大鼠坐骨神经用于 离体 神经生理学的制备
1Department of Electrical and Electronic Engineering,Imperial College London, 2Centre for Bioinspired Technology,Imperial College London, 3Care Research and Technology (CR&T),Imperial College London and the University of Surrey, 4Dementia Institute (UK DRI), 5Department of Bioengineering,Imperial College London
Summary
该协议描述了制备大鼠整个坐骨神经组织以进行 离体 电生理刺激并在环境调节的双隔室灌注盐水浴中记录。
Abstract
离体 制剂能够独立于身体其他部位研究许多神经生理过程，同时保留局部组织结构。这项工作描述了大鼠坐骨神经用于 离体 神经生理学的制备，包括缓冲液制备，动物程序，设备设置和神经生理学记录。这项工作概述了使用这种方法可能进行的不同类型的实验。概述的方法旨在在严格控制的条件下对提取的周围神经组织提供6小时的刺激和记录，以获得最佳的结果一致性。使用这种方法得到的结果是A纤维化合物动作电位（CAP），在整个实验过程中峰峰值幅度在毫伏范围内。CAP振幅和形状是一致和可靠的，使它们可用于测试和比较新电极与现有模型，或干预措施对组织的影响，例如使用化学品，手术改变或神经调节刺激技术。对传统的市售铂铱接触头袖带电极和定制导电弹性体电极进行了测试，并在神经刺激强度-持续时间响应方面给出了类似的结果。
Introduction
目前对 计算机 模拟的基本神经功能的理解在几个方面缺乏，特别是在躯体，轴突和树突之外的神经组织区室化的影响方面。Axon-髓鞘相互作用仍然知之甚少，因为即使是详细的计算神经模型（如MRG1 （用于哺乳动物神经））充分捕获常规电刺激反应，也无法捕获其他实验观察到的行为，例如高频阻滞携带2 或继发反应3。
该协议提供了一种在急性小型实验室动物模型中有效地研究神经水平神经生理学过程的方法，使用标准化的制备方案来分离神经，控制其环境，并将其从 体内 环境中移除到 离体 环境。这将防止 体内 神经刺激方案使用的其他身体过程或麻醉剂来改变神经行为并混淆测量结果或其解释4，5。这使得能够开发更现实的模型，仅关注对神经组织特有的影响，而这些效应知之甚少。该协议也可用于新的神经刺激和记录电极材料和几何形状的测试平台，以及新的刺激范式，如高频块2，3。该技术的变体以前已用于研究严格控制条件下的神经生理学6，例如，测量离子通道动力学和性质或局部麻醉剂7的影响。
与诸如急性 体内 小动物实验8的替代方案相比，该技术提供了几个优点。该技术消除了从体内提取组织时保持麻醉深度的需要，从而减少了所需设备的数量，例如麻醉扩散器，氧气浓缩器和加热垫。这简化了实验方案，降低了出错的风险。由于麻醉剂可以潜在地改变神经功能4，该技术可确保措施不会因这些麻醉化合物的副作用而混淆。最后，在研究神经毒性化合物（如河豚毒素）的作用时，这种技术比急性 体内 实验更合适，这些化合物会通过瘫痪杀死麻醉动物。
周围神经切片是一种独特的 离体 系统，因为负责记录的神经信号的纤维很有可能不包含任何体细胞。由于这些通常位于运动神经元，脊柱中以及脊柱旁边的背根神经节中的感觉神经元，因此哺乳动物神经的一部分的制备可以粗略地建模为具有离子通道的管状膜的集合，在两端打开9。代谢由组织解剖时位于轴突中的线粒体维持10。在提取后鼓励缝合轴突的开放端以关闭它们，从而有助于维持膜上现有的离子梯度，这对于正常的神经功能至关重要。
为了维持组织在体外的稳态，必须严格控制几个环境变量。这些是温度11，氧合12，渗透压，pH值13，14和获得葡萄糖以维持新陈代谢。对于该协议，方法是使用改良的克雷布斯 – 亨塞莱特缓冲液15，16（mKHB），连续曝气氧气和二氧化碳的混合物。mKHB属于心脏停搏缓冲液家族6，17，用于保存体外的解剖组织，例如，在离体实验中。这些缓冲液不含任何血红蛋白、抗生素或抗真菌药，因此仅适用于在有限时间内涉及少量组织的制剂。使用碳酸盐和二氧化碳氧化还原对实现pH控制，需要用二氧化碳对缓冲液进行恒定曝气以维持pH平衡。这是为了避免使用其它常见的缓冲剂，例如HEPES，它可以改变神经细胞18的功能。为了对缓冲液进行充氧并提供pH控制，使用称为碳化物（95%O 2，5%CO 2）的氧气中5%二氧化碳的混合物。使用加热搅拌器对缓冲容器进行温度控制，并将缓冲液通过神经浴灌注，然后再循环到起始容器中。典型的实验将持续6-8小时，直到神经失去其活力，并且不再对刺激有足够的反应，以代表健康组织的措施。
为了优化信噪比，使用氯化银电极进行记录，按照前面描述的方法19制备。对于刺激，可以使用商用现成的铂袖带电极和定制的导电聚合物袖带电极的组合。导电聚合物袖带电极具有显著更高的电荷容量，这在使用高振幅波形20刺激神经时是有用的。
该协议中使用的刺激器已在前面描述过20。文档，设计文件和软件脚本是公开的21.其他刺激器可用于执行该协议;然而，定制刺激器也能够进行高频替代电流（HFAC）阻断2，20，这使得更广泛的神经生理学实验成为可能。要使用HFAC阻滞，建议使用导电弹性体袖口，以避免对神经的损害。导电弹性体神经袖带是由导电弹性体作为导电组分和聚二甲基硅氧烷作为绝缘体22而制成的柔软和全聚合的电极阵列。设备采用传统的激光微加工技术以双极性配置制造。
Protocol
所有动物护理和程序均根据英国内政部根据“动物（科学程序）法”（1986年）颁发的适当许可证进行，并得到伦敦帝国理工学院动物福利和伦理审查委员会的批准。 1. 缓冲液的制备 注意：协议的这一部分可以在协议的其余部分之前进行，除了涉及制备1倍浓度的改性克雷布斯 – 亨塞莱特缓冲液（mKHB）的最后步骤。 准备1 M氯化钙2</…
Representative Results
该协议可以获得的代表性结果是坐骨神经内A型神经纤维的一致化合物动作电位。这些动作电位通常在电极处具有约1 mV的峰峰值幅度，因此一旦放大，则为100 mV（图2）。相似的刺激振幅和脉冲宽度应产生相似的CAP振幅。与市售的铂袖带电极相比，导电弹性体袖带电极通常需要略高的刺激振幅，以获得相同的CAP振幅。与刺激来自不同动物的神经所需的刺激幅度的变化相比，这?…
Discussion
在这项工作中，我们描述了一种为 离体 神经生理学准备大鼠坐骨神经的方案。组织提取大约需要30分钟，包括动物处理，麻醉，扑杀和解剖，而神经清洁，在浴缸中放置和电极植入应额外30分钟才能开始记录。缓冲液制备可以在30分钟内进行，但这可以在实验的其余部分之前完成。这种类型的制备和实验已经在过去的7，12中使用和描述，使用类似…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者感谢葛兰素史克制药公司、美国宾夕法尼亚州普鲁士国王的Gerald Hunsberger博士和加尔瓦尼生物电子学（英国斯蒂夫尼奇）与我们分享了他们最初的神经制备技术。作者承认罗伯特·托特的双室神经浴设计。作者承认工程和物理科学研究委员会（EPSRC）的医疗保健技术挑战奖（HTCA）资助的资金。作者承认伦敦帝国理工学院的高性能嵌入式和分布式系统博士培训中心（HiPEDS CDT）资助了阿德里安·拉波克斯（EP / L016796 / 1）。阿德里安·拉波目前由英国痴呆症研究所，护理研究和技术中心资助。作者感谢帝国理工学院生物工程系的Zack Bailey在制作JoVE视频文章期间为实验和获取动物组织提供了帮助。
Materials
| 1 L Glass bottle | VWR International Ltd | 215-1595 | Borosilicate glass |
| 1 L Glass graduated flask | VWR International Ltd | 612-3626 | Borosilicate glass |
| 2 L Glass bottle | VWR International Ltd | 215-1596 | Borosilicate glass |
| 2 L Glass graduated flask | VWR International Ltd | BRND937254 | Borosilicate glass |
| Adaptor, pneumatic, 8 mm to 1/4 NPT | RS UK | 536-2599 | push-to-fit straight adaptor between oxygen hose and gas dispersion tube |
| Alkoxy conformal coating | Farnell | 1971829 | ACC15 Alkoxy conformal coating for dissection petri dish preparation |
| Anesthetic | Chanelle | N/A | Isoflurane inhalation anesthetic, 250 mL bottle |
| Beaker, 2 L | VWR International Ltd | 213-0469 | Borosilicate glass |
| Bipolar nerve cuff | Cortec GMBH | N/A | 800 micron inner diameter, perpendicular lead out, no connector termination |
| Bossheads | N/A | N/A | Standard wet laboratory bossheads for attaching grippers to rods |
| Calcium Chloride dihydrate | Sigma Aldrich | C7902-500g | 500 g in plastic bottle |
| Carbogen canister | BOC | N/A | F-size canister |
| Centrifuge Tubes, 15 mL volume | VWR International Ltd | 734-0451 | Falcon tubes |
| Conductive elastomer nerve cuff | N/A | N/A | high charge capacity nerve cuff for stimulation, see protocol for fabrication reference |
| Connector, Termimate | Mouser UK | 538-505073-1100-LP | These should be soldered to wire terminated with crocodile clips (see entry 11) |
| Crocodile clip connectors | RS UK | 212-1203 | These should be soldered to wire terminated with TermiMate connectors (see entry 10) |
| Deionized Water | N/A | N/A | Obtained from deionized water dispenser |
| Forceps angled 45 degrees | InterFocus Ltd | 91110-10 | Fine forceps, student range |
| Forceps standard Dumont #7 | InterFocus Ltd | 91197-00 | Student range forceps |
| Gas Disperson Tube, Porosity 3 | Merck | 12547866 | N/A |
| Glucose anhydrous, powder | VWR International Ltd | 101174Y | 500 g in plastic bottle |
| Grippers | N/A | N/A | Standard wet laboratory rod-mounted grippers |
| Heating Stirrer | RS UK | 768-9672 | Stuart US152 |
| Hemostats | N/A | N/A | Any hemostat >12 cm in length is suitable |
| Insect Pins, stainless steel, size 2 | InterFocus Ltd | 26001-45 | N/A |
| Laptop computer | N/A | N/A | Any laboratory-safe portable computer with at least 2 unused USB ports is suitable |
| Line Noise Filter | Digitimer | N/A | Humbug noise eliminator (50 Hz line noise filter) |
| Low-Noise Preamplifier, SR560 | Stanford Research Systems | SR560 | Low-noise voltage preamplifier |
| Magnesium Sulphate salt | VWR International Ltd | 291184P | 500g in plastic bottle |
| MATLAB scripts | Github | https://github.com/Next-Generation-Neural-Interfaces/HFAC_Stimulator_4ch | Initialization, calibration and stimulation scripts for the custom stimulator |
| MATLAB software | Mathworks | N/A | Standard package |
| Microscope Light, PL-2000 | Photonic | N/A | Light source with swan necks. Product may be obtained from third party supplier |
| Microscope, SMZ 745 | Nikon | SM745 | Stereoscopic Microscope |
| Mineral oil, non-toxic | VWR International Ltd | 31911.A1 | Oil for nerve bath |
| Nerve Bath | N/A | N/A | Plexiglas machined nerve bath, see protocol for details. |
| Oscilloscope | LeCroy | N/A | 434 Wavesurfer. Product may be obtained from 3rd party suppliers |
| Oxygen Hose, 1 meter | BOC | N/A | 1/4" NPT terminations |
| Oxygen Regulator | BOC | C106X/2B:3.5BAR-BS3-1/4"NPTF 230Bar | N/A |
| Peristaltic Pump P-1 | Pharmacia Biotech | N/A | Product may be obtained from third party supplier |
| Petri Dish, Glass | VWR International Ltd | 391-0580 | N/A |
| Potassium Chloride salt | Sigma Aldrich | P5405-250g | 250 g in plastic bottle |
| Potassium Dihydrogen Sulphate salt | Merck | 1.04873.0250 | 250 g in plastic bottle |
| Rat | Charles River Laboratories | N/A | Sprague Dawley, 250-330 grams, female |
| Reference electrode, ET072 | eDaQ (Australia) | ET072-1 | Silver silver-chloride reference electrode |
| Rod | N/A | N/A | Standard wet laboratory rods with fittings for stands |
| Scale | Sartorius | N/A | M-Power scale, for weighing powders. Product may be obtained from third-party suppliers |
| Scissors straight 12 cm edge | InterFocus Ltd | 91400-12 | blunt-blunt termination, student range |
| Signal Acquisition Device | Cambridge Electronic Design | Micro3-1401 | Micro3-1401 Multichannel ADC |
| Silicone grease, non-toxic | Farnell | 3821559 | for sealing of bath partition |
| Silicone tubing, 2 mm inner diameter | N/A | N/A | N/A |
| Silicone tubing, 5 mm inner diameter | N/A | N/A | N/A |
| Silver wire | Alfa Aesar | 41390 | 0.5 mm, annealed |
| Sodium Bicarbonate salt | Sigma Aldrich | S5761-500g | 500 g in plastic bottle |
| Sodium Chloride salt | VWR International Ltd | 27810.295 | 1 kg in plastic bottle |
| Spring scissors angled 2 mm edge | InterFocus Ltd | 15010-09 | N/A |
| Stand | N/A | N/A | Standard wet laboratory stands with sockets for rods |
| Stimulator | Digitimer | DS3 | DS3 or Custom Stimulator (see references) |
| Stirring flea | VWR International Ltd | 442-0270 | For use with the heating stirrer |
| Syringe tip, blunt, 1 mm diameter | N/A | N/A | N/A |
| Syringe tip, blunt, 2 mm diameter | N/A | N/A | N/A |
| Syringe, plastic, 10 mL volume | N/A | N/A | syringe should have luer lock fitting |
| Tape, water-resistant | N/A | N/A | For securing tubing and wiring to workbench |
| Thermometer | VWR International Ltd | 620-0806 | glass thermometer |
| USB Power Bank | RS UK | 135-1000 | Custom Stimulator power supply, fully charge before experiment. Not needed if using DS3 |
| Valve, Leuer Lock, 3-Way | VWR International Ltd | 229-7440 | For attaching syringe to bath feed tube and priming siphon |
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Citar este artigo
Rapeaux, A., Syed, O., Cuttaz, E., Chapman, C. A. R., Green, R. A., Constandinou, T. G. Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology. J. Vis. Exp. (185), e63838, doi:10.3791/63838 (2022).