经颅直流电刺激 (tDCS) 在小鼠中的作用
Summary
经颅直流电刺激 (tDCS) 是一种治疗精神疾病的方法。动物模型是了解 tDCS 诱发的特定生物学变化的必要条件。本协议描述使用长期植入电极的 tDCS 鼠标模型。
Abstract
经颅直流电刺激 (tDCS) 是一种非侵入性调节技术, 作为一种替代或补充治疗几种神经精神疾病。tDCS 的生物学效应还没有完全理解, 这部分是由于难以获得人脑组织的原因。该协议描述了一个 tDCS 的小鼠模型, 使用长期植入电极允许研究 tDCS 的长期生物效应。在这个实验模型中, tDCS 改变皮质基因的表达, 并为理解其治疗使用的基本原理提供了显著的贡献。
Introduction
经颅直流电刺激 (tDCS) 是一种无创、低成本的治疗技术, 其重点是通过使用低强度连续电流1进行神经元调制。目前, tDCS 有两个设置 (anodal 和阴极)。当 anodal 刺激使电流电场太弱, 无法触发动作电位时, 电生理学研究表明, 这种方法能产生突触可塑性2的变化。例如, 证据表明, tDCS 诱发长期增强 (LTP) 效应, 如增加兴奋后突触电位的峰值振幅3,4和调节皮质兴奋性5。
反之, 阴极刺激诱导抑制, 导致膜极化6。这一机制的假说是基于生理学的发现, 其中 tDCS 被描述为调节动作电位频率和时间在神经元体3。值得注意的是, 这种效应并不能直接唤起动作电位, 尽管它可以改变退极化阈值, 促进或阻碍神经元发射7。这些对比效果以前已经证明。例如, anodal 和阴极刺激产生了相反的影响, 在条件反应登记通过肌电活动在兔子8。然而, 研究还表明, 延长 anodal 刺激疗程可能会降低兴奋性, 而增加阴极电流可能导致兴奋性, 呈现自我对比的效果3。
anodal 和阴极的刺激都聚集了电极对的使用。例如, 在 anodal 刺激中, “活性” 或 “阳极” 电极被置于大脑区域进行调制, 而 “参考” 或 “阴极” 电极位于一个区域, 而电流的影响假定为微不足道的9。在阴极刺激中, 电极的配置是反转的。有效 tDCS 的刺激强度取决于电流强度和电极尺寸, 对电场的影响不同于10。在大多数发表的研究中, 平均电流强度介于0.10 至 2.0 ma 和 0.1 ma 至 0.8 ma 之间, 分别为人和小鼠 (6、11)。虽然电极大小 35 cm2通常使用在人, 没有适当的理解关于电极维度为啮齿目动物和更加彻底的调查需要6。
tDCS 已被建议在临床研究, 试图提供一个替代或补充治疗几个神经和精神疾病11 , 如癫痫12, 双相障碍13, 中风5, 主要抑郁症14, 阿尔茨海默病15, 多发性硬化16 , 帕金森病17。尽管对 tDCS 及其在临床试验中的应用越来越感兴趣, 但对脑组织的详细细胞和分子诱发改变, 短期和长期的影响, 以及行为结果, 仍有待深入研究18,19. 由于直接人类方法彻底研究 tDCS 是行不通的, 使用 tDCS 动物模型可能会对 tDCS 的治疗机制所依据的细胞和分子事件提供有价值的见解, 因为可获得动物的脑组织。
现有的证据是有限的关于 tDCS 模型的小鼠。大多数报告的模型使用不同的植入布局, 电极尺寸和材料。例如, (2017) 植入头电极 (银/AgCl, 直径4毫米) 填充生理盐水, 并固定在颅骨与丙烯酸水泥和螺钉20。不同于我们的方法, 他们的胸电极被植入 (铂, 20 x 1.5 毫米)。Nasehi等。(2017) 使用的程序非常类似我们的, 虽然胸电极是由盐水浸泡海绵 (碳填充, 9.5 厘米2)21。另一项研究将两个电极植入了动物的头部, 这是通过使用固定板和用水凝胶导体22覆盖动物的头部实现的。在这里, 我们描述一个 tDCS 鼠标模型, 使用一个长期植入电极通过简单的手术程序和 tDCS 设置 (图 1)。
Protocol
本实验采用 C57BL/6 小鼠 (8-12 周) 的个体居住。动物在实验过程之前、期间和之后接受了适当的关心与食物和水广告随意.所有程序都是由米纳斯吉拉斯州联邦大学动物伦理委员会(59/2014 号议定书)批准的。 1. 电极放置 将动物镇静到立体定向装置上 消毒所有必要的手术器械。小说手术器械在440摄氏度消毒3分钟。棉拭子蒸压在 20 psi (英…
Representative Results
该手术方案提出的长期种植稳定性至少一个月, 没有炎症信号在受刺激的地方, 也没有任何其他不想要的效果。所有的动物幸存的手术过程和 tDCS 疗程 (n = 8)。在本实验中, tDCS 植入物位于 M1 和 M2 皮质 (+ 1.0 毫米前后方和0.0 毫米侧向 bregma)。一周后, tDCS (n = 3-4) 和假 (n = 3) 小鼠在10分钟的 0.35 mA 中连续五天被刺激。接触质量 (重庆) 的价值被注册, 以评估种植体的生存能力, 并?…
Discussion
近年来, 神经刺激技术已经进入临床实践, 作为治疗神经精神疾病的一个有希望的程序23。为了减少由于缺乏对神经刺激机制的了解而强加的约束, 我们在这里提出了一个 tDCS 的小鼠模型, 它携带一个能靶向大脑区域的电极。由于电极是长期植入的, 这种动物模型能够调查 tDCS (至少1月) 在复杂刺激模式诱发的持久生物学效应。所描述的 tDCS 动物模型具有较高的种植耐受性和很少的感…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢索萨先生协助维持老鼠的栖息地。l.a.v. M 是一个海角博士后研究员。这项工作得到赠款 PRONEX (FAPEMIG: APQ-00476-14) 的支持。
Materials
| BD Ultra-Fine 50U Syringe | BD | 10033430026 | For intraperitonially injection. |
| Shaver (Philips Multigroom) | Philips (Brazil) | QG3340/16 | For surgical site trimming. |
| Surgical Equipment | |||
| Model 940 Small Animal Stereotaxic Instrument with Digital Display Console | KOPF | 940 | For animal surgical restriction and positioning. |
| Model 922 Non-Rupture 60 Degree Tip Ear Bars | KOPF | 922 | For animal surgical restriction and positioning. |
| Cannula Holder | KOPF | 1766-AP | For implant positioning. |
| Precision Stereo Zoom Binocular Microscope (III) on Boom Stand | WPI | PZMIII-BS | For bregma localization and implant positioning. |
| Temperature Control System Model | KOPF | TCAT-2LV | For animal thermal control. |
| Cold Light Source | WPI | WA-12633 | For focal brightness |
| Tabletop Laboratory Animal Anesthesia System with Scavenging | VetEquip | 901820 | For isoflurane delivery and safety. |
| VaporGuard Activated Charcoal Adsorption Filter | VetEquip | 931401 | Delivery system safety measures. |
| Model 923-B Mouse Gas Anesthesia Head Holder | KOPF | 923-B | For animal restriction and O2 and isoflurane delivery. |
| Oxygen regulator, E-cylinder | VetEquip | 901305 | For O2 regulation and delivery. |
| Oxygen hose – green | VetEquip | 931503 | For O2 and isoflurane delivery. |
| Infrared Sterilizer 800 ºC | Marconi | MA1201 | For instrument sterilization. |
| Surgical Instruments | |||
| Fine Scissors – ToughCut | Fine Science Tools | 14058-11 | For incision. |
| Surgical Hooks | INJEX | 1636 | In House Fabricated – Used to clear the surgical site from skin and fur. |
| Standard Tweezers or Forceps | – | – | For skin grasping. |
| Surgical Consumables | |||
| Vetbond | 3M | SC-361931 | For incision closing. |
| Cement and Catalyzer KIT (Duralay) | Reliance | 2OZ | For implant fixation. |
| Sterile Cotton Swabs (Autoclaved) | JnJ | 75U | For surgical site antisepsis. |
| 24 Well Plate (Tissue Culture Plate) | SARSTEDT | 831,836 | For cement preparation. |
| Application Brush | parkell | S286 | For cement mixing and application. |
| Pharmaceutics | |||
| Xylazin (ANASEDAN 2%) | Ceva Pharmaceutical (Brazil) | P10160 | For anesthesia induction. |
| Ketamine (DOPALEN 10%) | Ceva Pharmaceutical (Brazil) | P30101 | For anesthesia induction. |
| Isoflurane (100%) | Cristália (Brazil) | 100ML | For anesthesia maintenance. |
| Lidocaine (XYLESTESIN 5%) | Cristal Pharma | – | For post-surgical care. |
| Ketoprofen (PROFENID 100 mg) | Sanofi Aventis | 20ML | For post-surgical care. |
| Ringer's Lactate Solution | SANOBIOL LAB | ############ | For post-surgical care. |
| TobraDex (Dexamethasone 1 mg/g) | Alcon | 631 | For eye lubrification and protection. |
| Stimulation | |||
| Animal Transcranial Stimulator | Soterix Medical | 2100 | For current generation. |
| Pin-type electrode Holder (Cylindrical Holder Base) | Soterix Medical | 2100 | Electrode support (Implant). |
| Pin-type electrode (Ag/AgCl) | Soterix Medical | 2100 | For current delivery (electrode). |
| Pin-type electrode cap | Soterix Medical | 2100 | For implant protection. |
| Body Electrode (Ag/AgCl Coated) | Soterix Medical | 2100 | For current delivery (electrode). |
| Saline Solution (0.9%) | FarmaX | ############ | Conducting medium for current delivery. |
| Standard Tweezers or Forceps | – | – | For tDCS setup. |
| Real Time Polymerase Chain Reaction | |||
| BioRad CFX96 Real Time System | BioRad | C1000 | For qPCR |
| SsoAdvancedTM Universal SYBR Green Supermix (5 X 1mL) | BioRad | 1725271 | For qPCR |
| Hard Shell PCR Plates PCT COM 50 p/ CFX96 | BioRad | HSP9601 | For qPCR |
| Microseal "B" seal pct c/ 100 | BioRad | MSB1001 | For qPCR |
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Cite This Article
de Souza Nicolau, E., de Alvarenga, K. A. F., Tenza-Ferrer, H., Nogueira, M. C. A., Rezende, F. D., Nicolau, N. F., Collodetti, M., de Miranda, D. M., Magno, L. A. V., Romano-Silva, M. A. Transcranial Direct Current Stimulation (tDCS) in Mice. J. Vis. Exp. (139), e58517, doi:10.3791/58517 (2018).