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Neurociência

非约束脑电遥测：硬膜外和脑深部脑电立体定位放置电极

Published: June 25, 2016
doi:
10.3791/54216
, , , , , , ,
1Department of Neuropsychopharmacology,Federal Institute for Drugs and Medical Devices (Bundesinstitut für Arzneimittel und Medizinprodukte, BfArM), 2Molecular and Cellular Cognition Lab,German Center for Neurodegenerative Diseases (Deutsches Zentrum für Neurodegenerative Erkrankungen, DZNE)

Summary

非约束脑电遥测是一种有价值的方法以自由移动啮齿类动物记录在体内长期脑电图。该详细协议描述了在不同脑区，以便获得CNS节律和CNS相关的行为的阶段可靠录音立体硬膜外和深脑电极放置。

Abstract

植入脑电图遥测是在神经精神病学和神经变性疾病，以及癫痫的转基因小鼠模型的神经学表征中央的相关性。这种强大的技术不仅提供了有价值的见解的基本病理生理机制， 即 ，中枢神经系统相关的疾病发病机理，这也促进了新的翻译， 即 ，治疗方法的发展。而竞争的技术，利用在外套或系留系统中使用的记录系统，从他们的非生理性抑制到半约束的性格受到影响，无线电遥测脑电图记录克服这些缺点。从技术上讲，植入脑电图遥测允许各种生理和病理条件下硬膜外深，脑脑电图精确和高度敏感的测量。首先，我们提出了一个直线前进的详细协议，成功，快速，高效的技术产生高品质electrocorticograms硬膜外（面）脑电图记录。其次，我们将演示如何植入深，脑电图脑电极， 例如 ，在海马（electrohippocampogram）。对于这两种方法，使用一个计算机化的三维立体电极注入系统。射频发射机本身被注入到在小鼠和大鼠皮下小袋。特别注意也必须支付给预，对实验动物的围绝经期和术后治疗。小鼠和大鼠，合适的麻醉的术前准备，以及术后治疗和疼痛管理中详细描述。

Introduction

遥测是用于测量各种行为和生理特性的不同大小的自觉，奔放的动物，特别是脑电图，心电图，肌电图，血压，身体核心温度和活动测量1-7的背景下，最有价值的方式方法。从理论上说，任何物种可使用可植入的EEG遥测从实验室啮齿动物如小鼠和大鼠，以猫，狗，猪和灵长类3,8进行分析。即使是鱼类，爬行动物和两栖动物都受到无线电遥测调查9。在过去的二十年中，可植入的EEG遥测已经被证明是人类疾病的各种转基因动物模型，如癫痫，睡眠障碍，神经变性和神经精神障碍7,10-12的表征有价值。在过去，很多方式方法收集生理数据，包括生物电势小鼠和大鼠一直递减ribed。穿夹克录像机系统，物理克制方法，非植入radiotransmitters和系留系统已收到主要关注在过去的13,14。现今，对于无线电遥测植入各种系统是市售的。然而，文学的屏幕还透露29出版物描述自制无线电遥测系统15-40的发展。而自制系统很可能更便宜和更用户适于市售系统是直线前进，安装比较容易，并可以快速地进行设置。

可植入的EEG遥测具有许多比同类技术如物理约束的方法，穿在护套系统或系留的方法的优点。后者是由定义约束， 即 ，动物是无法移动或它的正常行为受到损害。它甚至可能需要麻醉动物为采集重新负责数据。然而现代系留系统有可能是抑制少，但是这需要经过科学验证。另一方面遥测允许动物表现出其行为的完整全集而不时空限制，因此，被认为是优越的，以抑制方法和更预测，可以在人类1,3-获取的结果。它是已知的相当长一段时间了抑制方法可以显着地改变基本的生理参数， 例如 ，食物摄入量，身体核心体温，血压和心脏速率和用于实施例3的体力活动。系留系统代表一个仍然被广泛使用的经典限制的做法13,14。这要么硬膜外或深电极电极通常连接到锚定到头盖骨的微型插座。插座本身被暴露的电缆，其允许动物的相对自由运动的附着。 ALTHough时下系留系统已变得极为花丝和高度灵活的，它的主要缺点之一是，它仍然是半约束。此外，还有可能是感染了在电极植入部位的危险随​​着动物倾向于操纵从其主体（头部）始发任何外部设备。虽然无线遥测技术在不同物种已经在60年代末描述并因此存在了几十年，它只是在最近才负担得起的，可靠的，而且比较容易使用的10,41,42，特别是在小啮齿类实验动物如小鼠和大鼠。小型，微型植入脑电图发射机现在可商购，并且可以在小鼠中有20克（约10周）更大的植入。因此，特别是转基因小鼠模型的电生理学表征已成为可植入的EEG遥测这些天应用的一个主要领域。动物的大小不再是一个绝对的实验restric而化发射机“电池的寿命的确是。尽管其有限的生命时间，植入式发射系统能够最大限度地减少通过抑制系统相关的潜在的录音带来的压力最不利的。鼠害可呈现的生理行为，包括休息，运动活性（探索）和睡眠（REM，慢波睡眠）43,44的完整医疗设备。重要的是，植入式遥测能强烈地减少动物使用3。目前，对如何限制实验动物科学的数量和减少他们的痛苦激烈的讨论。显然，动物实验和人类和动物疾病的动物模型是我们的底线病理生理学和治疗取得进步的了解是必不可少的。此外，动物实验是新药研发的关键。他们基本上对药物临床前牌/毒物学研究贡献因此，致力于人类和动物保健。这是值得注意的是，目前没有替代品尚未有对动物的研究来了解这将是不可能的，否则将引起复杂的病理生理机制。与此同时，在3R， 即 ，更换，减少和细化战略在欧盟和美国大力鼓励研究补充和替代方法。遥测是一个成功的3R策略，因为它可以减少与其它技术相比实验动物的数量和他们的痛苦的一个重要例子。

在这里，我们提供详细的和连续的一步一步的方式在小鼠和大鼠进行射频发射机的皮下袋注入​​。此第一序列之后是立体硬膜外和深脑脑电图电极定位的描述。特别注意的是住房条件，麻醉，围绝经期和术后疼痛管理和可能的抗感染治疗。重点是计算机化的三维立体的方式可靠地瞄准硬膜外和深脑结构。我们也会在脑电图电极植入实验频繁陷阱和战略的术后恢复过程中减少损伤和疼痛管理的优化意见。最后，表面和深脑电图记录的实例。

Protocol

伦理学声明：根据当地和机构理事会关于动物保健（波恩大学，BfArM，LANUV，德国）的指导方针进行所有的动物实验。此外，所有的动物实验是按照上级的立法， 如进行。，的1986年11月24日（86/609 / EEC）或单个区域或国家立法欧洲共同体理事会指令。具体的努力来尽量减少使用动物和他们的痛苦的数量。 1.实验动物实验动物和品种选择在啮齿类动物中进行?…

Representative Results

本节说明了从表层和深层，颅内脑电图记录获得的例子。最初，它应该指出，在生理条件下的基线录音是以下如随后的录音，药物治疗前强制性的。这样的基线记录可能会提供有关不同的行为状态或睡眠/昼夜节律的大脑节律的功能相互依存的有价值的信息。这里，我们显示以下的proconvul​​sive / psychoenergetic药物急性施用记录，癫痫发作活动的例子。如上所?…

Discussion

可植入的EEG遥测是中央的相关性，因为它是一个非限制的技术，使实验动物来执行他们的行为1,3充分剧目。这是主要感兴趣的是遥测方法使得不仅自发脑电图记录但在认知任务和昼夜分析设置，如T-迷宫，迷宫，水迷宫，剥夺睡眠的任务或每当脑电图记录是必要的或有帮助也记录在复杂的认知和运动活力。

这个协议描述了在小鼠和大鼠并连接到可植入的EEG射频发射机?…

Declarações

The authors have nothing to disclose.

Acknowledgements

The authors would like to thank Dr. Christina Ginkel (German Center for Neurodegenerative Diseases, DZNE), Dr. Michaela Möhring (DZNE) and Dr. Robert Stark (DZNE) for assistance in animal breeding and animal health care. This work was financially supported by the Federal Institute for Drugs and Medical Devices (Bundesinstitut für Arzneimittel und Medizinprodukte, BfArM) Bonn, Germany.

Materials

Carprofen (Rimadyl VET – InjektionA2:D43slösung) Pfizer PZN 0110208 20 ml
binocular surgical magnification microscope  Zeiss Stemi 2000 0000001003877, 4355400000000, 0000001063306, 4170530000000, 4170959255000, 4551820000000, 4170959040000, 4170959050000
bulldog serrefine F.S.T. 18051-28 28mm
cages (Macrolon) Techniplast 1264C, 1290D
cold light source Schott KL2500 LCD 9.705 202 ordered at Th.Geyer
cotton tip applicators (sterile) Carl Roth  EH12.1
Dexpanthenole (Bepanthen Wund- und Heilsalbe) Bayer PZN: 1578818
drapes (sterile) Hartmann PZN 0366787
70% ethanol Carl Roth  9065.5
0.3% / 3% hydrogene peroxide solution Sigma 95321 30% stock solution 
gloves (sterile) Unigloves 1570
dental glas ionomer cement KentDental /NORDENTA 957 321
2% glutaraldehyde solution Sigma G6257
Graefe Forceps-curved, serrated F.S.T. 11052-10
Halsey Micro Needle Holder-Tungsten Carbide F.S.T. 12500-12 12.5 cm
heat-based surgical instrument sterilizer F.S.T. 18000-50
heating pad AEG HK5510 520010 ordered at myToolStore
high-speed dental drill Adeor SI-1708
Iris scissors extra thin  F.S.T. 14058-09 9 cm
Inhalation narcotic system (isoflurane) Harvard Apparatus GmbH 34-1352, 10-1340, 34-0422, 34-1041, 34-0401, 34-1067, 72-3044, 34-0426, 34-0387, 34-0415, 69-0230
Isoflurane Baxter 250 ml PZN 6497131
Ketamine Pfizer PZN 07506004
lactated Ringer’s solution (sterile) Braun L7502
Lexar-Baby Scissors-straight, 10 cm F.S.T. 14078-10 10 cm
Nissl staining solution Armin Baack BAA31712159
non-absorbable suture material 5-0/6-0 (sterile) SABANA (Sabafil) N-63123-45
Covidien (Sofsilk) S1172, S1173
Halsey Needle Holder F.S.T. 12001-13 13 cm
pads (sterile) ReWa Krankenhausbedarf 2003/01
0.9% saline (NaCl, sterile) Braun PZN:8609255
scalpel blades with handle (sterile) propraxis 2029/10
Standard Pattern Forceps F.S.T. 11000-12, 11000-14 12 cm and 14.5 cm length
Steel and tungsten electrodes parylene coated  FHC Inc., USA) UEWLGESEANND
stereotaxic frame Neurostar 51730M ordered at Stoelting
(Stereo Drive-New Motorized Stereotaxic)
tapes (sterile) BSN medical GmbH & Co. KG 626225
TA10ETA-F20  DSI 270-0042-001X Radiofrequency transmitter 3.9 g, 
3.9 g, 1.9 cc, input voltage range ± 2.5 mV,
channel bandwidth (B) 1-200 Hz, 
nominal sampling rate (f) 1000 Hz (f = 5B)
temperature operating range 34-41 °C
warranted battery life 4 months
TL11M2-F20EET  DSI 270-0124-001X Radiofrequency transmitter 
3.9 g, 1.9 cc, input voltage range ± 1.25 mV,
channel bandwidth (B) 1-50 Hz, 
nominal sampling rate (f) 250 Hz (f = 5B)
temperature operating range 34-41 °C
warranted battery life 1.5 months
Tissue Forceps- 1×2 Teeth 12 cm F.S.T. 11021-12 12 cm length
Tungsten carbide iris scissors F.S.T. 14558-11 11.5 cm
Vibroslicer 5000 MZ Electron Microscopy Sciences 5000-005
Xylazine (Rompun) Bayer PZN: 1320422
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Non-restraining EEGRadiotelemetryEpiduralDeep IntracerebralStereotaxicEEG Electrode PlacementFreely Moving RodentsDysrhythmiaNeurological DiseasesNeuropsychiatric DiseasesEpilepsyAnimal ModelsTransmitter LeadDeep ElectrodeAnesthesiaStereotaxic FrameBregmaLambdaPeriosteum
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Papazoglou, A., Lundt, A., Wormuth, C., Ehninger, D., Henseler, C., Soós, J., Broich, K., Weiergräber, M. Non-restraining EEG Radiotelemetry: Epidural and Deep Intracerebral Stereotaxic EEG Electrode Placement. J. Vis. Exp. (112), e54216, doi:10.3791/54216 (2016).
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