大鼠慢性植入的外周神经刺激电极的制备
Summary
现有的用于建造用于小型啮齿动物的可长期植入的外周神经袖口电极的方法通常需要专门的设备和/或训练有素的人员。在该协议中，我们演示了一种简单、低成本的制造慢性植入袖口电极的方法，并演示了它们对大鼠迷走神经刺激（VNS）的有效性。
Abstract
外周神经袖口电极早已用于神经科学和相关领域，以刺激，例如，迷走神经或坐骨神经。最近的几项研究表明，慢性VNS在提高中枢神经系统可塑性以改善运动康复、灭绝学习和感官歧视方面的有效性。由于大鼠体型小，长期植入装置的构建具有挑战性，典型的方案需要人员的广泛培训和耗时的微制造方法。或者，可以以更高的成本购买市售的植入式袖口电极。在该协议中，我们提出了一种简单、低成本的方法，用于构建用于大鼠的小型、慢性植入的外周神经袖口电极。我们验证了袖口电极的短期和长期可靠性，证明氯胺酮/xylazine麻醉大鼠的 VNS 在植入时和装置植入后 10 周内都会产生与激活 Hering-Breer 反射一致的呼吸速率降低。通过将 VNS 与熟练的杠杆压机性能配对以诱导电机皮质贴图可塑性，我们进一步演示了袖口电极在慢性刺激研究中的适用性。
Introduction
最近，对慢性植入袖口电极刺激周围神经的需求越来越大，因为研究越来越证明这种技术对治疗许多炎症,性疾病,,,,,,1、2、3,2和神经系统疾病44、5、6、7、8、9、10、11、12、13、14、15,14,,11,12的临床7,8105前有用。3 6913例如，慢性VNS已被证明可在各种学习环境中增强新皮质可塑性，改善运动康复,54，5，6，7，8，灭绝学习,6,7,810，11，12，13，14，,11,12,13,14和感官歧视15。10市售的外设神经袖口电极通常与订单履行时间延长和成本相对较高有关，从而限制其可访问性。另外，长期植入袖口电极的”内部”制造协议仍然有限，啮齿动物解剖由于其体积小而面临特殊挑战。目前为慢性啮齿动物实验构建袖口电极的协议通常需要使用复杂的设备和技术，以及经过广泛培训的人员。在该协议中，我们演示了一种基于以前发布和广泛使用的方法16、17,的袖口电极制造的简化方法。我们验证了我们长期植入大鼠电极的功能，证明在左颈椎迷走神经周围植入袖口时，对袖口电极的刺激成功地使SpO2的呼吸和下降停止。刺激的肺受体血管纤维是众所周知的从事赫林-布劳尔反射，其中抑制几个呼吸核在脑干的结果抑制灵感18。因此，停止呼吸与赫林-布劳尔反射一致，以及由此导致的 SpO2 下降，为麻醉大鼠正确植入电极和袖口功能提供了直接的测试。为了验证慢性植入袖口电极的长期功能，在植入时测量反射反应，并与植入后六周内在同一动物获得的反应进行比较。第二组大鼠在操纵杠杆任务的行为训练后植入了VNS袖口电极。在这些大鼠中，VNS与正确的任务性能配对，产生了皮质运动图的重组，与之前发表的,研究19、20、21、22,2021,一致。在麻醉下发生5~10周麻醉运动皮质映射时，通过确认VNS成功诱导呼吸停止和SpO2下降5%以上，进一步验证了VNS治疗动物的袖口功能。
最近出版的儿童类17和里奥斯等人的协议为简化袖口电极制造方法提供了一个经过验证的起点，因为这种流行的方法已被多个实验室使用,，在啮齿动物,,,,,,11、2、3、4、5、6、7、8、9、10、11中进行慢性,27,8610,VNS研究。349原始方法涉及几个高精度步骤来操纵细微线，使袖口电极制造需要一个多小时才能完成，并进行了广泛的培训才能可靠地执行。此处描述的简化方法所需的材料和工具要少得多，只需经过最少培训的人员，只需在一小时内完成。
Protocol
本议定书中描述的所有程序都按照《美国国家卫生和植物保护与使用指南》执行，并经达拉斯德克萨斯大学机构动物护理和使用委员会批准。 1. 刺激袖口电极制造 准备袖口管。 使用剃须刀刀片，切割一块 2.5 毫米长的聚合物管。插入钳子尖或回形针通过管子，并使用刀片使一个狭缝通过管壁的一侧袖口。 从管子上取下钳子，在袖口的中线插入一根大缝?…
Representative Results
根据先前公布的外科手术17、19、20、21、22，迷走神经,19袖口,电极和21头盖被长期植入大鼠体内。在植入之前，在袖口引线上测量 1 kHz 的阻抗，袖口管浸入盐水中（阻抗 = 1.2 = 0.17 kΩ [均值 = std];N = 9）。在盐水中只植入阻抗小于2 kΩ的袖口;所有袖口都符合此标准（不包括 0/9 ?…
Discussion
在这里，我们描述了一种简单、低成本的方法，用于组装用于啮齿动物的慢性植入刺激袖口电极，从而促进对这种新兴疗法的临床前研究。这种简化的方法不需要专门的培训或设备，并且使用少量的工具和用品，大多数研究实验室都,很容易获得，与其他方法16、26、27、28相比26,27，降低了设备制造的货币?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项工作由德克萨斯大学达拉斯分校和UT摄政委员会资助。我们感谢所罗门·戈尔丁、比拉尔·哈桑、玛吉·贾尼和清慈宗的技术援助。
Materials
| Biocompatible polyurethane-based polymer tubing, 0.080" OD x 0.040" ID | Braintree Scientific | MRE080 36 FT | |
| Dissecting microscope | AM Scopes | #SM-6T-FRL | |
| Fine Serrated Scissors, straight, 22mm cutting edge | Fine Science Tools | #14058-09 | for cutting Pt/Ir wire and suture thread |
| Forceps, #5 Dumont forceps, straight, 11 cm, 0.1 x 0.06 mm tip | Fine Science Tools | #11626-11 | |
| Forceps, ceramic tipped forceps, 0.3 mm x 30 mm tips | Electron Microscopy Sciences | #78127-71 | |
| Gold Pins, PCB Press Fit Socket | Mill-Max | #1001-0-15-15-30-27-04-0 | or similar small pins for connecting cuff leads to headcap |
| Isobutane lighter | BIC | #LCP21-AST | for de-insulating Pt/Ir wire |
| Micro strip connector with latch, 4-pin | Omnetics | A24002-004 / PS1-04-SS-LT | |
| Pipette tip, 10 uL | VWR | 89079-464 | |
| Platinum-Iridium (90/10%) Wire, 0.001" (diameter) x 9 strands, PTFE insulated | Sigmund Cohn | 10IR9/49T | |
| Razor Blade, Single Edge, Surgical Carbon Steel No.9 | VWR | #55411-050 | for cutting MicroRenathane tubing |
| Sewing needle, ca. 4.0 cm length x 0.7 mm diameter (size 6-7) | Singer | 00276 | Smaller needle for threading Pt/Ir wire |
| Sewing needle, ca. 4.5 cm length x 0.8 mm diameter (size 2-3) | Singer | 00276 | Larger needle for pinning cuff during assembly and for threading suture |
| Small foam board | Juvo+/Amazon | B07C9637SJ | for fabrication platform; our dimensions are ca. 2.5" x 3.5" x 1" (L x W x H) |
| Solder, multicore lead-free, 0.38mm diameter | Loctite/Multicore | #796037 | |
| Soldering station | Weller | WES51 | or similar soldering iron compatible with long conical tips (this part has been discontinued) |
| Soldering tip, long conical, 0.01" / 0.4 mm | Weller | 1UNF8 | |
| Suture, nonabsorbable braided silk ,size 6/0 | Fine Science tools | #18020-60 | |
| UV (405 nm) spot light | Henkel/Loctite | #2182207 | |
| UV Light Cure Adhesive 25 ml | Henkel/Loctite | AA 3106 | or similar biocompatible UV cure adhesive |
| Wire wrapping wire, 30 AWG | Digikey | K396-ND |
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Citar este artículo
Sanchez, C. A., Brougher, J., Rahebi, K. C., Thorn, C. A. Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats. J. Vis. Exp. (161), e61128, doi:10.3791/61128 (2020).