麻酔ラットにおける電図および視覚誘発電位の同時記録
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
電図（ERG）と視覚誘発電位（VEP）の測定は、視覚経路の完全性の有用な定量的評価を提供します。 VEPは、同じ光のイベント、次の一次視覚野への網膜から視覚経路の対応する機能の完全性を測定しながら、ERGは、光刺激に対する網膜の電気的応答を測定します。この原稿は、一般的に使用される実験用モデル、ラットにおけるERGおよびVEP応答の記録と分析のためのプロトコルについて説明します。
ERGは、光のフラッシュに網膜の総電気的応答を定量化することにより、キー網膜細胞クラスの数の機能的完全性の指標を提供します。イオンフラックスの協調一連の光開始によって開始され、オフセット、眼の外側に配置された表面電極を用いて測定することができる電圧の検出可能な変化を生じます。得られた波形がseの組み合わせを表し、振幅、タイミングおよび周波数の異なる明確に定義されたコンポーネントのリース、。研究の実質的な本体は、これらのコンポーネントは、比較的、多くの脊椎動物の網膜を横切って部品を互いに分離することができる保存されていることを示しています。思慮深く刺激（フラッシュ刺激、背景、刺激間隔）の条件を選択し、分析するために合成波形の特定の機能を選択することによって1は網膜細胞1,2の特定のグループの測定値を返すのを確信することができます。これらの特性は、網膜機能の非侵襲的測定として有用性ひいてはERGの広範なアプリケーションの基礎となります。この原稿が正（すなわち、光受容体（PIII成分）、双極細胞（PII成分）と網膜神経節細胞、ERGを測定し、網膜の主要な細胞のクラスのいくつかについての情報を返すために、その機能を解析するための方法論に焦点を当てて暗順応閾値応答またはPSTR）。
<p class= "jove_content"> VEPは、光に対する皮質応答のアッセイを提供します。最初の網膜から発信され、その後皮質3の領域V1に視神経、視索、視床（外側膝状核、LGN）と光学放射を介してシリアルに伝え。げっ歯類では、大多数-それぞれの目のX字形の4から視神経線維の（90〜95％）とは反対側の中脳を支配します。 ERGとは異なり、特定の細胞のクラスにVEPの異なるコンポーネントを帰するようまだできていない、どこにも視覚的な経路に沿って5こうして変更はVEP波形に影響を与える可能性があります。それにもかかわらず、VEPは、ビジュアル性能と視覚路の完全性の有用な非侵襲性の尺度です。 VEPは、ERGと組み合わせて使用される場合、視覚系（ すなわち、網膜/視覚経路）のより完全な評価を提供することができます。ERGおよびVEP記録は、アプリに応じて、単独で又は組み合わせて実施することができますカチオン。この論文に記載の方法は、網膜と皮質視覚誘発両眼からの電気生理学や麻酔したラットにおける両半球の同時評価を可能にします。これは、より包括的に網膜機能および網膜機能の変化が視覚誘発皮質機能に持つことができる上流の効果を評価するための便利な方法です。
Protocol
全ての実験手順は、オーストラリアで国立保健医療研究評議会が定めた科学的な目的のための動物の管理と使用に関する実践のオーストラリアの規範に従って行われました。倫理クリアランスはメルボルン大学、科学学部、動物倫理委員会（承認番号0911322.1）から入手しました。 1.慢性VEP電極の着床前注：同時ERGおよびVEP信号は外科的にVEPを移植しなければならない動物は、少なく…
Representative Results
ERG波（> -1.38ログcd.sm -2）、B波（> – 4.99ログcd.sm -2）のSTR（< – 4.99ログcd.sm -2）とVEPに（> – 0.52 cd.smを記録-2）（ 図1および3）を同時に記録しました。非常に薄暗い点滅で約220ミリ秒（ 図1および図2）では、正のSTR（PSTR）をフラッシュした後、約110ミリ秒を見られて、負のSTR（NSTR）。大B波とのERG、そのPI…
Discussion
ERGおよびVEPはそれぞれ網膜と皮質からの視覚機能の客観的な尺度です。同時記録の利点は、全体の視覚経路のより包括的なビューが与えられるということです。具体的には、それらの同時評価から補足情報は例えば、視神経障害がプライマリ脳萎縮19と共存することができる、ERGはまだ明確なVEPは18症状重複する障害のために（視覚路に損傷部位の鮮明な描写を提?…
Divulgazioni
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 |
Riferimenti
- Nguyen, C. T. O., Vingrys, A. J., Bui, B. V. Dietary omega-3 fatty acids and ganglion cell function. Invest Ophthalmol Vis Sci. 49, 3586-3594 (2008).
- Weymouth, A. E., Vingrys, A. J. Rodent electroretinography: methods for extraction and interpretation of rod and cone responses. Prog Retin Eye Res. 27, 1-44 (2008).
- Tsai, T. I., Bui, B. V., Vingrys, A. J. Effect of acute intraocular pressure challenge on rat retinal and cortical function. Invest Ophthalmol Vis Sci. 55, 1067-1077 (2014).
- Cowey, A., Franzini, C. The retinal origin of uncrossed optic nerve fibres in rats and their role in visual discrimination. Exp Brain Res. 35, 443-455 (1979).
- Weinstein, G. W., Odom, J. V., Cavender, S. Visually evoked potentials and electroretinography in neurologic evaluation. Neurol Clin. 9, 225-242 (1991).
- Odom, J. V., et al. Visual evoked potentials standard (2004). Doc Ophthalmol. 108, 115-123 (2004).
- Ridder, W. H., Nusinowitz, S., Heckenlively, J. R. Causes of cataract development in anesthetized mice. Exp Eye Res. 75, 365-370 (2002).
- Nixon, P. J., Bui, B. V., Armitage, J. A., Vingrys, A. J. The contribution of cone responses to rat electroretinograms. Clin Experiment Ophthalmol. 29, 193-196 (2001).
- Bui, B. V., et al. Using the electroretinogram to understand how intraocular pressure elevation affects the rat retina. J Ophthalmol. 2013, 262467 (2013).
- Nguyen, C. T., Vingrys, A. J., Bui, B. V. Dietary omega-3 fatty acids and ganglion cell function. Invest Ophthalmol Vis Sci. 49, 3586-3594 (2008).
- Hood, D. C., Birch, D. G. A quantitative measure of the electrical activity of human rod photoreceptors using electroretinography. Vis Neurosci. 5, 379-387 (1990).
- Birch, D. G., Hood, D. C., Locke, K. G., Hoffman, D. R., Tzekov, R. T. Quantitative electroretinogram measures of phototransduction in cone and rod photoreceptors – Normal aging, progression with disease, and test-retest variability. Arch Ophthalmol. 120, 1045-1051 (2002).
- Bui, B. V., Vingrys, A. J. Development of receptoral responses in pigmented and albino guinea-pigs (Cavia porcellus). Doc Ophthalmol. 99, 151-170 (1999).
- Robson, J. G., Saszik, S. M., Ahmed, J., Frishman, L. J. Rod and cone contributions to the a-wave of the electroretinogram of the macaque. J Physiol. 547, 509-530 (2003).
- Severns, M. L., Johnson, M. A. The care and fitting of Naka-Rushton functions to electroretinographic intensity-response data. Doc Ophthalmol. 85, 135-150 (1993).
- Bui, B. V., Fortune, B. Origin of electroretinogram amplitude growth during light adaptation in pigmented rats. Vis Neurosci. 23, 155-167 (2006).
- Bui, B. V., Fortune, B. Ganglion cell contributions to the rat full-field electroretinogram. J Physiol. 555, 153-173 (2004).
- Tremblay, F., Laroche, R. G., Debecker, I. The Electroretinographic Diagnosis of the Incomplete Form of Congenital Stationary Night Blindness. Vision Res. 35, 2383-2393 (1995).
- Bayer, A. U., Keller, O. N., Ferrari, F., Maag, K. P. Association of glaucoma with neurodegenerative diseases with apoptotic cell death: Alzheimer’s disease and Parkinson’s disease. Am J Ophthalmol. 133, 135-137 (2002).
- Wostyn, P., Audenaert, K., De Deyn, P. P. An abnormal high trans-lamina cribrosa pressure difference: A missing link between Alzheimer’s disease and glaucoma. Clinical Neurology and Neurosurgery. 110, 753-754 (2008).
- Yucel, Y. H., Zhang, Q. A., Weinreb, R. N., Kaufman, P. L., Gupta, N. Effects of retinal ganglion cell loss on magno-, parvo-, koniocellular pathways in the lateral geniculate nucleus and visual cortex in glaucoma. Prog Retin Eye Res. 22, 465-481 (2003).
- Gupta, N., Yucel, Y. H. What changes can we expect in the brain of glaucoma patients. Survey of Ophthalmology. 52, 122-126 (2007).
- Kong, Y. X., et al. Impact of aging and diet restriction on retinal function during and after acute intraocular pressure injury. Neurobiol Aging. 33, 1115-1125 (2012).
- Bui, B. V., Sinclair, A. J., Vingrys, A. J. Electroretinograms of albino and pigmented guinea-pigs (Cavia porcellus). Aust N Z J Ophthalmol. 26, 98-100 (1998).
- Jobling, A. I., Wan, R., Gentle, A., Bui, B. V., McBrien, N. A. Retinal and choroidal TGF-beta in the tree shrew model of myopia: isoform expression, activation and effects on function. Exp Eye Res. 88, 458-466 (2009).
- Robson, J. G., Frishman, L. J. Dissecting the dark-adapted electroretinogram. Doc Ophthalmol. 95, 187-215 (1998).
- Robson, J. G., Frishman, L. J. The rod-driven a-wave of the dark-adapted mammalian electroretinogram. Prog Retin Eye Res. 39, 1-22 (2014).
- Hudnell, H. K., Boyes, W. K. The comparability of rat and human visual-evoked potentials. Neurosci Biobehav Rev. 15, 159-164 (1991).
- Charng, J., et al. Conscious wireless electroretinogram and visual evoked potentials in rats. PLoS One. 8, e74172 (2013).
- Hetzler, B. E., Berger, L. K. Ketamine-Induced Modification of Photic Evoked-Potentials in the Superior Colliculus of Hooded Rats. Neuropharmacology. 23, 473-476 (1984).
Citazione di questo articolo
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).