the primary visual cortices according to Paxinos & Watson's 2007 rat brain atlas coordinates |
| Surgical blade | Swann-Morton Ltd. | 0206 | For incising the area of skin overlaying the primary visual cortex to implant the VEP electrodes |
| Suture | Shanghai Pudong Jinhuan Medical Products Co.,Ltd | 3-0 silk braided suture non-absorbable, for skin retraction during VEP electrode implantation surgery | |
| Tobramycine eye ointment 0.3% | Alcon Laboratories | CAS 32986-56-4 | Proprietary name: Tobrex. Prophylactic antibiotic ointment applied around the skin wound after surgery |
| Tropicamide 0.5% | Alcon Laboratories | CAS 1508-75-4 | Proprietary name: 0.5% Mydriacyl eye drop, Instilled to achieve mydriasis for ERG recording |
| Xylazine | Troy Laboratories | Ilium Xylazil-100 | Pharmaceutical-grade anesthetic for electrophysiological recording |
| Pipette tip | Eppendorf Pty Ltd | 0030 073.169 | Eppendorf epTIPS 100 – 5000 mL, for custom-made electrodes |
| Microsoft Office Excel | Microsoft | version 2010 | spreadsheet software for data analysis |
| Lethabarb Euthanazia Injection | Virbac (Australia) Pty Ltd | LETHA450 | 325 mg/mL pentobarbital sodium for rapid euthanazia |
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Citar este artigo
Nguyen, C. T., Tsai, T. I., He, Z., Vingrys, A. J., Lee, P. Y., Bui, B. V. Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats. J. Vis. Exp. (113), e54158, doi:10.3791/54158 (2016).