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
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?...

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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。 <p class="jove_conten...

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。

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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Computer</td>
			<td style="width:91px;">Toshiba</td>
			<td style="width:136px;">-</td>
			<td style="width:191px;">Any computer is actually compatible</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">MP-150 data acquisition system</td>
			<td style="width:91px;">Biopac Systems</td>
			<td rowspan="2" style="width:136px;">MP150WSW</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Acknowledge software</td>
			<td style="width:91px;">Biopac Systems</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Mice C57Bl/6</td>
			<td style="width:91px;">Charles River</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Anesthetic machine</td>
			<td style="width:91px;">Simtec Engineering</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Medical oxygen bottle</td>
			<td style="width:91px;">AGA</td>
			<td style="width:136px;">107563</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Medical air bottle&#160;</td>
			<td style="width:91px;">AGA</td>
			<td style="width:136px;">108639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Vetflurane (1000mg/g)</td>
			<td style="width:91px;">Virbac</td>
			<td style="width:136px;">137317</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">LPS</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:136px;">L2630</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Saline</td>
			<td style="width:91px;">Merck Millipore</td>
			<td style="width:136px;">1024060080</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">PBS 10X</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:136px;">P5493</td>
			<td>Diluted 10 times for used concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Syringe (1 ml)</td>
			<td style="width:91px;">BD Plastipak</td>
			<td style="width:136px;">303172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Needles 23G</td>
			<td style="width:91px;">KD-FINE</td>
			<td style="width:136px;">900284</td>
			<td>0.6 x 30 mm (blue)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Microdissecting forceps (curved)</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:136px;">F4142</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Dissecting scissors</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:136px;">Z265969</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Surgical suture 4-0</td>
			<td style="width:91px;">Ethicon</td>
			<td style="width:136px;">G667G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Euthanasia unit</td>
			<td style="width:91px;">Euthanex Smartbox</td>
			<td style="width:136px;">EA-32000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Cavilon No Sting Barrier Film</td>
			<td style="width:91px;">3M Health Care</td>
			<td style="width:136px;">3346N</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">TH1/TH2 9-Plex assay, ultrasensitive kit</td>
			<td style="width:91px;">MesoScale Discovery</td>
			<td style="width:136px;">K15013C-1</td>
			<td></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...

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 ...

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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 ...

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免疫与感染

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

尾部皮肤来研究异基因CD4 + T细胞应答的小鼠移植

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

高通量实时定量RT-PCR检测方法的确定I型和III型干扰素亚型的表达谱

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

的气管内管理<em&gt;流感嗜血杆菌</em&gt;在小鼠模型来研究气道炎症

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

辅助性T细胞作为一种遗传方法来研究逆转录病毒转导机制控制其分化和功能

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

IL-8瞬时转化的小鼠模型<em&gt;体内</em&gt;长期监测炎症反应

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

用于炎症反应的新内皮调节因子的筛选测定

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

一种研究人体 T 细胞召回反应的吸水泡协议

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...