Name: a reproducible intensive care unit-oriented endotoxin model in rats
Uploaded: 2021-02-20T15:00:00
Description: Watch this Scientific Journal Video about A Reproducible Intensive Care Unit-Oriented Endotoxin Model in Rats at JoVE.com

作者要感谢Beatrice Beck-Schimmer（医学博士）和Erik Schadde（医学博士）的批判性检查和他们对本手稿的宝贵贡献。

该协议中提出的所有实验均已获得瑞士苏黎世州兽医局的批准（批准号134/2014和ZH088/19）。此外，本实验中执行的所有步骤均符合瑞士医学科学院（SAMS）的《动物实验指南》和欧洲实验动物科学协会联合会（FELASA）的指南。
1. 麻醉诱导和动物监测
<ol><li>在没有病原体的条件下将体重为250-300克（g）的雄性Wistar大鼠保存在通风笼中。在22±1°C的环境温度下提供12-12小时的光/暗循...

<ol>
	<li>Hotchkiss, R. S., Karl, I. 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+pathophysiology+and+treatment+of+sepsis.">The pathophysiology and treatment of sepsis.</a> New England Journal of Medicine. 348 (2), 138-150 (2003).</li><li>Singer, M., 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=The+Third+International+Consensus+Definitions+for+Sepsis+and+Septic+Shock+(Sepsis-3).">The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3).</a> Journal of the American Medical Association. 315 (8), 801-810 (2016).</li><li>Vincent, J. 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Current Estimates and Limitations.</a> American Journal of Respiratory and Critical Care Medicine. 193 (3), 259-272 (2016).</li><li>Kumar, A., 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=Duration+of+hypotension+before+initiation+of+effective+antimicrobial+therapy+is+the+critical+determinant+of+survival+in+human+septic+shock.">Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock.</a> Critical Care Medicine. 34 (6), 1589-1596 (2006).</li><li>Gotts, J. E., Matthay, M. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+sepsis+and+shock:+a+review+and+lessons+learned.">Animal models of sepsis and shock: a review and lessons learned.</a> Shock. 9 (1), 1-11 (1998).</li><li>Buras, J. A., Holzmann, B., Sitkovsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+sepsis:+setting+the+stage.">Animal models of sepsis: setting the stage.</a> Nature Reviews Drug Discovery. 4 (10), 854-865 (2005).</li><li>Perretti, M., Duncan, G. S., Flower, R. J., Peers, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+corticosterone,+interleukin-1+and+tumour+necrosis+factor+in+rat+experimental+endotoxaemia:+comparison+between+Lewis+and+Wistar+strains.">Serum corticosterone, interleukin-1 and tumour necrosis factor in rat experimental endotoxaemia: comparison between Lewis and Wistar strains.</a> British Journal of Pharmacology. 110 (2), 868-874 (1993).</li><li>Marechal, X., 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=Endothelial+glycocalyx+damage+during+endotoxemia+coincides+with+microcirculatory+dysfunction+and+vascular+oxidative+stress.">Endothelial glycocalyx damage during endotoxemia coincides with microcirculatory dysfunction and vascular oxidative stress.</a> Shock. 29 (5), 572-576 (2008).</li><li>Thiemermann, C., Ruetten, H., Wu, C. C., Vane, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multiple+organ+dysfunction+syndrome+caused+by+endotoxin+in+the+rat:+attenuation+of+liver+dysfunction+by+inhibitors+of+nitric+oxide+synthase.">The multiple organ dysfunction syndrome caused by endotoxin in the rat: attenuation of liver dysfunction by inhibitors of nitric oxide synthase.</a> British Journal of Pharmacology. 116 (7), 2845-2851 (1995).</li><li>Osuchowski, M. F., 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=Minimum+quality+threshold+in+pre-clinical+sepsis+studies+(MQTiPSS):+an+international+expert+consensus+initiative+for+improvement+of+animal+modeling+in+sepsis.">Minimum quality threshold in pre-clinical sepsis studies (MQTiPSS): an international expert consensus initiative for improvement of animal modeling in sepsis.</a> Intensive Care Medicine Experimental. 6 (1), 26 (2018).</li><li>Fink, M. P., Heard, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+models+of+sepsis+and+septic+shock.">Laboratory models of sepsis and septic shock.</a> Journal of Surgical Research. 49 (2), 186-196 (1990).</li><li>Buras, J. A., Holzmann, B., Sitkovsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+sepsis:+Setting+the+stage.">Animal models of sepsis: Setting the stage.</a> Nature Reviews Drug Discovery. 4 (10), 854-865 (2005).</li><li>Balls, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+principles+of+humane+experimental+technique:+timeless+insights+and+unheeded+warnings.">The principles of humane experimental technique: timeless insights and unheeded warnings.</a> Altex-Alternatives to Animal Experimentation. 27 (2), 144-148 (2010).</li></ol>

这里描述的方案允许高度可重复，但简单易学的败血症模型，可以根据研究问题进行调整。可以连续收集涉及器官功能的基本体内数据，例如心率，血压和外周动脉血氧饱和度，并且可以在整个实验过程中重复进行血液采样。此外，可以安装有关液体置换方案和血管加压药支持的修改。鉴于动物的血流动力学稳定性，实验可以在几个小时内进行，如先前报道的8。
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脓毒症及其更严重的形式，脓毒性休克，是感染基础上的综合征，导致细胞因子释放的过度炎症反应，导致生理和生化变化，免疫防御受到抑制，致命结果1，2。这种不平衡的炎症反应导致器官功能障碍和各种重要器官（如肺，肾和肝）的器官衰竭。脓毒症为 37%3，是患者入住重症监护病房 （ICU） 的最常见原因之一。脓毒症的死亡率目前约为20-30%4。早期和有效的抗生素治疗至关重要5.液体和血管加压药需要及早安装复苏，除此之外，治疗是纯粹的支持性6.
脓毒症被定义为已证实或疑似细菌、真菌、病毒或寄生虫感染，伴有器官功能障碍。当进一步的心血管衰竭对单独液体治疗无反应，并且存在超过 2 毫摩尔/升的乳酸水平时，符合脓毒性休克标准2。脓毒症相关器官衰竭可能发生在任何器官中，但在心血管系统、大脑、肾脏、肝脏和肺部非常常见。大多数脓毒症患者需要气管插管，以保护患者的气道，防止误吸，并使用高比例的吸入氧气进行呼气末正向通气，以防止或克服缺氧。为了耐受气管插管和机械通气，患者通常需要镇静。
内毒素，如脂多糖（LPS）作为革兰氏阴性菌膜的组分，通过toll样受体（TLR）诱导强烈的炎症反应47。激活定义的途径可确保稳定的炎症反应。细胞因子诱导的中性粒细胞化学牵伸缩蛋白1（CINC-1），单核细胞化学吸引蛋白1（MCP-1）和白细胞介素6（IL-6）等细胞因子在该模型8中被称为严重程度和结局的预后因素。静脉注射LPS应用已成功用于研究大鼠脓毒症的各个方面8，9。
脓毒症的治疗仍然是一个挑战，特别是由于缺乏预测性动物模型。如果内毒素血症伴全身炎症的激活是开发药物治疗的充分模型是值得商榷的。然而，通过众所周知的LPS诱导TLR 4通路可以获得重要的知识。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-0 silk sutures</td>
			<td>Ethicon, Sommerville, NJ</td>
			<td>K833</td>
			<td>Standard surgical</td>
		</tr>
		<tr>
			<td>26 intravenous catheter</td>
			<td>Becton Dickinson, Franklin Lakes, NJ</td>
			<td>391349</td>
			<td>Standard anesthesia equipment</td>
		</tr>
		<tr>
			<td>6-0 LOOK black braided silk</td>
			<td>Surgical Specalities Corporation, Wyomissing, PA</td>
			<td>SP114</td>
			<td>Standard surgical</td>
		</tr>
		<tr>
			<td>Alaris Syringe Pump</td>
			<td>Bencton Dickinson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Mundipharma, Basel, Switzerland</td>
			<td>7.68034E+12</td>
			<td>GTIN-number</td>
		</tr>
		<tr>
			<td>Curved fine tips microforceps</td>
			<td>World precision instruments (WPI), Sarasota, FL</td>
			<td>504513</td>
			<td>Facilitates vascular preparation</td>
		</tr>
		<tr>
			<td>Fine tips microforceps</td>
			<td>World precision instruments (WPI), Sarasota, FL</td>
			<td>501976</td>
			<td>Tips need to be polished regularly</td>
		</tr>
		<tr>
			<td>Infinity Delta XL Anesthesia monitoring</td>
			<td>Draeger, L&#252;beck, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane, 250 mL bottles</td>
			<td>Attane, Piramal, Mumbai, India</td>
			<td>LDNI 22098</td>
			<td>Standard vet. equipment</td>
		</tr>
		<tr>
			<td>Ketamine (Ketalar)</td>
			<td>Pfitzer, New York, NY</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharide (LPS) from Escherichia coli, serotype 055:B5</td>
			<td>Sigma, Buchs, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Q-tips small</td>
			<td>Carl Roth GmbH, Karlsruhe, Germany</td>
			<td>EH11.1</td>
			<td>Standard surgical</td>
		</tr>
		<tr>
			<td>Ringerfundin</td>
			<td>Bbraun, Melsungen, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tec-3 Isofluorane Vaporizer</td>
			<td>Ohmeda, GE-Healthcare, Chicago, IL</td>
			<td>not available anymore</td>
			<td>Standard vet. equipment</td>
		</tr>
		<tr>
			<td>Xylazine (Xylazin Streuli)</td>
			<td>Streuli AG, Uznach, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a reproducible intensive care unit-oriented endotoxin model in rats

脓毒症和脓毒性休克仍然是重症监护病房的主要死因。尽管脓毒症管理有了显著改善，但死亡率仍在 20% 至 30% 之间。迫切需要新的治疗方法来减少脓毒症相关的多器官衰竭和死亡。强大的动物模型允许一种或多种治疗方法，以及测试它们对生理和分子参数的影响。在本文中，介绍了一个简单的动物模型。
首先，全身麻醉是通过使用挥发性或腹膜内麻醉在动物中诱导的。放置静脉导管（尾静脉）、气管切开术和插入动脉内导管（尾动脉）后，开始机械通气。记录平均动脉血压、动脉血氧饱和度和心率的基线值。
注射溶解在磷酸盐缓冲盐水中的脂多糖（1毫克/千克体重）通过toll样受体4诱导强烈且可重复的炎症反应。液体校正以及去甲肾上腺素的应用是根据完善的协议进行的。
本文中介绍的动物模型易于学习，并且强烈面向重症监护病房的临床脓毒症治疗，包括镇静，机械通气，连续血压监测和重复血液采样。此外，该模型是可靠的，允许根据动物研究的3R（减少，替换，精炼）原则，有限数量的动物提供可重复的数据。虽然脓毒症研究中的动物实验不能轻易被取代，但重复测量可以减少动物，并且保持脓毒性动物麻醉可以减轻痛苦。

所提出的系统允许血流动力学稳定动物的内毒素血症，如先前报道的9。虽然在有LPS刺激和没有LPS刺激的动物中，平均动脉压保持稳定，LPS处理的动物会出现脓毒症的特征，例如阴性碱过量和通过血浆细胞因子（应用后6小时）测量的强烈炎症反应，例如CINC-1（867 ng / mL），MCP-1（5027 ng / mL）和IL-6（867 ng / mL）8， 图 5.
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Watch this Scientific Journal Video about A Reproducible Intensive Care Unit-Oriented Endotoxin Model in Rats at JoVE.com

A Reproducible Intensive Care Unit-Oriented Endotoxin Model in Rats

在这里，我们提出了一种可重复的重症监护病房导向的大鼠内毒素模型。

一种可重复的重症监护病房导向内毒素模型在大鼠中的应用

Sepsis and septic shock remain the leading cause of death in intensive care units. Despite significant improvements in sepsis management, mortality still ...

该方案是内毒素血症的稳健且可重复的模型，其中可以重复评估生理和分子参数。这种方法可能有助于回答脓毒症研究中的问题。虽然我们的模型不能取代动物实验，但它通过连续镇静来完善败血症模型，并通过重复采样减少动物数量。初学者可能难以插入动脉和静脉导管，但只需有限的培训即可掌握这些技术。整个实验设置的可视化有助于研究人员设置或调整他们的模型，以更紧密地模拟ICU上的情况。首先将麻醉动物转移到加热垫上。将体温保持在36.5至37摄氏度之间。确保动物通过鼻锥提供氧气来自主呼吸。在安装气管切开术和动脉静脉导管之前，通过没有脚趾夹反射来确认麻醉水平。使用软膏来保护眼睛。准备无菌手术器械和导管，以及压力和氧饱和度监测，以获得足够的氧合。在大鼠的近端尾巴上涂抹止血带以方便静脉通路，并用酒精对尾巴进行三次消毒。将 26 号静脉导管引入两条侧尾静脉之一，避免空气喷射。放置静脉导管后解开止血带，并用胶带将静脉导管固定到位。将带三通旋塞阀的注射泵连接到静脉通路，以实现连续的液体和药物应用。通过剃除动物的前颈部区域开始气管切开术，然后用聚维酮碘溶液对剃光的皮肤进行三次消毒。使用10号手术刀刀片进行两厘米的纵向切口。用2-0条丝线缩回皮肤。然后钝地准备喉部和气管用手术剪刀打开。将无菌气管套管插入气管，小心不要穿透太深，以避免单侧通气。使用 2-O 丝缝线将套管固定到位，然后将套管连接到呼吸机以进行压力或容量控制通气。在设置实验之前，定义补液方案、血管收缩剂应用方案和流产标准。用聚维酮碘溶液对大鼠尾部进行消毒三次，然后用手术刀在腹侧纵向切开皮肤圈一厘米，注意不要切得太深，以免尾动脉受伤。使用手术显微镜仔细暴露动脉。用手术微剪刀切开动脉周围的筋膜。然后通过进行近端6-0丝缝合来连接动脉的远端部分，但不要收紧丝。将26号导管引入远端和近端丝缝之间的动脉，然后收紧近端丝缝线以将导管固定到位。将导管连接到压力传感器，以提供连续的动脉压测量。此外，在连接到压力传感器的导管和26号导管之间放置一个三通旋塞阀，以进行动脉血液采样。动物达到稳定状态后，通过注射LPS诱导脓毒症并采集血液样本。用Ringer的溶液以一到四的比例补充血液样品中的液体损失，始终避免空气注射，以防止空气栓塞。在有或没有LPS刺激的动物中，平均动脉压保持稳定。LPS处理的动物出现脓毒症的特征，例如负碱过量。LPS处理的动物也发展出强烈的炎症反应，其通过血浆细胞因子测量，例如化学牵拉肽蛋白-1，单核细胞化学吸引蛋白-1和白细胞介素-6。当尝试此方案时，请记住预定义液体和血管加压药方案以及定义终止标准，以获得可靠且可重复的研究数据。该方案可适用于其他脓毒症模型。因此，研究问题以最合适的方法回答，并遵循减少，完善，取代动物福利的原则。

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闭胸鼠心肌缺血再灌注模型

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

Lavage-induced Surfactant Depletion in Pigs As a Model of the Acute Respiratory Distress Syndrome (ARDS)

Lavage-induced Surfactant Depletion in Pigs As a Model of the Acute Respiratory Distress Syndrome ARDS

灌洗诱发的表面活性剂耗尽猪一样的急性呼吸窘迫综合征模型（ARDS）

Various animal models of lung injury exist to study the complex pathomechanisms of human acute respiratory distress syndrome (ARDS) and evaluate future ...

A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats

心脏重构通过大鼠腹主动脉缩窄的模型

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and ...

A Model to Simulate Clinically Relevant Hypoxia in Humans

一个模型来模拟人类临床相关性缺氧

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to ...

Use of a Central Venous Line for Fluids, Drugs and Nutrient Administration in a Mouse Model of Critical Illness

在重大疾病的小鼠模型中使用中心静脉导管的流体，药物和营养管理

This protocol describes a centrally catheterized mouse model of prolonged critical illness. We combine the cecal ligation and puncture method to induce ...

Observational Study Protocol for Repeated Clinical Examination and Critical Care Ultrasonography Within the Simple Intensive Care Studies

简单重症监护室内反复临床检查和重症监护室超声观察性研究方案

Longitudinal evaluations of critically ill patients by combinations of clinical examination, biochemical analysis and critical care ultrasonography (CCUS) ...

A Protocol to Set Up Needle-Free Connector with Positive Displacement on Central Venous Catheter in Intensive Care Unit

在重症监护病房的中央静脉导管上设置无针接头,带正位移

Needle-free connectors were initially designed and promoted to avoid blood exposure for healthcare workers. Some recent data suggest that the latest ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

通过直接内切内脂多糖灌输诱导小鼠急性肺损伤

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

Halogenated Agent Delivery in Porcine Model of Acute Respiratory Distress Syndrome via an Intensive Care Unit Type Device

重症监护病房型装置在猪模型急性呼吸窘迫综合征中的卤化剂递送

Acute respiratory distress syndrome (ARDS) is a common cause of hypoxemic respiratory failure and death in critically ill patients, and there is an urgent ...