Name: an ultrasonic tool for nerve conduction block in diabetic rat models
Uploaded: 2017-10-20T13:00:00.000+00:00
Duration: 10 min 27 s
Description: Watch this Scientific Journal Video about An Ultrasonic Tool for Nerve Conduction Block in Diabetic Rat Models at JoVE.com

这项研究得到了科学技术部 (最多 105-2221-400-001) 和台湾国家卫生研究院 (项目 BN-105-PP-10) 的支持。

台湾国家卫生研究院机构动物保育与使用委员会批准了所有动物协议. 
 1. 糖尿病模型在成年雄性大 (SD) 大鼠中的诱导作用 <ol> <li> 将从笼中取出的大鼠食品颗粒从笼子中移到快速雄性 SD 大鼠 (300-350 g), 用于6小时前的脲嘧啶诱导. </li>
	<li> 制备柠檬酸钠缓冲液 (0.1 米, pH 4.5)。
	<ol> <li> 溶解1.05 克柠檬酸一水合物 (C 6 h 8 o 7 和 #183; h 2 o; 摩尔. 210.14) 在50毫升蒸馏水中做一个0.1 米柠檬酸溶液. </li>
		<li> 溶解 1.47 g 三钠柠檬酸二水合物 (C 8 H 5 O 7 Na 3 和 #183; 2H 2 O; 摩尔. 294.12) 在50毫升蒸馏水中制造0.1 米柠檬酸钠溶液. </li>
		<li> 将25毫升柠檬酸溶液添加到25毫升枸橼酸钠溶液中。使用 ph 计监测柠檬酸钠缓冲 ph 值 (ph 值 4.5). 
		注: 通过加入适量的0.1 米枸橼酸钠溶液使缓冲液成为等渗. </li>
	</ol> </li> <li> 在0.1 米的柠檬酸钠缓冲液中溶解脲佐菌素, 生成50毫克/毫升的脲佐菌溶液. 
	注: 脲佐菌素溶液是光敏的, 因此, 在 15-20 min 的范围内用铝箔覆盖该溶液并使用. </li>
	<li> 在1毫升的胰岛素或结核菌素注射器中, 用26到28口径的针将50毫克/千克的脲佐菌素溶液抽出. #160; 用乙醇垫和腹腔将注射部位清洁, 将该溶液注入腹部下右象限, 以避免损伤腹部器官。和 #160; </li> <li> 在注射用10% 蔗糖水作为唯一的水源后48小时, 以防止低血糖. </li> </ol>
 2. 糖尿病的确认在脲佐菌素诱发大鼠中的作用 <ol> <li> 监视在 72 h 和葡萄糖米后注射的所有大鼠的空腹血糖浓度。
	<ol> <li> 在测量空腹血糖水平之前, 快速将糖尿病大鼠诱导为15小时. </li>
		<li> 在血糖测量过程中, 将老鼠放在一个约束袋里, 并露出尾巴进行血液收集. </li>
		<li> 用血柳叶刀刺破尾巴尖端, 以获得一滴血。将滴血放在葡萄糖试纸上。记录空腹血糖水平. 
		注: 葡萄糖仪检测并显示单位为毫克/dL 的血糖水平。在2周的诱导后, 将空腹血糖水平低于150毫克/dL 的大鼠排除在外. </li>
	</ol> </li> </ol> 3。糖尿病大鼠周围性糖尿病神经病变的评价 <ol> <li> 使用电子痛评估机械性的小弗雷。
	<ol> <li> 习惯在对1厘米直径金属网格地板上的笼中的糖尿病大鼠进行30分钟的观察后, 再评估后爪提取反应. </li>
		<li> 使用带有刚性尖端 (直径 0.8 mm) 的电子式弗雷探针, 手动将压力施加到大鼠后爪的足底表面, 并逐渐增加压力, 直到看到爪子的撤退反应. </li>
		<li> 记录在系统上显示的压力并重复测量每只老鼠5次, 每次测量间隔为三十年代. </li>
	</ol> </li> <li> 用热板评估热痛觉过敏。
	<ol> <li> 习惯在评估疼痛反应之前, 在热板 (24 和 #177; 0.5 和 #176; C) 中对糖尿病大鼠进行10分钟的实验. </li>
		<li> 在适应后将老鼠放回笼子里, 加热热板, 保持热板和 #39 温度在55和 #177; 0.5 和 #176; C. </li>
		<li> 将老鼠放在加热的热板上, 同时启动计时器. </li>
		<li> 当老鼠表现出明显的行为时, 如舔后爪或不正常地弹后腿, 停止计时器, 并记录撤退的潜伏期. 
		注: 如果鼠在二十年代 (二十年代截止时间) 后不表现出明显的行为, 请终止热板试验, 并将大鼠从热板中取出. </li>
	</ol> </li> </ol> 4。 在体内 用 HIFU 换能器进行神经传导阻滞 注意: 在50毫克/千克注射后, 体内 实验开始于5周. 
<ol> <li> 在用 HIFU 超声 CMAPs 之前执行动物程序。
	在手术前, <ol> <li> 在高压釜中消毒手术工具 (手术刀、剪刀、镊子和玻璃钩). </li>
		<li> 麻醉鼠腹腔注射 tiletamine/唑合剂 (40 毫克/千克) 和嗪 (10 毫克/千克) 或 通过 吸入1.75% 的异氟醚通过异氟醚蒸发器。将老鼠放在加热垫上以保持体温. </li>
		<li> 在腹卧床中放置大鼠进行手术。涂抹眼药膏。延长鼠腿, 用指甲捏足足底表面, 以确定麻醉深度。如果老鼠表现出戒断反应, 应用额外的麻醉. </li>
		<li> 用电动剪刀将老鼠的大腿和下背部的毛发移除。在手术部位用干净的纱布涂抹液体碘, 并在手术部位向外循环移动纱布。用酒精垫用相同的圆周运动擦拭液体碘。这个实验是在左侧和右侧的手术部位进行的。在执行下一步的步骤时, 在其他外科手术部位重复此步骤. </li>
		<li> 使用无菌手术剪刀或手术刀, 使背部大腿的皮肤切开。使用钝器手术剪刀仔细分离皮肤下面的组织和保护皮肤与皮肤挂钩. #160 股骨可以在肌肉中看到. </li>
		<li> 使用剪刀仔细分离肌肉平行于股骨, 直到大腿的坐骨神经纤维, 是嵌入在肌肉是可见的。小心地用玻璃钩把大腿坐骨神经从周围的结缔组织和肌肉中分离出来. </li>
	</ol> </li> <li> 使用 custom-made 神经固定器 ( 图 1 和 图 3 ) 在 HIFU 焦点区域定位坐骨神经. 
	注意: custom-made 神经固定器由3组件组成 ( 图 3 )。所有组件均由透明酯 (PMMA) 制成。组件顶部结构的外螺纹为 2.5 mm 高, M10XP0.7 ( 图 4A )。中央井是一个4.0 毫米直径孔通过组分 i。井的底部开口由一张胶带密封。槽的直径是1.2 毫米, 并且槽的中央平面和组分 I 的顶面之间的距离是3.1 毫米. 组分 II 包括一个主要身体, 四条腿和底部结构 ( 图 4B )。底部结构的内螺纹为2.5 毫米高, M10XP0.7, 以适应组件的外部螺纹。中心孔的直径与构件 i. 的中心井相同主体的尺寸为32毫米直径和5.4 毫米的厚度。四条腿对称地被部署。两个相同的短腿设计为对齐和两个相同的长腿工作挂钩的组件 III。组分 III 的外径和高度是41毫米和9.2 毫米。内螺纹为 M36XP1.0, 通孔直径为27.5 毫米 (强类 = "xfig">> 图 4C )。锥是一个中空的锥形, 顶部开口直径84毫米, 直径27.5 毫米的底部开口。高度为 57.5 mm ( 图 5A )。
	<ol> <li> 在实验前, 将 custom-made 丙烯酸神经固定器浸泡在漂白液中 30-60 分钟, 然后浸泡在无菌水中. </li>
		<li> 使用玻璃钩, 小心地提起神经, 并把它放在组件的插槽 i. </li>
		<li> 螺丝组件 II 到组件 i. 用林格和 #39 的方法来填充中心井, 用于超声传播和神经保护. </li>
		<li> 螺杆组件 III 至 HIFU 外壳锥。通过组件 II. 的四条腿将组件 III 与组件 II 结合起来 
		注: 三元件的几何中心和传感器对齐。神经和换能器之间的距离等于焦距, 从而保证了神经在 HIFU 聚焦区的内部. </li>
	</ol> </li> <li> 将一双针刺针插入坐骨神经的原点, 另一对进入腓肠肌。通过一根同轴电缆将每对针连接到电生理采集系统 ( 图 1 ). 
	注: 在电缆的一端是两个鳄鱼夹子分别夹住两根针, 另一端是连接系统的 BNC 连接器。在坐骨神经和腓肠肌的记录电极上, 对针灸针的作用是刺激电极。
	<ol> <li> 将电生理学采集系统的采样率和带宽分别设置为50赫和 70 Hz-3 赫。应用一个超极大刺激与脉冲宽度0.1 毫秒的刺激电极在坐骨神经的起源. </li>
		<li> 记录 CMAPs 从记录电极和放大 CMAPs 与内置放大器的电生理采集系统. 
		注: 在电生理采集系统中使用内置放大器, 放大神经信号, 并通过电生理采集系统记录电极 CMAPs. </li>
	</ol> </li> <li> 使用商用 2.68 MHz HIFU 传感器来抑制糖尿病神经病理性大鼠的 CMAPs. 
	注: 换能器的规格说明如下: 元素球形碗, 孔径直径为6厘米, 焦距为5厘米, 在自由场中有4毫米深的椭圆形焦带和0.8 毫米的宽度。
	<ol> <li> 将球形锥、HIFU 换能器和锥盖浸没在装有脱水的水箱中。将 HIFU 换能器放入球面锥中, 用6头螺钉 ( 图 5B ) 将锥盖固定在球面锥的顶部开口上。在球形锥体的气泡自然地被逐出了由于低气泡的密度与水相比, 封印 front-end 打开锥体由一个透明0.03 毫米厚实的磁带。螺丝组件 III 到球面锥上. </li>
		<li> 从脱水箱中取出球面锥和组件 III 的 HIFU 传感器. 
		注: 研究中使用的反渗透水是由反渗透过程净化的水。反渗透水煮沸, 以排出气体。冷却后, 脱水在单个密封罐中获得. </li>
	</ol> </li> <li> 将组件 i 放入神经和肌肉之间的空间, 并将神经放置在组件的间隙中. 执行步骤4.2.3 和 4.2.4, 以确保神经在 HIFU 的焦点区域内 ( 图 3 a/强 >>). </li>
	<li> 连接函数发生器和射频功率放大器。将功率放大器与 hifu 换能器连接, 以产生 hifu 光束。手动设置功能发生器的电压输出到 HIFU 传感器 通过 功率放大器。在 HIFU 曝光时间过后, 手动关闭函数发生器。使用计时器观察时间. 
	注: 本研究中使用的 HIFU 光束的强度和能量分别为2810瓦特/厘米 2 和 84 J/mm 2 . </li>
	<li> 同时, 通过电生理采集系统 (步骤 4.3) 和 hifu 光束通过 hifu 系统 (步骤 4.6) 向坐骨神经传递刺激, 同时记录 CMAPs。从 3 s、5 s 到8秒逐渐增加 HIFU 对坐骨神经的照射, 直到 CMAPs 的振幅减小或抑制。
	<ol> <li> 在 HIFU 光束交付期间每秒记录一次 CMAPs。观察了 CMAPs 振幅的变化, 关闭了 HIFU 系统, 并手动点击了电生理学采集软件上的记录图标, 记录 CMAPs 在10分钟的每2分钟, 每5分钟在连续30分钟, 每10分钟最后一个阶段, 直到录制时间达到 2 h. </li>
	</ol> </li> <li> 分离神经固定器的第二和第三部分 ( 图 3 ), 从切口部位卸下 HIFU 传感器。分离的第二部分和 i 释放安全的坐骨神经。在记录 CMAPs 后, 用4-0 张铬线缝线缝合糖尿病大鼠的手术部位。将液体碘应用于手术部位以防止感染。
	<ol> <li> 将笼子放在加热垫上, 让老鼠在笼子里恢复, 然后再将它们送回动物设施。在饮用水中提供布洛芬3天或腹腔注射丁丙诺啡 (0.05-0.1 毫克/千克). </li>
	</ol> </li> <li> 在最初的 HIFU 超声后的4.1.2 和4.3 天7、14和28中的步骤中描述了在麻醉性糖尿病神经病鼠坐骨神经和腓肠肌的起源中插入刺激和记录电极。重复步骤4.3.1 到4.3.2。
	<ol> <li> 将笼子放在加热垫上, 允许老鼠在将笼子移到动物设施之前在笼子里恢复. </li>
	</ol> </li> <li> 在实验后安乐老鼠。把老鼠放在二氧化碳室里。等待约5分钟的老鼠停止呼吸。确保心脏停止跳动. </li>
</ol>

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B., 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=Single-pulse+transcranial+magnetic+stimulation+for+acute+treatment+of+migraine+with+aura:+a+randomised,+double-blind,+parallel-group,+sham-controlled+trial.">Single-pulse transcranial magnetic stimulation for acute treatment of migraine with aura: a randomised, double-blind, parallel-group, sham-controlled trial.</a> Lancet Neurol. 9 (4), 373-380 (2010).</li></ol>

作者没有什么可透露的。

对糖尿病大鼠神经病理性神经作用电位的局部和暂时性抑制, 并观察了 HIFU 治疗后的阻滞作用的即刻发生. 为期28天的 CMAPs 随访研究表明, 可以在适当的 HIFU 照射下进行神经传导的安全阻断。因此, HIFU 治疗的上述协议可以为糖尿病大鼠坐骨神经可逆传导阻滞提供一种替代方案。
在这种方法中, 没有神经变性, 感觉神经可以完全恢复在一个小时到几天的轻度神经损伤14。对于严重的损伤, 感觉恢复的时间过程通常需要几个月, 如果它发生在所有。此外, 当神经管和雪旺细胞基底板在粉碎伤后完好无损,15后, 周围神经纤维再生更完整。因此, 我们推断 hifu 治疗导致轻度但可逆的神经损伤的情况下, 因为抑制 CMAPs 后, hifu 处理返回基线随着时间的推移。对于严重的神经损伤, CMAPs 只恢复部分, 即使在28天之后。
本研究的技术为研究 HIFU 对周围神经的影响提供了实验平台。由于本研究中所开发的结构成分和协议, 可以精确地对准目标神经, 从而解决了以往的定位问题。除正常神经外, HIFU 阻滞技术也可应用于病变神经。然而, 目前的技术的局限性包括缺乏温度监测 (导致周围组织的损伤) 和短暂的阻滞作用, 虽然重复 HIFU 暴露可能延长镇痛。为了将 hifu 阻断技术转化为临床试验, 需要对 hifu 聚焦区进行无创导引, 如超声成像或 mr 成像, 以确定靶组织的位置并实时监测温度。
现有的口服药物治疗结果只有1/4 例神经病理性疼痛经验超过50% 疼痛缓解, 并造成一些重要的副作用, 如嗜睡, 头晕和嗜16。物理模式的发展, 希望提高镇痛功效, 如针灸, 电和磁刺激。然而, 针灸的功效很大的依赖于临床医生的经验, 而这一过程是具有侵入性的。无创电刺激或磁刺激的功效约为2小时疼痛自由的40%。这两种刺激都不集中在本地站点上, 这会产生一些不良事件17。因此, 为了满足临床未满足的周围疼痛缓解的需要, HIFU 阻断技术是一种有前途的工具, 因为即时的效果, 可逆的效果, 物理治疗, 非侵入性治疗, 和潜在的家用。
准确地瞄准坐骨神经的 HIFU 聚焦区是至关重要的。图 3演示了将神经定位到焦点区域的示意图过程。第一步是用一个玻璃钩稍微抬起坐骨神经, 并把我的神经下的组成部分, 然后放下神经到插槽的组成部分 i (图 3A)。通过第一步, 神经通过了组件 I 的中心点。第二步是组装组件 II 与组件 i通过螺钉盖结构。第一和第二部分的组装是图 2B中显示的神经固定器。组件 II 扮演连接组件 I 和 III. 的角色在组合第二和 III 组分之前, 他们充满了脱气林格的解答传送超声并且保留神经。最后一步是插入组件 III front-end 结构的 HIFU 传感器锥耦合器到组件 II。二对灵活的长和短的支柱组分 II 提供足够的固定力。根据榫榫原理设计了第二和 III 组件的装配, 保证了三元件的中心点在轴上。HIFU 换能器的焦距等于换能器中心点和元件中心点之间的距离。因此, 神经肯定是在焦带内, 这是一个椭圆形, 宽度为0.8 毫米, 深度为4毫米。

口服药物, 针灸1, 和电神经刺激2已被用于治疗痛苦的糖尿病神经病。然而, 口服药物的副作用、针刺的侵入性操作、电神经刺激对治疗效果和患者的依从性都有影响。超声在动物模型中的周围神经阻滞已被调查了几十年3,4,5。对 0.4-1.0 s 6的 10-20 脉冲超声照射后, 大绿蛙的坐骨神经进行了可逆性抑制。阻断神经传导的一个因素是超声7引起的温升。对于神经病的患者, 在低强度超声照射下, 腓神经对复合肌肉动作电位 (CMAPs) 的抑制作用为 2 min8。完全恢复时间在5分钟以内。
最近, 美国食品和药物管理局批准 HIFU 作为非侵入性治疗子宫肌瘤肿瘤9, 疼痛 palliations 骨转移的10, 和前列腺癌11。HIFU 传感器在体外发出声束, 光束在不同的组织介质中传播, 并会聚在靶肿瘤的焦点上。在不损害周围组织的情况下, 立即形成病灶区, 对靶向肿瘤产生局部效应。HIFU 也已被用于抑制神经传导或导致神经失神经在体内实验正常的大 (SD) 大鼠12。此外, 研究了 HIFU 对神经病理性神经的短期和远期影响,13。以往的结果表明, 可逆转或永久块的感觉神经传导可以实现的 HIFU 与适当的参数。除了镇痛应用外, HIFU 还可作为研究神经传导阻滞的重要因素, 以此为基础, 探讨神经病学和疼痛药物的发展。因此, 需要在动物模型中专门针对周围神经的 HIFU 阻断技术平台。本文旨在通过 HIFU 对糖尿病神经病理性大鼠部分或完全阻断外周神经动作电位的方法进行研究。建立糖尿病大鼠模型, 评价周围神经病理性症状。本文介绍了一种专门用于治疗大鼠坐骨神经的 HIFU 平台和实验过程。

<table><tbody><tr><td>streptozotocin</td><td>Sigma</td><td>85882</td><td></td></tr><tr><td>citric acid monohydrate&amp;nbsp;</td><td>Sigma</td><td>C1909</td><td></td></tr><tr><td>trisodium citrate dihydrate</td><td>Sigma</td><td>W302600</td><td></td></tr><tr><td>glucose meters</td><td>Roche Accu-Check Active</td><td>GC</td><td></td></tr><tr><td>electronic von Frey device</td><td>IITC Life Science</td><td>2390</td><td></td></tr><tr><td>hot plate</td><td>IITC Life Science</td><td></td><td></td></tr><tr><td>Biopac MP36 acquisition system</td><td>Biopac Systems, Inc.</td><td></td><td></td></tr><tr><td>HIFU transducer</td><td>Sonic Concepts</td><td>H108</td><td></td></tr><tr><td>function generator</td><td>Agilent</td><td>33250A</td><td></td></tr><tr><td>power amplifier</td><td>Electronics &amp;amp; Innovation</td><td>1040L</td><td></td></tr><tr><td>Rats&amp;nbsp;</td><td>Biolasco taiwan</td><td>Sprague-Dawley</td><td></td></tr><tr><td>Puralube vet ointment</td><td>Dechra</td><td></td><td></td></tr><tr><td>isoflurane vaporizer</td><td>Parkland Scientific</td><td>V3000PS</td><td></td></tr><tr><td>Isoflurance</td><td>Attane</td><td></td><td></td></tr><tr><td>Restraint bag (Decapicones)</td><td>Braintree Scientific</td><td>DC 200</td><td></td></tr></tbody></table>

an ultrasonic tool for nerve conduction block in diabetic rat models

近年来, 在正常和糖尿病动物模型中, 采用高强度聚焦超声 (HIFU) 传感器进行神经传导阻滞。HIFU 在使用适当的超声参数时, 可以在不损伤神经的情况下可逆地阻断周围神经的传导。神经活动电位的暂时和部分阻滞表明, HIFU 有潜力是一个有用的临床治疗疼痛缓解。这项工作演示了用 HIFU 换能器抑制糖尿病大鼠神经病理性神经的动作电位的方法在体内。第一步是通过注射链脲佐菌素生成成年男性糖尿病神经病理性大鼠。第二步是通过电子弗雷探针和热板对糖尿病大鼠周围糖尿病神经病变进行评价。最后一步是记录在体内暴露于 HIFU 超声的神经的胞外动作电位。该方法可为超声镇痛应用研究带来有益的结果。

在体内研究表明, HIFU 剂量为 3 s 超声在强度为2810瓦特/厘米2, CMAPs 被抑制20% 的基线, 但他们完全恢复后, 30 分钟 (图 2A, 钻石) 和几乎在28天 (图 2B, 菱形) 期间的常量。对于 5 s HIFU 暴露在相同强度, CMAPs 下降到 65.4% (9.5%) 的基线在4分钟, 恢复到 73.7% (12.6%) 的基线, 120 分钟 (图 2A, 正方形)。CMAPs 直到14天 (图 2B、正方形) 才返回到基线水平。当 HIFU 超声时间在相同强度下增加到8秒时, CMAPs 在 4 min (图 2A, 三角形) 下降到 26.0% (14.1%) 基线, 但在 38.0% min 时增加到 12.0% (120) 的基线, 并逐渐增加到74% 的基线, 按天28 (图 2B, 三角形)。有关其他详细信息, 请参见 Lee, 许贤,13 。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55675/55675fig1.jpg"> 
图 1: 实验性设置在体内高强度聚焦超声 (HIFU) 的神经传导阻滞.(a)用反渗透脱水与神经固定器结合在一起的 custom-made 丙烯酸球形锥, 以确保 HIFU 传感器的焦点区域位于神经的焦平面上。图中显示了(B)刺激和记录电极。神经固定器定位在 HIFU 束的焦平面上的坐骨神经。<a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55675/55675fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55675/55675fig2.jpg"> 
图 2: 在中的 CMAPs 响应在体内糖尿病神经病理性大鼠 3 s, 5 s, 和 8 s 的 HIFU 超声.(A) CMAPs 在1天录音期间的时间路线。在 3 s、5 s 和 8 s 的 HIFU 超声后, CMAPs 在10分钟内减少, 并在120分钟后恢复, (B)在7、14和28录音中的 CMAPs 的时间路线。CMAPs 在 3 s 和 5 s hifu 超声在天1以后完全地被恢复了14天和部份地增加了 28 s hifu 超声。金刚石: 3 s hifu 超声, 正方形: 5 s hifu 超声, 和三角形: 8 s 的 hifu 超声。n = 6 用于每个 HIFU 参数。* Signiﬁcantly 与1天不同。数据被表示为中值 (范围), 其中误差条是该范围的一半。此图是从 Lee, 许贤, 201513中修改的。<a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55675/55675fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55675/55675fig3.jpg"> 
图 3: 在 HIFU 热斑中确保坐骨神经的组装过程.(A)三装配步骤如下所示: (1) 仔细解决元件 i 的插槽中的神经, (2) 组装元件 II 和元件 i, (3) 将 front-end 的部件与 HIFU 传感器锥结构插入元件 II。(B)与大鼠神经结合的 HIFU 传感器示意图。<a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55675/55675fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55675/55675fig4.jpg"> 
图 4: 第一部分 (a)、第二 (B) 和第三 (C) 项的绘图.<a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55675/55675fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55675/55675fig5.jpg"> 
图 5: 球面锥 (a) 和锥盖 (B) 的图形.<a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55675/55675fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about An Ultrasonic Tool for Nerve Conduction Block in Diabetic Rat Models at JoVE.com

An Ultrasonic Tool for Nerve Conduction Block in Diabetic Rat Models

本工作提出了应用高强度聚焦超声阻断糖尿病神经病理性神经动作电位的方法。

糖尿病大鼠模型神经传导阻滞的超声工具

Nerve conduction block with a high intensity-focused ultrasound (HIFU) transducer has been performed in normal and diabetic animal models recently. HIFU ...

an-ultrasonic-tool-for-nerve-conduction-block-in-diabetic-rat-models

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

7.3K 次观看.  National Health Research Institutes. 本工作提出了应用高强度聚焦超声阻断糖尿病神经病理性神经动作电位的方法。

视频: An Ultrasonic Tool for Nerve Conduction Block in Diabetic Rat Models

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

糖尿病大鼠的热痛觉阈值的快速测定

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

科研

神经科学

The Sciatic Nerve Cuffing Model of Neuropathic Pain in Mice

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in injured sensory neurons and along the entire nociceptive pathway within the central nervous system. It is usually chronic and challenging to treat. In order to study neuropathic pain and its treatments, different models have been developed in rodents. These models derive from known etiologies, thus reproducing peripheral nerve injuries, central injuries, and metabolic-, infectious- or chemotherapy-related neuropathies. Murine models of peripheral nerve injury often target the sciatic nerve which is easy to access and allows nociceptive tests on the hind paw. These models rely on a compression and/or a section. Here, the detailed surgery procedure for the &#34;cuff model&#34; of neuropathic pain in mice is described. In this model, a cuff of PE-20 polyethylene tubing of standardized length (2 mm) is unilaterally implanted around the main branch of the sciatic nerve. It induces a long-lasting mechanical allodynia, i.e., a nociceptive response to a normally non-nociceptive stimulus that can be evaluated by using von Frey filaments. Besides the detailed surgery and testing procedures, the interest of this model for the study of neuropathic pain mechanism, for the study of neuropathic pain sensory and anxiodepressive aspects, and for the study of neuropathic pain treatments are also discussed.

Neuropathic pain is a consequence of a lesion or disease affecting the somatosensory system. The &#8220;cuff model&#8221; of neuropathic pain in mice consists of the implantation of a polyethylene cuff around the main branch of the sciatic nerve. Mechanical allodynia is tested using von Frey filaments.

神经病理性疼痛小鼠坐骨神经掴型号

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in ...

医学

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

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 Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and latency detect chronic axon loss and demyelination, respectively. Axonal excitability techniques &#34;by threshold tracking&#34; expand upon these measures by providing information regarding the activity of ion channels, pumps and exchangers that relate to acute function and may precede degenerative events. As such, the use of axonal excitability in animal models of neurological disorders may provide a useful in vivo measure to assess novel therapeutic interventions. Here we describe an experimental setup for multiple measures of motor axonal excitability techniques in the rat ulnar nerve.
The animals are anesthetized with isoflurane and carefully monitored to ensure constant and adequate depth of anesthesia. Body temperature, respiration rate, heart rate and saturation of oxygen in the blood are continuously monitored. Axonal excitability studies are performed using percutaneous stimulation of the ulnar nerve and recording from the hypothenar muscles of the forelimb paw. With correct electrode placement, a clear compound muscle action potential that increases in amplitude with increasing stimulus intensity is recorded. An automated program is then utilized to deliver a series of electrical pulses which generate 5 specific excitability measures in the following sequence: stimulus response behavior, strength duration time constant, threshold electrotonus, current-threshold relationship and the recovery cycle.
Data presented here indicate that these measures are repeatable and show similarity between left and right ulnar nerves when assessed on the same day. A limitation of these techniques in this setting is the effect of dose and time under anesthesia. Careful monitoring and recording of these variables should be undertaken for consideration at the time of analysis.

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

Axonal excitability techniques provide a powerful tool to examine pathophysiology and biophysical changes that precede irreversible degenerative events. This manuscript demonstrates the use of these techniques on the ulnar nerve of anesthetized rats.

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 ...

Establishing a Mouse Model of a Pure Small Fiber Neuropathy with the Ultrapotent Agonist of Transient Receptor Potential Vanilloid Type 1

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to degeneration and injury of small-diameter sensory neurons, referred to as small fiber neuropathy. Present animal models of small fiber neuropathy affect both large- and small-diameter sensory fibers and thus create a neuropathology too complex to properly assess the effects of injured small-diameter sensory fibers. Therefore, it is necessary to develop an experimental model of pure small fiber neuropathy to adequately examine these issues. This protocol describes an experimental model of small fiber neuropathy specifically affecting small-diameter sensory nerves with resiniferatoxin (RTX), an ultrapotent agonist of transient receptor potential vanilloid type 1 (TRPV1), through a single dose of intraperitoneal injection, referred to as RTX neuropathy. This RTX neuropathy showed pathological manifestations and behavioral abnormalities that mimic the clinical characteristics of patients with small fiber neuropathy, including intraepidermal nerve fiber (IENF) degeneration, specifically injury in small-diameter neurons, and induction of thermal hypoalgesia and mechanical allodynia. This protocol tested three doses of RTX (200, 50, and 10 &#181;g/kg, respectively) and concluded that a critical dose of RTX (50 &#181;g/kg) is required for the development of typical small fiber neuropathy manifestations, and prepared a modified immunostaining procedure to investigate IENF degeneration and neuronal soma injury. The modified procedure is fast, systematic, and economic. Behavioral evaluation of neuropathic pain is critical to reveal the function of small-diameter sensory nerves. The evaluation of mechanical thresholds in experimental rodents is particularly challenging and this protocol describes a customized metal mesh that is suitable for this type of assessment in rodents. In summary, RTX neuropathy is a new and easily established experimental model to evaluate the molecular significance and intervention underlying neuropathic pain for the development of therapeutic agents.

This study establishes an experimental model of pure small fiber neuropathy with resiniferatoxin (RTX). A unique dose of RTX (50 &#181;g/kg) is optimal for developing a small fiber neuropathy model that mimics patient characteristics and could help investigate the nociceptive molecular significance underlying neuropathic pain.

利用瞬态受体电位辣椒1型 Ultrapotent 激动剂建立单纯小纤维神经病变的小鼠模型

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to ...

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

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

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 ...

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular mechanism of neuropathic pain in mice and rats. Currently, there are two types of SNI model, one to cut and ligate the common peroneal and the tibial nerves with intact sural nerve, which is defined as SNIs in this study, and another to cut and ligate the common peroneal and the sural nerves with intact tibial nerve, which is defined as SNIt in this study. Because the sural nerve is purely sensory whereas the tibial nerve contains both motor and sensory fibers, the SNIt model has much less motor deficit than the SNIs model. In the traditional SNIt mouse model, the common peroneal and the sural nerves are cut and ligated separately. Here a modified SNIt surgery method is described to damage both common peroneal and sural nerves with only one ligation and one cut with a shorter procedure time, which is easier to perform and reduces the potential risk of stretching the sciatic or tibial nerves, and produces similar mechanical hypersensitivity as the traditional SNIt model.

<meta charset="utf8"></head><body>改良的小鼠神经性疼痛备用神经损伤手术模型

The modified surgery is a simplified method for mouse or rat spared nerve injury model that requires only one ligation and one cut to injure both common peroneal and sural nerves.

改良的小鼠神经性疼痛备用神经损伤手术模型

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular ...

Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology

Ex vivo preparations enable the study of many neurophysiological processes in isolation from the rest of the body while preserving local tissue structure. This work describes the preparation of rat sciatic nerves for ex vivo neurophysiology, including buffer preparation, animal procedures, equipment setup and neurophysiological recording. This work provides an overview of the different types of experiments possible with this method. The outlined method aims to provide 6 h of stimulation and recording on extracted peripheral nerve tissue in tightly controlled conditions for optimal consistency in results. Results obtained using this method are A-fibre compound action potentials (CAP) with peak-to-peak amplitudes in the millivolt range over the entire duration of the experiment. CAP amplitudes and shapes are consistent and reliable, making them useful to test and compare new electrodes to existing models, or the effects of interventions on the tissue, such as the use of chemicals, surgical alterations, or neuromodulatory stimulation techniques. Both conventional commercially available cuff electrodes with platinum-iridium contacts and custom-made conductive elastomer electrodes were tested and gave similar results in terms of nerve stimulus strength-duration response.

Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology

This protocol describes the preparation of rat whole sciatic nerve tissue for ex vivo electrophysiological stimulation and recording in an environmentally-regulated, two-compartment, perfused saline bath.

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. ...

Open-Source Real-Time Closed-Loop Electrical Threshold Tracking for Translational Pain Research

Nociceptors are a class of primary afferent neurons that signal potentially harmful noxious stimuli. An increase in nociceptor excitability occurs in acute and chronic pain conditions. This produces abnormal ongoing activity or reduced activation thresholds to noxious stimuli. Identifying the cause of this increased excitability is required for the development and validation of mechanism-based treatments. Single-neuron electrical threshold tracking can quantify nociceptor excitability. Therefore, we have developed an application to allow such measurements and demonstrate its use in humans and rodents. APTrack provides real-time data visualization and action potential identification using a temporal raster plot. Algorithms detect action potentials by threshold crossing and monitor their latency after electrical stimulation. The plugin then modulates the electrical stimulation amplitude using an up-down method to estimate the electrical threshold of the nociceptors. The software was built upon the Open Ephys system (V0.54) and coded in C++ using the JUCE framework. It runs on Windows, Linux, and Mac operating systems. The open-source code is available (<a data-saferedirecturl="https://www.google.com/url?q=https://github.com/Microneurography/APTrack&amp;source=gmail&amp;ust=1680789829274000&amp;usg=AOvVaw0nMk9J_EI2jUn8SlmC5JJm" href="https://github.com/Microneurography/APTrack" target="_blank">https://github.com/Microneurography/APTrack</a>). The electrophysiological recordings were taken from nociceptors in both a mouse skin-nerve preparation using the teased fiber method in the saphenous nerve and in healthy human volunteers using microneurography in the superficial peroneal nerve. Nociceptors were classified by their response to thermal and mechanical stimuli, as well as by monitoring the activity-dependent slowing of the conduction velocity. The software facilitated the experiment by simplifying the action potential identification through the temporal raster plot. We demonstrate real-time closed-loop electrical threshold tracking of single-neuron action potentials during&#160;in vivo&#160;human microneurography, for the first time, and during&#160;ex vivo&#160;mouse electrophysiological recordings of C-fibers and A&#948;-fibers. We establish proof of principle by showing that the electrical threshold of a human heat-sensitive C-fiber nociceptor is reduced by heating the receptive field. This plugin enables the electrical threshold tracking of single-neuron action potentials and allows the quantification of changes in nociceptor excitability.

APTrack is a software plugin developed for the Open Ephys platform that enables real-time data visualization and the closed-loop electrical threshold tracking of neuronal action potentials. We have successfully used this in microneurography for human C-fiber nociceptors and mouse C-fiber and A&#948;-fiber nociceptors.

用于转化疼痛研究的开源实时闭环电阈值跟踪

Nociceptors are a class of primary afferent neurons that signal potentially harmful noxious stimuli. An increase in nociceptor excitability occurs in ...

糖尿病大鼠模型神经传导阻滞的超声工具

摘要

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此视频中的章节

糖尿病大鼠的热痛觉阈值的快速测定

神经病理性疼痛小鼠坐骨神经掴型号

在对小鼠坐骨神经体内的电测量

在体内轴突兴奋性试验对大鼠尺神经的电生理测量

利用瞬态受体电位辣椒1型 Ultrapotent 激动剂建立单纯小纤维神经病变的小鼠模型

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

改良的小鼠神经性疼痛备用神经损伤手术模型

改良的小鼠神经性疼痛备用神经损伤手术模型

大鼠坐骨神经用于离体神经生理学的制备

用于转化疼痛研究的开源实时闭环电阈值跟踪

糖尿病大鼠模型神经传导阻滞的超声工具

摘要

探索更多视频

此视频中的章节

糖尿病大鼠的热痛觉阈值的快速测定

神经病理性疼痛小鼠坐骨神经掴型号

在对小鼠坐骨神经体内的电测量

在体内轴突兴奋性试验对大鼠尺神经的电生理测量

利用瞬态受体电位辣椒1型 Ultrapotent 激动剂建立单纯小纤维神经病变的小鼠模型

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

改良的小鼠神经性疼痛备用神经损伤手术模型

改良的小鼠神经性疼痛备用神经损伤手术模型

大鼠坐骨神经用于 离体 神经生理学的制备

用于转化疼痛研究的开源实时闭环电阈值跟踪

大鼠坐骨神经用于离体神经生理学的制备