植入和无线电图记录和视觉诱发电位清醒大鼠
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…
Disclosures
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 |
References
- Frishman, L. J. . Origins of the Electroretinogram. , (2006).
- Granit, R. The components of the retinal action potential in mammals and their relation to the discharge in the optic nerve. J Physiol. 77, 207-239 (1933).
- Brown, K. T. The eclectroretinogram: its components and their origins. Vision Res. 8, 633-677 (1968).
- Brown, K. T., Murakami, M. Biphasic Form of the Early Receptor Potential of the Monkey Retina. Nature. 204, 739-740 (1964).
- Kline, R. P., Ripps, H., Dowling, J. E. Generation of b-wave currents in the skate retina. Proc Natl Acad Sci U S A. 75, 5727-5731 (1978).
- Krasowski, M. D., et al. Propofol and other intravenous anesthetics have sites of action on the gamma-aminobutyric acid type A receptor distinct from that for isoflurane. Mol Pharmacol. 53, 530-538 (1998).
- Stockton, R. A., Slaughter, M. M. B-wave of the electroretinogram. A reflection of ON bipolar cell activity. J Gen Physiol. 93, 101-122 (1989).
- 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).
- Weinstein, G. W., Odom, J. V., Cavender, S. Visually evoked potentials and electroretinography in neurologic evaluation. Neurol Clin. 9, 225-242 (1991).
- Sand, T., Kvaloy, M. B., Wader, T., Hovdal, H. Evoked potential tests in clinical diagnosis. Tidsskr Nor Laegeforen. 133, 960-965 (2013).
- Brankack, J., Schober, W., Klingberg, F. Different laminar distribution of flash evoked potentials in cortical areas 17 and 18 b of freely moving rats. J Hirnforsch. 31, 525-533 (1990).
- Creel, D., Dustman, R. E., Beck, E. C. Intensity of flash illumination and the visually evoked potential of rats, guinea pigs and cats. Vision Res. 14, 725-729 (1974).
- Herr, D. W., Boyes, W. K., Dyer, R. S. Rat flash-evoked potential peak N160 amplitude: modulation by relative flash intensity. Physiol Behav. 49, 355-365 (1991).
- Guarino, I., Loizzo, S., Lopez, L., Fadda, A., Loizzo, A. A chronic implant to record electroretinogram, visual evoked potentials and oscillatory potentials in awake, freely moving rats for pharmacological studies. Neural Plast. 11, 241-250 (2004).
- Szabo-Salfay, O., et al. The electroretinogram and visual evoked potential of freely moving rats. Brain Res Bull. 56, 7-14 (2001).
- Valjakka, A. The reflection of retinal light response information onto the superior colliculus in the rat. Graefes Arch Clin Exp Ophthalmol. 245, 1199-1210 (2007).
- Lapray, D., Bergeler, J., Dupont, E., Thews, O., Luhmann, H. J. A novel miniature telemetric system for recording EEG activity in freely moving rats. J Neurosci Methods. 168, 119-126 (2008).
- Lim, K., Burke, S. L., Armitage, J. A., Head, G. A. Comparison of blood pressure and sympathetic activity of rabbits in their home cage and the laboratory environment. Exp Physiol. 97, 1263-1271 (2012).
- Nguyen, C. T., Brain, P., Ivarsson, M. Comparing activity analyses for improved accuracy and sensitivity of drug detection. J Neurosci Methods. 204, 374-378 (2012).
- Ivarsson, M., Paterson, L. M., Hutson, P. H. Antidepressants and REM sleep in Wistar-Kyoto and Sprague-Dawley rats. Eur J Pharmacol. 522, 63-71 (2005).
- He, Z., Bui, B. V., Vingrys, A. J. The rate of functional recovery from acute IOP elevation. Invest Ophthalmol Vis Sci. 47, 4872-4880 (2006).
- Lamb, T. D., Pugh, E. N. A quantitative account of the activation steps involved in phototransduction in amphibian photoreceptors. J Physiol. 449, 719-758 (1992).
- Hood, D. C., Birch, D. G. Rod phototransduction in retinitis pigmentosa: estimation and interpretation of parameters derived from the rod a-wave. Invest Ophthalmol Vis Sci. 35, 2948-2961 (1994).
- Charng, J., et al. Conscious wireless electroretinogram and visual evoked potentials in rats. PLoS Onez. 8, e74172 (2013).
- Galambos, R., Szabo-Salfay, O., Szatmari, E., Szilagyi, N., Juhasz, G. Sleep modifies retinal ganglion cell responses in the normal rat. Proc Natl Acad Sci U S A. 98, 2083-2088 (2001).
- Meeren, H. K., Van Luijtelaar, E. L., Coenen, A. M. Cortical and thalamic visual evoked potentials during sleep-wake states and spike-wave discharges in the rat. Electroencephalogr Clin Neurophysiol. 108, 306-319 (1998).
- Nair, G., et al. Effects of common anesthetics on eye movement and electroretinogram. Doc Ophthalmol. 122, 163-176 (2011).
- Amouzadeh, H. R., Sangiah, S., Qualls, C. W., Cowell, R. L., Mauromoustakos, A. Xylazine-induced pulmonary edema in rats. Toxicol Appl Pharmacol. 108, 417-427 (1991).
- Charng, J., et al. Retinal electrophysiology is a viable preclinical biomarker for drug penetrance into the central nervous system. J Ophthalmol. , (2016).
Cite This Article
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).