Summary

植入和无线电图记录和视觉诱发电位清醒大鼠

Published: June 29, 2016
doi:

Summary

我们显示手术植入和记录程序,以测量从眼睛(视网膜电流图)和清醒大鼠脑(视觉诱发电位),这是更类似于在那里记录被无麻醉的困惑进行的人的条件视觉电生理信号。

Abstract

满场电图(ERG)和视觉诱发电位(VEP)是评估在实验室和临床环境视网膜和视觉通路的完整性的有用工具。目前,临床前ERG和VEP测量是在麻醉下进行,以确保稳定的电极位置。然而,麻醉的非常存在已经显示出污染正常的生理反应。为了克服这些麻醉的困惑,我们开发了一个新的平台,以测定ERG和VEP清醒大鼠。电极植入手术子conjunctivally对眼睛的测定ERG和硬膜外在视觉皮层测量VEP。振幅和灵敏度的范围/时序参数在提高发光能量测定两个ERG和VEP。在ERG和VEP信号被证明是稳定的和可重复的至少4周后手术植入。该记录ERG和VEP信号,无需麻醉能力混淆了临床前小号ETTING应以临床数据提供的高级翻译。

Introduction

在ERG和VEP 在体内工具微创以评估分别在实验室和临床既视网膜和视觉通路的完整性。全视野ERG产生的特性波形可被分解成不同的组件,与代表视网膜通路1,2的不同细胞类的每个元素。经典的全视野ERG波形由一个初始负斜率(a波),这已被证明是表示感光体的活性交曝光2-4。在一个波之后是反映中央视网膜,主要是接通双极细胞5-7的电活动的实质性利好波形(B波)。此外,一个可以改变光的能量,相互刺激间隔从杆反应8隔离锥。

闪光VEP代表了视觉皮层和脑干的电势响应视网膜光刺激9,10。该波形可以被分解成早期和晚期的组件,与早期部件反射的眼膜-geniculo具条纹通路11-13和表示在各种V1薄片大鼠11,13进行皮质加工后期成分的神经元的活性。因此,ERG和VEP的同时测量返回参与视觉通路结构的综合评估。

目前,为了记录在动物电,麻醉采用使电极稳定放置。已经出现了以测量ERG和VEP清醒大鼠14-16尝试但这些研究中采用的有线设置,这可能是麻烦的,并且可以通过限制动物的运动和自然行为17导致动物的压力。随着无线技术的最新进展,包括改进的小型化和电池寿命,现在有可能实现对ERG的遥测方法ðVEP记录,减少与有线录音和提高长期存活率带来的压力。遥测探针内部充分稳定注入已被证明是成功的为体温,血压18,活动19的慢性监测以及脑电图20。技术的这种进步还将协助重复性和意识录音稳定性,提高了平台的实用程序,用于慢性研究。

Protocol

伦理学声明:动物实验均按照与动物科学为目的的护理和使用(2013年),澳大利亚代码进行。从动物伦理委员会,澳大利亚墨尔本大学,获得动物伦理委员会批准。此处所用材料是用于实验室实验而已,并非用于医疗或兽医用途。 1.准备电极注意:一个三通道发射机被用于手术植入使2 ERG和1 VEP记录可以同时进行。三种活性和三个非活性电极需要被预先制作成在植入前的环?…

Representative Results

感光体响应由在顶部2的发光能量(1.20,1.52日志CSM -2)对于每个动物拟合延迟高斯到ERG响应的初始下行肢的前缘分析的基础上,羊肉和普格的模型22日 ,胡德和桦木23制定。此公式返回一个幅度和灵敏度参数,( 图1C和1D,分别)。双曲线函数拟合为每个动物杆双极细胞的光能量响应,这也返回一个幅度和灵敏度参数,(?…

Discussion

由于视觉电生理的微创性,在人类患者ERG和VEP录音意识的条件下进行,只需要放置电极使用局部麻醉剂的。与此相反,在动物模型中视觉电全身麻醉下常规地进行通过消除自愿眼睛和身体动作,以使稳定的电极放置。然而,常用的全身麻醉药改变如由我们以前出版24和 ​​其他25-27 ERG和VEP反应。在啮齿动物模型有意识ERG和VEP平台的这种发展提供了在动物模型中的生理反应,其可以…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

JC would like to acknowledge the David Hay Memorial Fund, The University of Melbourne for financial support in writing this manuscript. Funding for this project was provided by an ARC Linkage grant 100200129 (BVB, AJV, CTON).

Materials

Bioamplifier ADInstruments ML 135 Amplifies ERG and VEP signals
Carboxymethylcellulose sodium 1.0% Allergan CAS 0009000-11-7 Maintain corneal hydration during surgery
Carprofen 0.5% Pfizer Animal Health Group CAS 53716-49-7 Post-surgery analgesia, given with injectable saline for fluid replenishment
Chlorhexadine 0.5% Orion Laboratories 27411, 80085 Disinfection of surgical instrument
Cyanoacrylate gel activator RS components 473-439 Quickly dries cyanoacrylate gel
Cyanocrylate gel  RS components 473-423 Fix stainless screws to skull
Dental burr Storz Instruments, Bausch and Lomb E0824A 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 Automatic drill for trepanning
Enrofloxin Troy Laboratories Prophylactic antibiotic post surgey
Ganzfeld integrating sphere Photometric Solutions International Custom designed light stimulator: 36 mm diameter, 13 cm aperture size
Gauze swabs Multigate Medical Products Pty Ltd 57-100B Dries surgical incision and exposed skull surface during surgery
Isoflurane 99.9% Abbott Australasia Pty Ltd CAS 26675-46-7 Proprietory Name: Isoflo(TM) Inhalation anaaesthetic. Pharmaceutical-grade inhalation anesthetic mixed with oxygen gas for VEP electrode implant surgery
Kenacomb ointment Aspen Pharma Pty Ltd To reduce skin irritation and itching after surgery
Luxeon LEDs Phillips Lighting Co. For light stimulation, twenty 5 watt and one 1 watt LEDs, controlled by Scope software
Needle (macrosurgery) World Precision Instruments 501959 for suturing abdominal and head surgery, used with 3-0 suture, eye needle, cutting edge 5/16 circle Size 1, 15mm
Needle holder (macrosurgery) World Precision Instruments 500224 To hold needle during abdominal and head surgery
Needle holder (microsurgery) World Precision Instruments 555419NT To hold needle during ocular surgery
Optiva catheter Smiths Medical International LTD 16 or 21 G Guide corneal active electrodes from skull to conjunctiva
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 Acquire signal from telemetry transmitter, paired to telemetry data converter
Proxymetacaine 0.5% Alcon Laboratories  CAS 5875-06-9 Topical ocular analgesia
Restrainer cutom made Front of the restrainer is tapered to minimize head movement, length can be adjusted to accommodate different rat length, overall diameter is 60 mm. 
Scapel blade R.G. Medical Supplies SNSM0206 For surgical incision
Scissors (macrosurgery) World Precision Instruments 501225 for cutting tissue on the abodmen and forhead
Scissors (microsurgery) World Precision Instruments 501232 To dissect the conjunctiva for electrode attachment
Scope Software ADInstruments version 3.7.6 Simultaneously triggers the stimulus via the ADI Powerlab system and collects data
Shaver Oster Golden A5 Shave fur from surgical areas
Stainless streel screws  MicroFasteners L001.003CS304 0.7 mm shaft diameter, 3 mm in length 
Stereotaxic frame David Kopf Model 900 A small animal stereotaxic instrument for locating the implantation landmarks on the skull
Surgical drape Vital Medical Supplies GM29-612EE Ensure sterile enviornment during surgery
Suture (macrosurgery) Ninbo medical needles 3-0 for suturing abdominal and head surgery, sterile silk braided, 60cm
Suture needle (microsurgery) Ninbo medical needles 8-0 or 9-0 for ocular surgery including, suturing electrode to sclera and closing conjunctival wound, nylon suture, 3/8 circle 1×5, 30cm
Telemetry data converter  DataSciences International R08 allows telemetry signal to interface with data collection software
Telemetry Data Exchange Matrix DataSciences International Gathers data from transmitters, pair with receiver
Telemetry data receiver DataSciences International RPC-1 Receives telemetry data from transmitter
Telemetry transmitter DataSciences International F50-EEE 3 channel telemetry transmitter
Tropicamide 0.5% Alcon Laboratories  Iris dilation
Tweezers (macrosurgery) World Precision Instruments 500092 Manipulate tissues during abdominal and head surgery
Tweezers (microsurgery) World Precision Instruments 500342 Manipulate tissues during ocular surgery

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Charng, J., He, Z., Bui, B., Vingrys, A., Ivarsson, M., Fish, R., Gurrell, R., Nguyen, C. Implantation and Recording of Wireless Electroretinogram and Visual Evoked Potential in Conscious Rats. J. Vis. Exp. (112), e54160, doi:10.3791/54160 (2016).

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