Name: increased recovery time and decreased lps administration to study the vagus nerve stimulation mechanisms in limited inflammatory responses
Uploaded: 2017-03-29T14:00:00.000+00:00
Duration: 6 min 43 s
Description: Watch this Scientific Journal Video about Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses at JoVE.com

The study was supported by the Swedish Research Council, the Swedish Rheumatism Asociation, Karolinska Institute Foundations, Stockholm County Council, The Wallenberg Foundation, and the GV 80 Years' Foundation for research. The authors would also like to thank Hannah Aucott for proofreading the manuscript.

所有的动物实验均根据照顾和使用由当地伦理委员会Karolinska研究所，斯德哥尔摩批准动物的指导进行。当地伦理委员会如下动物保健的欧盟指令。 注意:从原始协议的主要变化是恢复时间后手术（6小时对 1小时）和LPS的水平注射（2mg / kg的与 15毫克/千克）。否则，与手术本身不同的设置没有被改变。 1.准备材料刺激<ol><li>打开计算机和连接到刺激电极（ 图1A）中的数据采集系统上。 </li><li>进入应答程序。 </li><li>以5毫克/毫升在1×PBS中，等分它制备浓度LPS的储备溶液，并且将其存储在-20℃。在实验的当天，解冻等分并准备ADE为了根据重量至约100微升注入到动物，LPS（0.5毫克/毫升）的样品quate。 </li></ol> 2.准备麻醉动物<ol><li>使用C57BL / 6小鼠。保持它们与12小时亮/暗循环的气候受控条件下，喂它们标准啮齿动物食物，并给予他们随意饮水 。 </li><li>对周围25克打压实验当天小鼠进行手术。 注:当炎症反应引起的，它进行定期检查，并在动物临床反应的观察就显得尤为重要。如果动物条件不满足道德标准需要通过CO 2吸入过早安乐死。 </li><li>设置麻醉机。 <ol><li>确保油管连接是否正确，并以任何方式不被损坏。确保通风正常工作。关键过滤器连接到异氟醚瓶，并填补了VAPOrizer用异氟烷的足够量。 </li><li>打开气源（空气和氧气），并确保该瓶含有足够的天然气用于实验。三通连接器，然后能够将异氟醚流发送到所述吸气室或掩模。 </li></ol></li><li>打开三路连接器的感应室。匹克从家笼一个鼠标和插入动物进入室内。调节流量调节器，以1.0升/分钟的氧气和1.0升/分钟的空气。调节异氟烷浓度为4 - 5％。 </li><li>当达到麻醉的期望的水平，当达到麻醉的期望电平，剃手术区和从所述腔室到所述掩模移动的动物。打开三路连接到面罩流。调节流量调节器，以0.25升/分钟的氧气和0.25升/分钟的空气。 <ol><li>调节异氟烷浓度至1.5 - 2.5％。在开始手术再修改前检查通过神经反射控制和呼吸速率麻醉水平即</li></ol></li><li>修复鼠标使用胶带工作台的腿。确保动物的鼻子还在仔细位于掩模。 </li></ol>迷走神经的3外科和刺激<ol><li>消毒用70％乙醇手术区。 </li><li>使用手术刀，仔细在颈部（约1至1.5厘米的切口）的电平切开皮肤。 注:在该协议中，手术过程结束这里假手术的动物。事实上，这已经表明，刚刚接触到神经用金属工具已经能够刺激到一定程度。因此，使用LPS少量时获得更准确的手术控制动物，停止在这一步的手术。 </li><li>用显微镜（12.5X物镜）的帮助下，从隔离使用解剖镊颈动脉左侧迷走神经。首先通过去除皮肤和脂肪层定位胸锁乳突肌，然后收回它为了把镊子神经和动脉都落后。 注:以下步骤是非常棘手的，因为神经和动脉贴紧对方。因此，这是很容易切断血管，杀死动物。但是，通过将镊子非常仔细的神经和动脉之间，他们最终分开，并有可能对神经隔离。 </li><li>放置电极（ 图1B）的迷走神经下。针状电极是很长，所以即使神经刺激过程中轻微移动，它总是会在与电极接触。 </li><li> （关于细胞因子水平对所读出的， 即，为了测量下调效应）进行LPS的腹膜内（ip）注射（2毫克/千克）与注射器的帮助。 </li><li>在开始刺激前等待5分钟。 </li><li>通过在确认程序按下启动按钮刺激5分钟的迷走神经在5V和1Hz。 </li><li>回覆移动电极和缝合手术缝合线的动物的伤口。 </li><li>喷在伤口无刺阻隔薄膜（NSBF），以提高治疗和感染保护。 </li></ol> 4.动物的恢复<ol><li>手术后，将动物返回其家笼觉醒和恢复。在为了保持体温的红外光，确保监测动物，直到全意识已经恢复。 </li><li>让动物在笼子里收回牺牲分析前6小时。 注:迷走神经刺激的效果是非常快，也被证明是持久（可达48小时），所以恢复时间可以由实验者根据研究的需要进行设置。 </li></ol> 5.牺牲作进一步的分析<ol><li>放置动物在链接到一个CO 2管理装置的保持架。 </li><li>在设备上设置一个5CO 2吸入-min周期。 </li><li>当安乐死完成后，收集感兴趣的器官，并直接将它们冷冻用于进一步的分析干冰（ 例如，细胞因子水平的中脾提取物使用鼠标TH1测量/ TH2 9-Plex的测定）3。 </li></ol>

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作者什么都没有透露。

自21世纪初发现的，盖的机制已经被彻底研究。我们现在有途径的好照片，特别是，靶器官，脾，其中NE，记忆性T细胞，乙酰胆碱，和巨噬细胞作为一种非常有效的团队工作，下调炎症介质2。我们最近还发表了关于功能性前列腺素系统的小鼠中的重要性，特别是PGE 2，这显然是对乙酰胆碱的释放脾脏中盖3的活化后的强制性成分数据。 实验技术，被称为迷走神经刺激，可以很容易地在实验室中进行，但一些重要的协议必须关于动物和手术过程中观察到。我们使用正弦波刺激信号，这应该是一个"电荷平衡"的刺激，以避免建立在电荷神经可能摧毁它（而不是单相刺激）。同时刺激神经，这是不可能的传入从传出信号，这只能用于其它目的与迷走神经切断实验来完成分离。用于实验的持续时间和频率已知对细胞因子释放的影响下炎症的挑战，例如，但不会影响心脏率或给健康对照组明显的副作用。 首先，实验者必须始终牢记，处理，手术和动物的恢复必须按照动物护理的伦理规则来完成。另一点是，练习手术是非常重要的。分离所述迷走神经是一个困难的步骤，它可以在发生血管损伤的动物致死。随着经验的积累，手术可在15内完成 - 20分钟，这意味着动物没有被麻醉的很长一段时间，这帮助恢复。 讨论也可以就一个好的假手术动物的选择提出。在我们的协议中，我们使用的动物，只有在颈部的水平进行了手术，没有放置电极迷走神经下。之所以选择此选项是，只是将所述电极的神经下将机械刺激神经在一定程度上（如先前已观察到的）19，创建掩蔽发生在炎症的非常低的水平的潜在机制的风险。如果使用非常高剂量的LPS，最好假手术对照很可能是放置神经下电极，作为假手术的效果将不干扰或掩模上的细胞因子的突发中的明显的电刺激的效果。 我们已经开发出了目前的协议，以适应我们的研究领域中所遇到的各种问题。正如我们在前面的论述了，我们看着前列腺素系统在CAP可能参与。由于酶代谢，我们认为原始协议的1小时的恢复时间将不适合我们的实验。因此，我们扩展了手术后的时间间隔6小时。这样做，我们也不得不向下滴定LPS的剂量，以符合道德标准。我们选择了6小时，我们认为足够长的时间用于此目的的延迟的恢复时间。我们还从15mg / kg的（原始协议）至0毫克/千克滴定LPS。以2mg / kg时，我们看到了VNS的对细胞因子的最后效果（ 图2），即消失在较低剂量的效果。 虽然VNS的脾脏中的主要作用是已知的，其特征在于，记忆性T细胞和活化后，巨噬细胞，在允许的降低细胞因子，我们这里也表明，VNS似乎直接诱导介质释放细胞（它仍然是从细胞，necessitati不清楚纳克进一步研究），以便从对炎症的一次响应招募其他的细胞类型。事实上，虽然VNS强烈强烈的炎症诱导（15mg / kg的LPS; 即，原始协议）后KC / GRO下降时，它激活KC / GRO在没有炎症的一个非常快的释放。趋化因子KC / GRO，一个IL-8相关蛋白具有很强的趋化性质，已知具有在白细胞发展中起重要作用（ 例如，驱动成熟和活化），贩卖（ 例如，吸引和嗜中性粒细胞的募集），和功能15 。例如，中性粒细胞是先天免疫系统的吞噬系统的重要成员。他们充当主机抵御病原体入侵的第一线，但同时又是炎症引起的损伤16的重要介质。有趣的是，在我们试图改善该协议，我们然后在此观察，突出直销来到在迷走神经功能的脾其它细胞类型的吨参与。 所有在该领域的开拓性工作集中于脾，CAP的靶器官，并且在该途径的响应于败血症或外周全身性炎症的免疫调节作用。重要的是，这项工作用的是迷走神经的刺激急性完成，如在本文所提出的一个。这可以被用于或（在手术后24至48小时的最大值）在涉及炎症的慢性动物模型中的功能性研究观察到的短期影响特定器官的分子分析。 然而，迷走神经游荡当然也意味着CAP可显著使用治疗涉及不同的神经支配的器官13慢性炎症性疾病。一个在这方面最深入研究的器官是肠，重点对炎症性肠疾病（IBD），包括克罗恩<html> ＃39病和溃疡性结肠炎，而且术后引起肠梗阻17。此外，涉及肝，肾和肺的上限监管在许多其他炎症性疾病的重要性也正在调查18。尽管如此，目前的协议不能被在此上下文中使用的，如迷走神经激活的长期效果将需要当然可以只用体内植入电极来完成重复刺激。为了这个目的，所述动物应进行手术，其中刺激袖带电极置于围绕迷走神经20。同样重要的是要注意，后一种技术在人类或更大尺寸的动物更频繁地描述。刺激迷走神经的另一种方法是机械的，非侵入性和经皮刺激迷走神经的显示内毒素血症小鼠模型的免疫抑制作用REF"&gt; 21。然而，这种技术的主要关切是结果的可重复性，以及如何确保迷走神经受到刺激的动物模型的长期评估的方法相同。装置已经被开发为人类如反式耳VNS刺激或经宫颈VNS刺激，即发送电脉冲，这是深受主题21的耐受性。他们已经主要用于治疗癫痫症和偏头痛，并表现出乐观的结果，可能导致未来走动和外源性治疗。 总之尤其是在体内迷走神经的广泛支配的，因为，该上限的监管研究了广泛的炎症性疾病的寻找有趣的分子机械性能，可能导致未来潜在治疗靶点的希望。在这里，我们提出了一个尖锐的方法来刺激迷走神经。它允许CAP的研究在有限flammatory响应由于中等炎症刺激与VNS和分析（允许酶代谢的发生，例如）之间的较长时间范围内组合。 理解的分子机制的基础的迷走神经刺激的效果，以及在炎性疾病的治疗中其有效地使用，是非常重要的，特别是从临床角度。事实上，该方法的优点是广阔的。由于速度极快的神经信号的特性，它是快速和有效的;上的细胞因子水平的观察到的效果，例如，发生在小于1个小时。 4此外，神经的机械的，非侵入性的，和经皮刺激，可以使用给予希望未来的走动和容易给药的疗法。最后，违背了正规治疗，迷走神经刺激使用内源性途径。因此，与所有的药物治疗，没有新的代理人在介绍到主体，从而避免了任何潜在的副作用。

先天免疫提供了针对感染和疾病在广泛的生物体防御的立即第一行。它不仅启动初次免疫应答以消除威胁，但它也起着活化和教育，其执行在病原体特异性方式二次免疫应答的适应性免疫中起关键作用。炎症细胞因子和趋化因子过多，这反过来又吸引其他免疫细胞对感染部位的能力和诱发炎症的主要体征，如红，肿，痛，功能丧失，发热策划。的持续时间和炎症的强度取决于几个因素，但解决炎症和恢复稳态是避免慢性炎症性疾病的发病的关键步骤。在神经科学和免疫学领域的最新进展没有完全揭开特定的神经机制，与巨大的治疗潜能控制INFL无论是在中枢神经系统和外周ammation。一这些机制之一是胆碱能抗炎通路（CAP），也被称为炎性反射，这是由自主神经系统4,5驱动。 这是目前认为炎症介质激活感觉神经和发送有关炎症的状态，以中枢神经系统的信号。一种反射反应，然后通过传出迷走神经活性。在CAP的解剖细节进行了广泛的研究揭示了两个神经组成的副交感神经，交感神经模型，迷走神经和神经脾，分别为6。在CAP中，活化胆碱能传出迷走神经在腹腔 - 肠系膜神经节结束，导致肾上腺素能神经脾的活化的机制尚待探索。脾神经，从而激活，已知的是内瓦泰岛接近免疫细胞在白色的纸浆，边缘区，和脾，主要和强制器官CAP 7,8的红髓。去甲肾上腺素（NE）从脾神经末梢结合于脾T淋巴细胞上表达相应的β2肾上腺素能受体。这引起胆碱乙酰转移酶（ChAT的）介导的乙酰胆碱（ACh）的释放，这反过来又激活巨噬细胞上α7烟碱型乙酰胆碱受体（α7nACh），从而限制了细胞因子的产生和炎症2。因此，现在很清楚，神经系统能够调节炎症外周组织和恢复局部免疫稳态。 由于通路顾名思义，乙酰胆碱系统是至关重要的这种神经免疫调节通路的功能。有趣的是，在所涉及的激活的机制的CAP似乎是在周边和在中枢神经系统中的不同。而烟碱受体（α7nAChR）在脾的重要性在前面已经证明9，毒蕈碱受体（的mAChR）是强制性的通路10，11的中心活化。最近，一个中枢作用M1毒蕈碱激动剂的外周施用显著抑制血清和脾脏肿瘤坏死因子α（TNFα）致死小鼠内毒素血症期间，所要求的完整迷走神经，脾神经信令12的动作。最近，我们还表明，缺乏前列腺素E 2小鼠（PGE 2）无法对迷走神经刺激作出反应，并没有下调血清和脾脏3中的细胞因子的LPS诱导释放。因此，CAP也可能比主乙酰胆碱pathw其他系统调节唉。 迷走神经已被命名为这样的，因为其在体内游荡当然，支配主要器官，包括肝，肺，脾，肾和肠13。考虑到这个大神经支配和迷走神经的非常有效的免疫效果，CAP的治疗潜力可以覆盖广泛的炎症性疾病。迷走神经可电（或机械）活化，具有过电压和频率，并与传统的治疗控制，无药物加入到所述主体。试验目前在风湿病患者进行，例如，以测试在治疗慢性炎症14 VNS的临床意义。总之，神经·免疫通信和炎症的调节目前正在研究中，这将提供一个可能的替代治疗对常规治疗。因此，迷走神经的分析stimulatio在不同的神经支配的器官n效果，同时也表征在慢性炎症的动物模型中的潜在治疗作用的，肯定会给予的见解，并希望新的潜在治疗靶点。 由特蕾西和他的同事4开发的原始方法不能移位，我们的研究领域，由于炎症反应（由LPS的致死剂量）的过度刺激和CAP激活和读出之间太短的时间范围。在本论文中，我们将提出对原始协议所做的修改，对细胞因子水平比较两个不同的方法，并突出于靶器官（脾）的新和相反观察。

<table><tbody><tr><td>Computer</td><td>Toshiba</td><td>-</td><td>Any computer is actually compatible</td></tr><tr><td>MP-150 data acquisition system</td><td>Biopac Systems</td><td>MP150WSW</td><td></td></tr><tr><td>Acknowledge software</td><td>Biopac Systems</td><td></td><td></td></tr><tr><td>Mice C57Bl/6</td><td>Charles River</td><td></td><td></td></tr><tr><td>Anesthetic machine</td><td>Simtec Engineering</td><td></td><td></td></tr><tr><td>Medical oxygen bottle</td><td>AGA</td><td>107563</td><td></td></tr><tr><td>Medical air bottle&amp;nbsp;</td><td>AGA</td><td>108639</td><td></td></tr><tr><td>Vetflurane (1,000 mg/g)</td><td>Virbac</td><td>137317</td><td></td></tr><tr><td>LPS</td><td>Sigma-Aldrich</td><td>L2630</td><td></td></tr><tr><td>Saline</td><td>Merck Millipore</td><td>1024060080</td><td></td></tr><tr><td>PBS 10x</td><td>Sigma-Aldrich</td><td>P5493</td><td>Diluted 10 times for used concentration</td></tr><tr><td>Syringe (1 ml)</td><td>BD Plastipak</td><td>303172</td><td></td></tr><tr><td>Needles 23 G</td><td>KD-FINE</td><td>900284</td><td>0.6 x 30 mm (blue)</td></tr><tr><td>Microdissecting forceps (curved)</td><td>Sigma-Aldrich</td><td>F4142</td><td></td></tr><tr><td>Dissecting scissors</td><td>Sigma-Aldrich</td><td>Z265969</td><td></td></tr><tr><td>Surgical suture 4-0</td><td>Ethicon</td><td>G667G</td><td></td></tr><tr><td>Euthanasia unit</td><td>Euthanex Smartbox</td><td>EA-32000</td><td></td></tr><tr><td>Cavilon No Sting Barrier Film</td><td>3M Health Care</td><td>3346N</td><td></td></tr><tr><td>TH1/TH2 9-Plex assay, ultrasensitive kit</td><td>MesoScale Discovery</td><td>K15013C-1</td><td></td></tr><tr><td>Stimulating electrode device</td><td>Biopac Systems</td><td>STIMSOC</td><td></td></tr><tr><td>Aesculap Isis shaver</td><td>Agnthos</td><td>GT420</td><td></td></tr><tr><td>R70</td><td></td><td></td><td>Rodent diet from Lantmannen, Stockholm, Sweden</td></tr></tbody></table>

increased recovery time and decreased lps administration to study the vagus nerve stimulation mechanisms in limited inflammatory responses

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release cytokines and other mediators of inflammation. For a long time, humoral and cellular mechanisms have been studied for their role in regulating the immune response, but recent advances in the field of immunology and neuroscience have also unraveled specific neural mechanisms with interesting therapeutic potential. The so-called cholinergic anti-inflammatory pathway (CAP) has been described to control innate immune responses and inflammation in a very potent manner. In the early 2000s, Tracey and collaborators developed a technique that stimulates the vagus nerve and mimics the effect of the pathway. The methodology is based on the electrical stimulation of the vagus nerve at low voltage and frequency, in order to avoid any side effects of overstimulation, such as deregulation of heart rate variability. Electrical devices for stimulation are now available, making it easy to set up the methodology in the laboratory. The goal of this research was to investigate the potential involvement of prostaglandins in the CAP. Unfortunately, based on earlier attempts, we failed to use the original protocol, as the induced inflammatory response either was too high or was not suitable for enzymatic metabolism properties. The different settings of the original surgery protocol remained mostly unchanged, but the conditions regarding inflammatory induction and the time point before sacrifice were improved to fit our purposes (i.e., to investigate the involvement of the CAP in more limited inflammatory responses). 
The modified version of the original protocol, presented here, includes a longer time range between vagus nerve stimulation and analysis, which is associated with a lower induction of inflammatory responses. Additionally, while decreasing the level of lipopolysaccharides (LPS) to inject, we also came across new observations regarding mechanistic properties in the spleen.

手术后增加时间流逝和减少LPS的剂量后TNFα和白介素1β（IL-1β）的水平如前所示，使用原来的协议，VNS降低TNFα的水平（169.3±24.9皮克/在SHAM mg相比在VNS 39.7±10.8皮克/毫克，P &lt;0.001）和IL-1β（360.0±40.21微克/毫克SHAM对191.7±27.2皮克/在VNS腹膜内注射LPS（15毫克/千克）（ 图2A）以后毫克，p &lt;0.01）在脾。改变用于分析的时间为1〜6小时，降低LPS剂量为15〜2毫克/公斤后，我们仅观察到对TNFαVNS的效果（13.67±1.81皮克在SHAM / mg相比在8.82±1.20皮克/毫克VNS，p &lt;0.05），而IL-1β并没有受到影响（368.62±35.65皮克在SHAM / mg相比304.99±43.54皮克/毫克）（ 图2B）。没有任何的注射LPS挠度（ 即，盐水），无差异VNS（ 图2C）后进行检测。 使用的不同状况角质形成细胞趋化/人生长调节致癌基因（KC / GRO）的水平在脾VNS后与原来的协议中，我们表明，KC / GRO是强烈VNS（在SHAM 597.4±17.8皮克/ mg相比在VNS 416.4±29.7皮克/毫克，P &lt;0.001）（ 图3A）下调。 SHAM和VNS动物（ 图3B）之间使用LPS的6小时的时间点和2毫克/千克，没有观察到变化，而VNS仍然是强有力的上TNFα，例如。然而，当没有LPS注射了6个小时的时间点上，我们观察到KC / GRO的以下VNS强烈上调（59.37±4.29皮克在SHAM / mg相比在VNS 141.22±10.56微克/毫克，P &lt;0.001 ）（ 图3C）。正如预期的那样，大部分其它细胞因子（TNFα，IL-1β，IL-2，IL-4，IL-5，IL-10，IL-12，和IFNγ）是在非常低的水平（如果检测到），以及无差异SHAM之间可以看到和VNS刺激的动物。 <img alt="图1" src="/files/ftp_upload/54890/54890fig1.jpg" /> 图1: 以执行迷走神经刺激所需的材料照片。 A）ⅰ）计算机，ⅱ）数据采集系统，以及iii）刺激电极装置。 B）被放置在迷走神经下的电极的特写。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54890/54890fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54890/54890fig2.jpg" /> 图2:TNFα的水平和IL-1β在脾ëSHAM和VNS治疗的动物的xtracts。动物进行SHAM或VNS手术，根据下列条件:A）15毫克/公斤LPS，B）6小时的2毫克/千克LPS腹膜内注射后的恢复的腹膜内注射后1小时恢复，和C） 6小时恢复无LPS注射。水平上的促炎症细胞因子包被的板上测量并表示为组织的皮克/毫克。数据表示为平均值±SEM。一个星号表示（每组n = 8）和VNS SHAM小鼠之间的统计学差异。斯氏t检验，* P &lt;0.05; ** P &lt;0.01; *** P &lt;0.001。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54890/54890fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54890/54890fig3.jpg" /> 图3: 在SP KC / GRO的水平SHAM和利恩提取物VNS治疗的幼鼠。动物进行SHAM或VNS手术，根据下列条件:A）15毫克/公斤LPS，B）6小时的2毫克/千克LPS腹膜内注射后的恢复的腹膜内注射后1小时恢复，和C） 6小时恢复无LPS注射。水平上的促炎症细胞因子包被的板上测量并表示为组织的皮克/毫克。数据表示为平均值±SEM。一个星号表示（每组n = 8）和VNS SHAM小鼠之间的统计学差异。学生的t检验*** P &lt;0.001。

Watch this Scientific Journal Video about Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses at JoVE.com

Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses

Vagus nerve stimulation has proven to have a strong efficacy for decreasing peripheral inflammation. Here, we present a modified vagus nerve stimulation protocol that allows for further examinations of the cholinergic anti-inflammatory mechanisms in limited inflammatory responses.

增加了恢复时间，并减少LPS管理研究迷走神经刺激机制的有限炎症反应

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release ...

increased-recovery-time-decreased-lps-administration-to-study-vagus

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

9.8K 次观看.  Karolinska University Hospital. Vagus nerve stimulation has proven to have a strong efficacy for decreasing peripheral inflammation. Here, we present a modified vagus nerve stimulation protocol that allows for further examinations of the cholinergic anti-inflammatory mechanisms in limited inflammatory responses.

视频: Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses

Vagus Nerve Stimulation as a Tool to Induce Plasticity in Pathways Relevant for Extinction Learning

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been maintaining the behavior. There is close correspondence between fear and human anxiety, and therefore studies of extinction learning might provide insight into the biological nature of anxiety-related disorders such as post-traumatic stress disorder, and they might help to develop strategies to treat them. Preclinical research aims to aid extinction learning and to induce targeted plasticity in extinction circuits to consolidate the newly formed memory. Vagus nerve stimulation (VNS) is a powerful approach that provides tight temporal and circuit-specific release of neurotransmitters, resulting in modulation of neuronal networks engaged in an ongoing task. VNS enhances memory consolidation in both rats and humans, and pairing VNS with exposure to conditioned cues enhances the consolidation of extinction learning in rats. Here, we provide a detailed protocol for the preparation of custom-made parts and the surgical procedures required for VNS in rats. Using this protocol we show how VNS can facilitate the extinction of conditioned fear responses in an auditory fear conditioning task. In addition, we provide evidence that VNS modulates synaptic plasticity in the pathway between the infralimbic (IL) medial prefrontal cortex and the basolateral complex of the amygdala (BLA), which is involved in the expression and modulation of extinction memory.

Vagus nerve stimulation (VNS) has emerged as a tool to induce targeted synaptic plasticity in the forebrain to modify a range of behaviors. This protocol describes how to implement VNS to facilitate the consolidation of fear extinction memory.

迷走神经刺激作为一种工具来诱导可塑性相关途径灭绝学习

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been ...

科研

行为学

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

肺炎链球菌在鼻内殖民期间炎症反应的特征

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

免疫与感染

A Method of Nodose Ganglia Injection in Sprague-Dawley Rat

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located in the organs of the abdomen and thorax. The vagus nerve communicates information from stimuli such as heart rate, blood pressure, bronchopulmonary irritation, and gastrointestinal distension to the nucleus of solitary tract of the medulla. The cell bodies of the vagus nerve are located in the nodose and petrosal ganglia, of which the majority are located in the former. The nodose ganglia contain a wealth of receptors for amino acids, monoamines, neuropeptides, and other neurochemicals that can modify afferent vagus nerve activity. Modifying vagal afferents through systemic peripheral drug treatments targeted at the receptors on nodose ganglia has the potential of treating diseases such as sleep apnea, gastroesophageal reflux disease, or chronic cough. The protocol here describes a method of injection neurochemicals directly into the nodose ganglion. Injecting neurochemicals directly into the nodose ganglia allows study of effects solely on cell bodies that modulate afferent nerve activity, and prevents the complication of involving the central nervous system as seen in systemic neurochemical treatment. Using readily available and inexpensive equipment, intranodose ganglia injections are easily done in anesthetized Sprague-Dawley rats.

Afferent vagal signaling transmits important information to central nervous system from receptors located in organs of the abdomen and thorax. The nodose ganglia of vagus nerves contain many types of receptors that modulate vagal activity. This protocol describes a method of local injections of neurochemicals into the nodose ganglia.

结状神经节注射液只SD大鼠的方法

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located ...

神经科学

Laboratory Administration of Transcutaneous Auricular Vagus Nerve Stimulation (taVNS): Technique, Targeting, and Considerations

Non-invasive vagus nerve stimulation (VNS) may be administered via a novel, emerging neuromodulatory technique known as transcutaneous auricular vagus nerve stimulation (taVNS). Unlike cervically-implanted VNS, taVNS is an inexpensive and non-surgical method used to modulate the vagus system. taVNS is appealing as it allows for rapid translation of basic VNS research and serves as a safe, inexpensive, and portable neurostimulation system for the future treatment of central and peripheral disease. The background and rationale for taVNS is described, along with electrical and parametric considerations, proper ear targeting and attachment of stimulation electrodes, individual dosing via determination of perception threshold (PT), and safe administration of taVNS.

Laboratory Administration of Transcutaneous Auricular Vagus Nerve Stimulation taVNS: Technique, Targeting, and Considerations

A methodological description of the technique, potential targets, and proper administration of transcutaneous auricular vagus nerve stimulation (taVNS) on the human ear is described.

经皮耳穴神经刺激实验室管理 (tavns): 技术、定位和考虑

Non-invasive vagus nerve stimulation (VNS) may be administered via a novel, emerging neuromodulatory technique known as transcutaneous auricular vagus ...

Nerve Stimulator-guided Injection of Autologous Stem Cells Near the Equine Left Recurrent Laryngeal Nerve

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent laryngeal nerve shows lesions of demyelination. The benefit of applying stem cells to demyelinated nerves has been demonstrated in various animal models. The aim of the study was to test the feasibility and safety of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to the left recurrent laryngeal nerve in healthy horses by using an electrical nerve stimulator.
Muscle-derived stems cell are obtained from five healthy Standardbred horses by sampling 20 mg of muscle tissue with a semi-automatic 14 G biopsy needle from the triceps muscle. Movements of the larynx are monitored via upper-airway video endoscopy. The left recurrent laryngeal nerve is approached with an insulated nerve block needle. Nerve stimulation is applied, starting at 2 mA, and the successful abduction of the left arytenoid is monitored. The stimulation intensity is reduced progressively. When a loss of the motor response is observed at 0.5 mA, 107 autologous muscle-derived stem cells are injected. Two examiners, who are blinded to the time point, score the laryngeal function of the horses prior to the treatment and at day 1, day 7, and day 28 after the injection of the cells. In a sixth horse, 1 mL of 2% lidocaine is injected to further confirm the correct positioning of the needle. This leads to a temporary paralysis of the left arytenoid cartilage.
This study proves that the recurrent laryngeal nerve can be approached with the help of an electrical nerve stimulator and that the electrical stimulation of the nerve is well tolerated by the horses. No modification of the laryngeal function was observed in any of the horses after the injection of the stem cells. Further studies should be conducted to describe the effects of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to horses suffering from RLN.

Here, we present a protocol to inject autologous muscle-derived stem cells in the proximity to the recurrent laryngeal nerve with the help of an electrical nerve stimulator. This novel technique may become useful for the treatment of equine recurrent laryngeal neuropathy.

神经刺激器引导注射自体干细胞近马左喉返神经

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent ...

医学

Vagus Nerve Stimulation As an Adjunctive Neurostimulation Tool in Treatment-resistant Depression

Vagus nerve stimulation (VNS) is an approved neurostimulation therapy. The purpose of the method is to treat patients with therapy-resistant depression (TRD). VNS exhibits antidepressive and stabilizing effects. This method is particularly useful as a long-term treatment, in which up to two-thirds of patients respond. The vagus nerve stimulator is positioned on the left vagus nerve during a surgical procedure and is activated telemetrically by a wand connected to a handheld computerized device. The treating physician can perform various adjustments of the vagus nerve stimulator during in-office visits (e.g., by modifying stimulation intensity or stimulation frequency) to achieve maximum therapeutic effects with low side effects. Set-up of the device usually takes several months. Typical side effects include wound infection, temporary salivation, coughing, paralysis of the vocal cords, bradycardia, or even asystole. The patient can stop the VNS by placing a magnet over the generator. The current protocol describes delivery of the specific stimulation tool and methods for adjusting the tuning parameters to achieve the best remission rates in patients with TRD.

Vagus nerve stimulation (VNS) has been shown to be effective as an adjunct treatment for treatment-resistant depression (TRD). VNS leads to antidepressive and antisuicidal effects and quality of life improvements. This protocol offers a step-by-step guide to managing and adjusting a vagus nerve stimulator for efficient treatment of TRD.

迷走神经刺激作为抗抑郁抑郁症的辅助神经刺激工具

Vagus nerve stimulation (VNS) is an approved neurostimulation therapy. The purpose of the method is to treat patients with therapy-resistant depression ...

Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. Several recent studies have demonstrated the effectiveness of chronic VNS in enhancing central nervous system plasticity to improve motor rehabilitation, extinction learning, and sensory discrimination. Construction of chronically implantable devices for use in such studies is challenging due to rats&#8217; small size, and typical protocols require extensive training of personnel and time-consuming microfabrication methods. Alternatively, commercially available implantable cuff electrodes can be purchased at a significantly higher cost. In this protocol, we present a simple, low-cost method for construction of small, chronically implantable peripheral nerve cuff electrodes for use in rats. We validate the short and long-term reliability of our cuff electrodes by demonstrating that VNS in ketamine/xylazine anesthetized rats produces decreases in breathing rate consistent with activation of the Hering-Breuer reflex, both at the time of implantation and up to 10 weeks after device implantation. We further demonstrate the suitability of the cuff electrodes for use in chronic stimulation studies by pairing VNS with skilled lever press performance to induce motor cortical map plasticity.

Existing approaches for constructing chronically implantable peripheral nerve cuff electrodes for use in small rodents often require specialized equipment and/or highly trained personnel. In this protocol we demonstrate a simple, low-cost approach for fabricating chronically implantable cuff electrodes, and demonstrate their effectiveness for vagus nerve stimulation (VNS) in rats.

大鼠慢性植入的外周神经刺激电极的制备

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. ...

经皮耳迷走神经刺激的神经调控测量

Understanding the Effects of Non&#45;Invasive Transauricular Vagus Nerve Stimulation on EEG and HRV

Several studies have demonstrated promising results of transcutaneous auricular vagus nerve stimulation (taVNS) in treating various disorders; however, no mechanistic studies have investigated this technique's neural network and autonomic nervous system effects. This study aims to describe how taVNS can affect EEG metrics, HRV, and pain levels. Healthy subjects were randomly allocated into two groups: the active taVNS group and the sham taVNS group. Electroencephalography (EEG) and Heart Rate Variability (HRV) were recorded at baseline, 30 min, and after 60 min of 30 Hz, 200-250 &#181;s taVNS, or sham stimulation, and the differences between the metrics were calculated. Regarding vagal projections, some studies have demonstrated the role of the vagus nerve in modulating brain activity, the autonomic system, and pain pathways. However, more data is still needed to understand the mechanisms of taVNS on these systems. In this context, this study presents methods to provide data for a deeper discussion about the physiological impacts of this technique, which can help future therapeutic investigations in various conditions.

作者聚焦：探索经耳迷走神经刺激的效果

This protocol provides information on how to apply transcutaneous auricular vagus nerve stimulation (taVNS) in a clinical trial, including potential biomarkers such as EEG metrics and heart rate variability (HRV) to measure the effect of this treatment on the autonomic nervous system.

了解非侵入性耳迷走神经刺激对脑电图和 HRV 的影响

Several studies have demonstrated promising results of transcutaneous auricular vagus nerve stimulation (taVNS) in treating various disorders; however, no ...

增加了恢复时间，并减少LPS管理研究迷走神经刺激机制的有限炎症反应

摘要

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此视频中的章节

迷走神经刺激作为一种工具来诱导可塑性相关途径灭绝学习

肺炎链球菌在鼻内殖民期间炎症反应的特征

结状神经节注射液只SD大鼠的方法

经皮耳穴神经刺激实验室管理 (tavns): 技术、定位和考虑

神经刺激器引导注射自体干细胞近马左喉返神经

迷走神经刺激作为抗抑郁抑郁症的辅助神经刺激工具

大鼠慢性植入的外周神经刺激电极的制备

了解非侵入性耳迷走神经刺激对脑电图和 HRV 的影响

了解非侵入性耳迷走神经刺激对脑电图和 HRV 的影响

了解非侵入性耳迷走神经刺激对脑电图和 HRV 的影响