Name: preparation of rat sciatic nerve for ex vivo neurophysiology
Uploaded: 2022-07-12T13:00:00
Description: Watch this Scientific Journal Video about Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology at JoVE.com

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。

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。
<ol><li>准备1 M氯化钙2</...

<ol>
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W., Nazarpour, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separability+of+neural+responses+to+standardised+mechanical+stimulation+of+limbs.">Separability of neural responses to standardised mechanical stimulation of limbs.</a> Scientific Reports. 7 (1), 11138 (2017).</li><li>Schmalbruch, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiber+composition+of+the+rat+sciatic+nerve.">Fiber composition of the rat sciatic nerve.</a> The Anatomical Record. 215 (1), 71-81 (1986).</li><li>Kagiava, A., Theophilidis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+permeability+of+the+rat+sciatic+nerve+epineural+sheath+against+compounds+with+local+anesthetic+activity:+an+ex+vivo+electrophysiological+study.">Assessing the permeability of the rat sciatic nerve epineural sheath against compounds with local anesthetic activity: an ex vivo electrophysiological study.</a> Toxicology Mechanisms and Methods. 23 (8), 634-640 (2013).</li><li>Motori, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+metabolic+rewiring+promotes+resilience+to+neurodegeneration+caused+by+mitochondrial+dysfunction.">Neuronal metabolic rewiring promotes resilience to neurodegeneration caused by mitochondrial dysfunction.</a> Science Advances. 6 (35), 8271 (2020).</li><li>Schwarz, J. R., Eikhof, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na+currents+and+action+potentials+in+rat+myelinated+nerve+fibres+at+20+and+37&#176;+C.">Na currents and action potentials in rat myelinated nerve fibres at 20 and 37&#176; C.</a> Pfl&#252;gers Archiv. 409 (6), 569-577 (1987).</li><li>Cranefield, P. F., Brink, F., Bronk, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+oxygen+uptake+of+the+peripheral+nerve+of+the+rat.">The oxygen uptake of the peripheral nerve of the rat.</a> Journal of Neurochemistry. 1 (3), 245-249 (1957).</li><li>Lehmann, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+changes+in+pH+on+the+action+of+mammalian+A+nerve+fibers.">The effect of changes in pH on the action of mammalian A nerve fibers.</a> American Journal of Physiology-Legacy Content. 118 (3), 600-612 (1937).</li><li>Hamm, L. L., Nakhoul, N., Hering-Smith, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acid-base+homeostasis.">Acid-base homeostasis.</a> Clinical Journal of the American Society of Nephrology: CJASN. 10 (12), 2232-2242 (2015).</li><li>Minasian, S. M., Galagudza, M. M., Dmitriev, Y. V., Kurapeev, D. I., Vlasov, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+protection+against+global+ischemia+with+Krebs-Henseleit+buffer-based+cardioplegic+solution.">Myocardial protection against global ischemia with Krebs-Henseleit buffer-based cardioplegic solution.</a> Journal of Cardiothoracic Surgery. 8, 60 (2013).</li><li>Bailey, L. E., Ong, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Krebs-Henseleit+solution+as+a+physiological+buffer+in+perfused+and+superfused+preparations.">Krebs-Henseleit solution as a physiological buffer in perfused and superfused preparations.</a> Journal of Pharmacological Methods. 1 (2), 171-175 (1978).</li><li>Miller, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sydney+Ringer:+physiological+saline,+calcium+and+the+contraction+of+the+heart.">Sydney Ringer: physiological saline, calcium and the contraction of the heart.</a> The Journal of Physiology. 555, 585-587 (2004).</li><li>Yamamoto, D., Suzuki, N., Miledi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blockage+of+chloride+channels+by+HEPES+buffer).">Blockage of chloride channels by HEPES buffer).</a> Proceedings of the Royal Society of London. Series B. Biological Sciences. 230 (1258), 93-100 (1987).</li><li>Janz, G. J., Taniguchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+silver-silver+halide+electrodes.+Preparation,+stability,+and+standard+potentials+in+aqueous+and+non-aqueous+media.">The silver-silver halide electrodes. Preparation, stability, and standard potentials in aqueous and non-aqueous media.</a> Chemical Reviews. 53 (3), 397-437 (1953).</li><li>Rapeaux, A., Constandinou, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+HFAC+block-capable+and+module-extendable+4-channel+stimulator+for+acute+neurophysiology.">An HFAC block-capable and module-extendable 4-channel stimulator for acute neurophysiology.</a> Journal of Neural Engineering. 17 (4), 046013 (2020).</li><li>Next-Generation-Neural-Interfaces/HFAC_Stimulator_4ch. Next Generation Neural Interfaces Available from: <a target="_blank" href="https://github.com/Next-Generation-Neural-Interfaces/HFAC_Stimulator_4ch&gt">https://github.com/Next-Generation-Neural-Interfaces/HFAC_Stimulator_4ch&gt</a> (2021)</li><li>Cuttaz, E. A., Chapman, C. A. R., Syed, O., Goding, J. A., Stretchable Green, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fully+polymeric+electrode+arrays+for+peripheral+nerve+stimulation.">fully polymeric electrode arrays for peripheral nerve stimulation.</a> Advanced Science. 8 (8), 2004033 (2021).</li><li>Lossi, L., Merighi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+ex+vivo+rodent+platforms+in+neuroscience+translational+research+with+attention+to+the+3Rs+philosophy.">The use of ex vivo rodent platforms in neuroscience translational research with attention to the 3Rs philosophy.</a> Frontiers in Veterinary Science. 5, 164 (2018).</li></ol>

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...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 L Glass bottle</td>
			<td>VWR International Ltd</td>
			<td>215-1595</td>
			<td>Borosilicate glass</td>
		</tr>
		<tr>
			<td>1 L Glass graduated flask</td>
			<td>VWR International Ltd</td>
			<td>612-3626</td>
			<td>Borosilicate glass</td>
		</tr>
		<tr>
			<td>2 L Glass bottle</td>
			<td>VWR International Ltd</td>
			<td>215-1596</td>
			<td>Borosilicate glass</td>
		</tr>
		<tr>
			<td>2 L Glass graduated flask</td>
			<td>VWR International Ltd</td>
			<td>BRND937254</td>
			<td>Borosilicate glass</td>
		</tr>
		<tr>
			<td>Adaptor, pneumatic, 8 mm to 1/4 NPT</td>
			<td>RS UK</td>
			<td>536-2599</td>
			<td>push-to-fit straight adaptor between oxygen hose and gas dispersion tube</td>
		</tr>
		<tr>
			<td>Alkoxy conformal coating</td>
			<td>Farnell</td>
			<td>1971829</td>
			<td>ACC15 Alkoxy conformal coating for dissection petri dish preparation</td>
		</tr>
		<tr>
			<td>Anesthetic</td>
			<td>Chanelle</td>
			<td>N/A</td>
			<td>Isoflurane inhalation anesthetic, 250 mL bottle</td>
		</tr>
		<tr>
			<td>Beaker, 2 L</td>
			<td>VWR International Ltd</td>
			<td>213-0469</td>
			<td>Borosilicate glass</td>
		</tr>
		<tr>
			<td>Bipolar nerve cuff</td>
			<td>Cortec GMBH</td>
			<td>N/A</td>
			<td>800 micron inner diameter, perpendicular lead out, no connector termination</td>
		</tr>
		<tr>
			<td>Bossheads</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Standard wet laboratory bossheads for attaching grippers to rods</td>
		</tr>
		<tr>
			<td>Calcium Chloride dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>C7902-500g</td>
			<td>500 g in plastic bottle</td>
		</tr>
		<tr>
			<td>Carbogen canister</td>
			<td>BOC</td>
			<td>N/A</td>
			<td>F-size canister</td>
		</tr>
		<tr>
			<td>Centrifuge Tubes, 15 mL volume</td>
			<td>VWR International Ltd</td>
			<td>734-0451</td>
			<td>Falcon tubes</td>
		</tr>
		<tr>
			<td>Conductive elastomer nerve cuff</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>high charge capacity nerve cuff for stimulation, see protocol for fabrication reference</td>
		</tr>
		<tr>
			<td>Connector, Termimate</td>
			<td>Mouser UK</td>
			<td>538-505073-1100-LP</td>
			<td>These should be soldered to wire terminated with crocodile clips (see entry 11)</td>
		</tr>
		<tr>
			<td>Crocodile clip connectors</td>
			<td>RS UK</td>
			<td>212-1203</td>
			<td>These should be soldered to wire terminated with TermiMate connectors (see entry 10)</td>
		</tr>
		<tr>
			<td>Deionized Water</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Obtained from deionized water dispenser</td>
		</tr>
		<tr>
			<td>Forceps angled 45 degrees</td>
			<td>InterFocus Ltd</td>
			<td>91110-10</td>
			<td>Fine forceps, student range</td>
		</tr>
		<tr>
			<td>Forceps standard Dumont #7</td>
			<td>InterFocus Ltd</td>
			<td>91197-00</td>
			<td>Student range forceps</td>
		</tr>
		<tr>
			<td>Gas Disperson Tube, Porosity 3</td>
			<td>Merck</td>
			<td>12547866</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Glucose anhydrous, powder</td>
			<td>VWR International Ltd</td>
			<td>101174Y</td>
			<td>500 g in plastic bottle</td>
		</tr>
		<tr>
			<td>Grippers</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Standard wet laboratory rod-mounted grippers</td>
		</tr>
		<tr>
			<td>Heating Stirrer</td>
			<td>RS UK</td>
			<td>768-9672</td>
			<td>Stuart US152</td>
		</tr>
		<tr>
			<td>Hemostats</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Any hemostat &#62;12 cm in length is suitable</td>
		</tr>
		<tr>
			<td>Insect Pins, stainless steel, size 2</td>
			<td>InterFocus Ltd</td>
			<td>26001-45</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Laptop computer</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Any laboratory-safe portable computer with at least 2 unused USB ports is suitable</td>
		</tr>
		<tr>
			<td>Line Noise Filter</td>
			<td>Digitimer</td>
			<td>N/A</td>
			<td>Humbug noise eliminator (50 Hz line noise filter)</td>
		</tr>
		<tr>
			<td>Low-Noise Preamplifier, SR560</td>
			<td>Stanford Research Systems</td>
			<td>SR560</td>
			<td>Low-noise voltage preamplifier</td>
		</tr>
		<tr>
			<td>Magnesium Sulphate salt</td>
			<td>VWR International Ltd</td>
			<td>291184P</td>
			<td>500g in plastic bottle</td>
		</tr>
		<tr>
			<td>MATLAB scripts</td>
			<td>Github</td>
			<td>https://github.com/Next-Generation-Neural-Interfaces/HFAC_Stimulator_4ch</td>
			<td>Initialization, calibration and stimulation scripts for the custom stimulator</td>
		</tr>
		<tr>
			<td>MATLAB software</td>
			<td>Mathworks</td>
			<td>N/A</td>
			<td>Standard package</td>
		</tr>
		<tr>
			<td>Microscope Light, PL-2000</td>
			<td>Photonic</td>
			<td>N/A</td>
			<td>Light source with swan necks. Product may be obtained from third party supplier</td>
		</tr>
		<tr>
			<td>Microscope, SMZ 745</td>
			<td>Nikon</td>
			<td>SM745</td>
			<td>Stereoscopic Microscope</td>
		</tr>
		<tr>
			<td>Mineral oil, non-toxic</td>
			<td>VWR International Ltd</td>
			<td>31911.A1</td>
			<td>Oil for nerve bath</td>
		</tr>
		<tr>
			<td>Nerve Bath</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Plexiglas machined nerve bath, see protocol for details.</td>
		</tr>
		<tr>
			<td>Oscilloscope</td>
			<td>LeCroy</td>
			<td>N/A</td>
			<td>434 Wavesurfer. Product may be obtained from 3rd party suppliers</td>
		</tr>
		<tr>
			<td>Oxygen Hose, 1 meter</td>
			<td>BOC</td>
			<td>N/A</td>
			<td>1/4&#34; NPT terminations</td>
		</tr>
		<tr>
			<td>Oxygen Regulator</td>
			<td>BOC</td>
			<td>C106X/2B:3.5BAR-BS3-1/4&#34;NPTF 230Bar</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Peristaltic Pump P-1</td>
			<td>Pharmacia Biotech</td>
			<td>N/A</td>
			<td>Product may be obtained from third party supplier</td>
		</tr>
		<tr>
			<td>Petri Dish, Glass</td>
			<td>VWR International Ltd</td>
			<td>391-0580&#160;</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Potassium Chloride salt</td>
			<td>Sigma Aldrich</td>
			<td>P5405-250g</td>
			<td>250 g in plastic bottle</td>
		</tr>
		<tr>
			<td>Potassium Dihydrogen Sulphate salt</td>
			<td>Merck</td>
			<td>1.04873.0250</td>
			<td>250 g in plastic bottle</td>
		</tr>
		<tr>
			<td>Rat</td>
			<td>Charles River Laboratories</td>
			<td>N/A</td>
			<td>Sprague Dawley, 250-330 grams, female</td>
		</tr>
		<tr>
			<td>Reference electrode, ET072</td>
			<td>eDaQ (Australia)</td>
			<td>ET072-1</td>
			<td>Silver silver-chloride reference electrode</td>
		</tr>
		<tr>
			<td>Rod</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Standard wet laboratory rods with fittings for stands</td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>Sartorius</td>
			<td>N/A</td>
			<td>M-Power scale, for weighing powders. Product may be obtained from third-party suppliers</td>
		</tr>
		<tr>
			<td>Scissors straight 12 cm edge</td>
			<td>InterFocus Ltd</td>
			<td>91400-12</td>
			<td>blunt-blunt termination, student range</td>
		</tr>
		<tr>
			<td>Signal Acquisition Device</td>
			<td>Cambridge Electronic Design</td>
			<td>Micro3-1401</td>
			<td>Micro3-1401 Multichannel ADC</td>
		</tr>
		<tr>
			<td>Silicone grease, non-toxic</td>
			<td>Farnell</td>
			<td>3821559</td>
			<td>for sealing of bath partition</td>
		</tr>
		<tr>
			<td>Silicone tubing, 2 mm inner diameter</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Silicone tubing, 5 mm inner diameter</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Silver wire</td>
			<td>Alfa Aesar</td>
			<td>41390</td>
			<td>0.5 mm, annealed</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate salt</td>
			<td>Sigma Aldrich</td>
			<td>S5761-500g</td>
			<td>500 g in plastic bottle</td>
		</tr>
		<tr>
			<td>Sodium Chloride salt</td>
			<td>VWR International Ltd</td>
			<td>27810.295</td>
			<td>1 kg in plastic bottle</td>
		</tr>
		<tr>
			<td>Spring scissors angled 2 mm edge</td>
			<td>InterFocus Ltd</td>
			<td>15010-09</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Stand</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Standard wet laboratory stands with sockets for rods</td>
		</tr>
		<tr>
			<td>Stimulator</td>
			<td>Digitimer</td>
			<td>DS3</td>
			<td>DS3 or Custom Stimulator (see references)</td>
		</tr>
		<tr>
			<td>Stirring flea</td>
			<td>VWR International Ltd</td>
			<td>442-0270</td>
			<td>For use with the heating stirrer</td>
		</tr>
		<tr>
			<td>Syringe tip, blunt, 1 mm diameter</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Syringe tip, blunt, 2 mm diameter</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Syringe, plastic, 10 mL volume</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>syringe should have luer lock fitting</td>
		</tr>
		<tr>
			<td>Tape, water-resistant</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>For securing tubing and wiring to workbench</td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td>VWR International Ltd</td>
			<td>620-0806</td>
			<td>glass thermometer</td>
		</tr>
		<tr>
			<td>USB Power Bank</td>
			<td>RS UK</td>
			<td>135-1000</td>
			<td>Custom Stimulator power supply, fully charge before experiment. Not needed if using DS3</td>
		</tr>
		<tr>
			<td>Valve, Leuer Lock, 3-Way</td>
			<td>VWR International Ltd</td>
			<td>229-7440</td>
			<td>For attaching syringe to bath feed tube and priming siphon</td>
		</tr>
	</tbody>
</table>

preparation of rat sciatic nerve for ex vivo neurophysiology

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。

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?...

Watch this Scientific Journal Video about Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology at JoVE.com

Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology

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。

大鼠坐骨神经用于 离体 神经生理学的制备

Ex vivo preparations enable the study of many neurophysiological processes in isolation from the rest of the body while preserving local tissue structure. ...

该协议可以观察许多不太清楚的神经生理现象，例如高频/高振幅电流注射后的神经阻滞残留，同时防止来自活体中其他过程的干扰。该技术产生高度可重复的可靠结果，并且比体内电生理学更实用，因为麻醉深度等复杂变量不必控制，因此不会影响测量。麻醉下进行安乐死手术后，将雌性大鼠放在解剖台上，将脚踝牢牢地握在拇指，食指和中指之间，并用12厘米直的钝剪刀切断跟骨肌腱。使用细镊子和剪刀，通过靠近腿部后部中间的肌肉层仔细切开，直到坐骨神经暴露出来。一旦神经可见，使用冰冷的改良克雷布斯 - 亨塞莱特缓冲液或MKHB滋润腔，以防止神经干燥。使用止血剂，拉动每侧的皮肤瓣，并保持切口打开以进行更精细的解剖。从跟骨肌腱切口位置开始，用细剪刀打断腿内侧肌肉以释放神经。继续用冰冷的MKHB保持该地区的湿度水平。当神经在沿着腿向上移动时暴露时，解剖上覆的肌肉组织。将靠近脊柱的神经从结缔组织中释放出来，直到到达脊柱裂，此时神经中存在扭结。不要清洁神经，因为在这个阶段，速度是必不可少的。为了使解剖更容易，使用镊子，轻轻地拉动脚踝附近的神经末端。然后，用细剪刀，使靠近脊柱的神经严重。将解剖的神经放入装有MKHB的15毫升离心管中，然后将管放回冰上，直到清洁程序开始。用冷却的含氧MKHB填充涂覆的培养皿的一半，然后将一个解剖的坐骨神经放入培养皿中，并将神经的两端固定到培养皿上，使神经是直的，没有扭结，扭转或扭曲。使用6-0丝缝合线或细线，在神经两端打一个双结，以防止细胞质基质泄漏到缓冲液中。将结放在靠近神经中心的一侧的昆虫针旁边，以防止从神经组织泄漏到缓冲区中。在显微镜下，使用两毫米角弹簧剪刀和细镊子，从神经中去除脂肪，血管和肌肉组织。修剪任何不会用于刺激和记录的神经分支。每五分钟清洁一次后，用新鲜、冷却、含氧的MKHB替换缓冲液。清洁后，将神经放回充满缓冲液的运输管中，并将管子放在冰上。准备一个干净的双室神经浴。使用标准的实验室凸台头和夹具，将神经浴置于放置在加热搅拌器上的两升瓶的水平下方。将浴槽的排水口连接到蠕动泵入口。将蠕动泵的出口连接到通回缓冲瓶的管子上。使用可调节的流量阀将浴缸入口连接到管子上，然后将管子放在瓶子内。使用三通阀，注射器连接到中间出口，以帮助将管子作为重力辅助缓冲器流入的虹吸管。通过拉动注射器来启动虹吸管，直到缓冲区流入其中。配置阀门，使进入浴槽的缓冲流速为每分钟五到六毫米。最初可以增加流速以填充浴槽。一旦浴液缓冲液水平达到排水管，将神经放入浴槽中。使用昆虫针，将神经末端固定在充满缓冲液的浴室的角落。使用45度角的细镊子并仅在末端捏住神经，小心地通过两个浴室之间隔板上的孔来穿刺要刺激的神经。使用昆虫针，将神经的另一端固定在浴池的油室中，确保神经是直的而不会被拉伸，并且没有扭结和扭曲。使用硅脂，做一个密封，以防止缓冲液从缓冲室泄漏到油室用硅油或矿物油填充油室。将银/氯化银记录电极钩放在油浴室中，并使用凸台头和夹具固定它们。然后，将油浴中的神经部分覆盖在钩子上，而不会拉紧神经。调整或修复硅脂密封，如果在移动神经后观察到任何泄漏。将参比银/氯化银电极连接到放大器接地，并使用实验室夹具固定电极，将其置于充满缓冲液的浴槽室中。准备用于刺激的干净神经袖带电极后，将电极置于充满缓冲液的浴室中。使用带有钝头或倾斜尖端的镊子或细镊子，在浴缸中打开电极以弄湿袖口内部。如果仍有气泡，请使用精细的注射器从浴中抽出缓冲液，并将气泡从袖口中取出。使用镊子，轻轻打开袖口并将其滑到神经下方。关闭神经周围的袖带，避免神经的任何扭结或扭曲。将刺激电极和电流回流电极连接到刺激器。如果使用方形铂片作为电流回流电极，请将片材放置在远离浴槽中神经的位置。用胶带固定电极的引线。将激励器TTL信号输出连接到示波器的通道4。在示波器屏幕上，按“触发通道”选项卡，并将通道 4 指定为触发通道。使用电平旋钮将触发电平设置为 1 伏。在示波器上，将时间分辨率设置为每分 1 毫秒，将电压分辨率设置为每分 10 毫伏。及时将触发参考值居中，并将触发电平设置为 1 伏。将刺激器连接到计算机后，通过将电池电源连接到电源输入来打开刺激器。然后，在计算机上启动 MATLAB 软件。执行自定义的 MATLAB 脚本。然后，打开指示的 MATLAB 脚本。编辑脚本并将参数刺激器脉冲幅度设置为负300微安，刺激器脉冲宽度设置为300微秒，刺激器脉冲数设置为10，刺激器脉冲间隔时间设置为1秒。通过单击软件中的“运行”来启动刺激方案。复合动作电位或CAP在电极处的峰峰值幅度为1毫伏，放大时为100毫伏。标准铂神经袖带和定制导电弹性体神经袖带的典型神经兴奋性如下所示。这里显示了使用两种坐骨神经进行一天实验后离体获得的CAP。用右坐骨神经观察到最小的CAP振幅减小。相比之下，左坐骨神经CAP振幅在大约三毫伏时与实验开始时右坐骨神经的振幅相似，神经拔除后六个多小时。强调解剖、清洁和实施技术的精度和可重复性。注意浴缸里发生的事情。如果神经信号丢失，可能只是电极没有很好地接触或浴油分区中有盐水，而不是神经死亡。第一次使用的用户可能会忍不住匆匆忙忙，尤其是在解剖阶段。这可能会导致错误并损害神经。精度和一致性是这里的关键，并将带来更高质量的结果。还应特别注意缓冲液制备的一致性。

Research

JoVE Journal

Neuroscience

Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology

Axoplasm Isolation from Rat Sciatic Nerve

从大鼠坐骨神经轴浆隔离

Isolation of pure axonal cytoplasm (axoplasm) from peripheral nerve is crucial for biochemical studies of many biological processes. In this article, we ...

科研

神经科学

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

在对小鼠坐骨神经体内的电测量

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

体内基因转移到雪旺细胞在啮齿动物坐骨神经电穿孔

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

在体内轴突兴奋性试验对大鼠尺神经的电生理测量

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

使用 Vivo 单纤维记录和带附骨神经的固定背道根结条，以检查传导失败机制

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...

Preparation of Newborn Rat Brain Tissue for Ultrastructural Morphometric Analysis of Synaptic Vesicle Distribution at Nerve Terminals

新生大鼠脑组织在神经端子突形圆锥花序分布超微结构形态分析中的制备

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial ...

Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth

来自胚胎GFP-表达小鼠的Ex Vivo Oculo运动切片培养,用于对Oculo运动神经外生长进行时移成像

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

坐骨神经粉碎损伤大鼠模型中功能评估的3D运动学分析

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Ex Vivo Pressurized Hippocampal Capillary-Parenchymal Arteriole Preparation for Functional Study

Ex Vivo 加压希马卡毛细管- 功能研究准备

From subtle behavioral alterations to late-stage dementia, vascular cognitive impairment typically develops following cerebral ischemia. Stroke and ...

大鼠坐骨神经用于 离体 神经生理学的制备

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