Name: lipopolysaccharide infusion as a porcine endotoxemic shock model
Uploaded: 2023-12-08T13:40:00.000+00:00
Duration: 5 min 52 s
Description: Watch this Scientific Journal Video about Lipopolysaccharide Infusion as a Porcine Endotoxemic Shock Model at JoVE.com

作者要感谢 Dagmar Dirvonskis 的出色技术支持。

该协议中的实验已获得国家和机构动物护理委员会（Landesuntersuchungsamt Rheinland-Pfalz，Koblenz，Germany，TVA G21-1-080）的批准。实验是根据ARRIVE指南进行的。在这项研究中，使用了六头年龄在2-3个月大、体重30-35公斤的健康雄性德国长白猪。实验时间线总结在 图1中。与本协议中使用的所有材料和仪器相关的详细信息列 在材料表中。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66039/66039fig01.jpg"> 图 1:实验时间表。 在动物制备和30分钟的稳定期后进行基线健康测量。LPS注射诱导内毒素血症超过30分钟，再过30分钟进行0小时测量;之后，每小时继续测量 4 小时。缩写:BLH = 基线健康;LPS = 脂多糖。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66039/66039fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
1.动物准备
<ol><li>尽可能长时间地将动物留在通常的环境中，以尽量减少压力。在麻醉前停止进食 6 小时，同时允许自由饮水。</li>
	<li>在正常环境中，用肌内注射氮杂哌酮（3mg ∙kg-1）和咪达唑仑（0.5mgkg-1）镇静动物。</li>
	<li>一旦镇静剂生效，通常在给药后约15-20分钟内发生，将猪运送到实验室。 注意:确保在整个转移过程中保持持续镇静至关重要;根据地区立法机构的不同，这可能需要兽医的永久监督。</li>
	<li>在运输过程中密切注意保持猪的正常体温（~38°C）。例如，考虑用毯子盖住动物以防止体温过低。 注意:重要的是要限制运输时间不超过镇静的持续时间，通常从30分钟到60分钟不等。</li>
	<li>消毒后，通过在耳静脉中插入 22 G 导管来建立静脉通路。在对猪进行任何进一步的移动或麻醉诱导之前，请确保导管牢固固定，以防止任何突然移动导致脱位。</li>
	<li>使用夹在尾巴或耳朵上的传感器连续监测外周血氧饱和度。</li>
</ol>2.麻醉和机械通气
<ol><li>静脉注射芬太尼（4μgkg-1）和丙泊酚（3mgkg-1）以诱导麻醉。</li>
	<li>将猪置于仰卧位。</li>
	<li>施用阿曲库铵（0.5mg kg-1）作为肌肉松弛剂，并立即使用狗通气面罩开始无创通气。将面罩放在鼻子上，用拇指用力按压，同时用中指/无名指向前拉下颌。将呼吸机设置为以下参数: 吸气氧分数 （FiO2 ） = 100%，潮气量 = 6-8 mL kg-1，呼气末正压 （PEEP） = 5 mbar，峰值吸气压 ≤ 20 mbar，呼吸频率 = 18-20 min-1。</li>
	<li>通过开始连续输注平衡电解质溶液（5 mL kg-1 h-1）、芬太尼（10 μg kg-1 h-1）和丙泊酚（6 mg kg-1 h-1）来维持麻醉。</li>
	<li>使用标准气管插管（内径 6-7 mm）、导丝和配备 Macintosh 刀片（4 号）的喉镜进行气管插管。<ol><li>让助手张开嘴巴，将舌头放在左侧。</li>
		<li>插入 Macintosh 刀片，直到看到会厌。然后，向上抬起喉镜，使会厌向腹侧移动并观察声带。有时，会厌可能会粘在软腭上;在这种情况下，通过用管子或布吉轻轻向侧面滑动来动员它。</li>
		<li>小心地将气管插管插入声带并取出诱导器。如果遇到困难，请尝试在不施加过大力的情况下旋转管子。如有必要，请使用较小的管子。管子就位后，用 10 mL 空气给袖带充气。</li>
	</ol></li>
	<li>将气管插管连接到呼吸机并开始通气。通过检测呼气末 CO2 并进行双侧听诊来确认管子的正确定位。使用以下通气设置: FiO2 = 40%，潮气量 = 6-8 mL kg-1，PEEP = 5 mbar，吸气呼气比 = 1:2，呼吸频率 = 调整以达到呼气末 CO2 水平 <45 mmHg，通常为 30-40 min-1 。 注意: 如果管子错误地放置在食道中，空气会使胃充气，导致明显的隆起。在这种情况下，请立即取出管子，进行无创通气1-2分钟，并正确重新定位管子。</li>
	<li>插入胃管以防止反流或呕吐。如果插入被证明具有挑战性，请使用喉镜更好地观察食管入口。</li>
</ol>3. 仪器仪表
<ol><li>在股动脉和静脉中分别放置动脉和中心静脉管路，用于血流动力学监测和静脉容量治疗。</li>
	<li>使用绷带缩回并固定后腿，以便更好地进入股血管。</li>
	<li>在仪器之前准备所有必要的材料。用生理盐水填充所有导管，并确保易于接触电线和导管，以尽量减少多次导管插入尝试和不必要的失血。</li>
	<li>将酒精消毒剂涂抹在腹股沟区域，然后用无菌拭子擦拭。重复此过程两次。再次涂抹消毒剂，无需擦拭，等待 3 分钟。在腹股沟区域放置无菌开窗窗帘。</li>
	<li>使用超声波识别股血管。使用平面超声引导的 Seldinger 技术进行导管插入术，以最大限度地减少组织损伤和失血。</li>
	<li>纵向观察股动脉。用连接到针头的注射器刺穿动脉以进行连续抽吸。鲜红色的搏动血液证实动脉穿刺。取出注射器并插入准备好的电线。取下针头，同时将电线留在原位。</li>
	<li>对股静脉重复相同的过程。静脉穿刺通过缓慢流动的暗红色血液确诊。</li>
	<li>通过使用超声波观察两条股血管来确认两条导线的正确位置。</li>
	<li>使用 Seldinger 技术先插入动脉引诱导器鞘，然后插入静脉引引子鞘。通过对从两条线抽取的血样进行血气分析来确认正确定位。</li>
	<li>确保可以从所有管路抽出血液。用生理盐水冲洗所有管路，以防止血栓形成。</li>
	<li>使用手术缝合线将线条牢固地固定在皮肤上，以防止脱位。</li>
	<li>将动脉和中心静脉管路连接到换能器，以测量血流动力学参数。</li>
	<li>将脉搏轮廓心输出量 （PiCCO） 导管插入动脉导引器鞘，并将其连接到动脉压力传感器和 PiCCO 监护仪的温度接口电缆。</li>
	<li>将 Swan-Ganz 导管连接到换能器。<ol><li>在连续测量压力的同时，将导管插入中心静脉导引器鞘中。在大约 30 cm 后，当中心静脉压力曲线变得可见时，将球囊充气。</li>
		<li>在监测压力曲线的同时缓慢推进导管。当导管进入右心室时，寻找收缩压高、舒张压低的脉搏曲线。导管的进一步推进将导致一致的收缩压值和舒张压值的增加，表明位于肺动脉中。</li>
		<li>将导管固定在此位置（通常在 50 到 70 厘米之间）。将 PiCCO 系统的注射液温度传感器连接到 Swan-Ganz 导管的近端管腔。</li>
	</ol></li>
	<li>剃掉猪的额头，并用粘性传感器电极测量脑区域血氧饱和度。</li>
	<li>麻醉诱导和仪器仪表后，让动物稳定30分钟或直到血流动力学参数稳定，然后进行基线测量并诱导内毒素休克。</li>
</ol>4. 电击感应
注意: 使用 LPS 时，请始终戴上手套、护目镜、口罩和实验室外套。避免直接接触LPS。
<ol><li>通过将 5 mg LPS 溶解在 50 mL 0.9% NaCl 中，制备浓度为 100 μg mL-1 的 LPS 溶液。</li>
	<li>在开始 LPS 输注之前立即获得基线血流动力学测量值。</li>
	<li>在30分钟内施用150μkg-1剂量的LPS（相当于300μkg-1∙h-1连续输注速率30分钟）。</li>
	<li>30分钟后，在实验的剩余时间内将输注速率降低至15μg∙kg-1∙h-1 。</li>
	<li>持续监测血流动力学参数，包括动脉和肺动脉血压、心率和通气参数。持续监测体温以维持正常体温。</li>
</ol>5.血流动力学不稳定的治疗
<ol><li>当平均动脉血压低于 60 mmHg 时，使用 PiCCO 测量心脏指数 （CI）、整体舒张末期容积指数 （GEDI） 和血管外肺水指数 （ELWI）。根据 图2流程图中的建议治疗低血压。<ol><li>在 PiCCO 显示器上，按热稀释按钮 （TD）。</li>
		<li>按下 中心静脉压 （CVP） 输入 按钮并输入当前 CVP 值。</li>
		<li>按 "开始 "按钮。</li>
		<li>当指示这样做时，将 10 mL 冷盐水溶液 注入连接到 Swan-Ganz 导管的注射液温度传感器中。 注意: 不要在 PiCCO 测量之前或期间直接注入任何其他东西，因为这会影响测量。</li>
	</ol></li>
	<li>获得 CI、GEDI 和 ELWI 的测量值后，根据图 2 中的流程图治疗血流动力学不稳定。如果建议容量加载，请快速输注 200 mL 平衡电解质溶液。如果推荐儿茶酚胺治疗，将去甲肾上腺素输注速率增加1μgkg-1h-1。</li>
	<li>每当平均动脉血压低于 60 mmHg 时，重复此过程。在严重血流动力学不稳定的情况下，选择快速升级治疗。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66039/66039fig02.jpg"> 图 2:PiCCO 引导的血流动力学不稳定治疗。 获得 CI、GEDI 和 ELWI 的测量值后，根据图表进行治疗。此图改编自 PiCCO 用户指南12。缩写:PiCCO = 脉搏轮廓心输出量;V+ = 体积加载;猫=儿茶酚胺疗法;V- = 体积减少;CI = 心脏指数;GEDI = 全球舒张末期容积指数;ELWI = 血管外肺水指数。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66039/66039fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
6. 实验结束和安乐死
<ol><li>静脉注射 0.5 mg 芬太尼。等待 5 分钟。注射200mg异丙酚。</li>
	<li>通过中心静脉管路快速注射 40 mL 1 M 氯化钾对猪实施安乐死。</li>
</ol>

通过 LPS 输注诱导猪内毒素血症的剂量依赖性

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近红外光谱设备由美国美敦力公司无条件提供，用于实验研究目的。亚历山大·齐巴特（Alexander Ziebart）获得了美敦力公司（Medtronic PLC）的演讲费。没有作者报告任何财务或其他利益冲突。稿件由ChatGPT®（Python软件，版本:2023年5月24日）校对和编辑。

我们提出了一种通过LPS输注诱导猪实验性内毒素血症的方案，旨在可靠地诱导脓毒症和感染性休克中常见的变化。该协议中需要考虑几个关键步骤。在运输前对猪进行充分的镇静对于防止应激引起的儿茶酚胺水平升高至关重要，因为儿茶酚胺水平可能会影响结果。与人类相比，猪的插管可能会带来挑战，因为它们的鼻子细长的解剖学特征。为了解决这个问题，我们建议使用 Macintosh 刀片进行插管，并且气管插管应配备直引导器。会厌粘附在软腭上是很常见的，有时，由于动物气管的声门下狭窄，可能需要较小的气管插管，因此能够穿过声带的管子可能仍然太大。
在输注LPS之前，精确制备LPS浓度是必不可少的。给予较高剂量的LPS可导致严重的血流动力学不稳定甚至死亡，而较低剂量的LPS可能无法产生预期的效果。此外，应该注意的是，不同的LPS电荷可能表现出不同程度的功效。我们建议对每次试用使用相同的 LPS 费用。可以进行剂量探索试验以确定每项研究的适当剂量。在开始输注 LPS 时，由于可能出现快速不稳定，因此持续监测血流动力学参数至关重要。可能需要及时干预以控制任何不良反应。
PiCCO用于高级血流动力学测量。该技术也经常用于在ICU接受治疗的人类患者。它是为人类开发的，在猪身上的使用可能会带来一些挑战。体表面积 （BSA） 用于计算血流动力学参数。这是在输入患者的身高和体重时自动计算的。虽然这里使用的公式（对于人类）对于计算猪的BSA并不理想，但不幸的是，没有其他方法可以进入BSA。根据我们的经验，通过为猪输入 130 厘米的高度解决了这个问题，这为 BSA 产生了最充分的结果。但是，在解释 PiCCO 结果时应牢记此限制。
以前的研究已经描述了使用LPS来模拟猪的感染性休克。在这些研究中，描述了在脓毒症患者中经常观察到的变化，例如低血压、外周血管舒张、肺动脉压升高和全身摄氧量增加 13,14,15。还描述了诱导猪实验性脓毒症和脓毒性休克的替代方法。一种模型涉及通过腹腔内施用粪便诱发腹膜炎16、17、18、19。另一种方法是将活细菌直接注射到动物的血液中19,20。与LPS注射相比，利用腹膜炎或菌血症诱导实验性败血症的方案具有更逼真的优势。这些方法通过细菌感染诱发实际的脓毒症，而LPS注射仅代表潜在致病机制的一个方面。
然而，LPS输注方法也有其优点。与腹膜炎模型相比，该方案需要更少的努力和专业知识，因为它仅涉及静脉注射，无需腹膜内通路。此外，休克症状比其他模型更快地表现出来，从而缩短了观察时间并降低了资源利用率。此外，由于每头猪接受相同的LPS剂量，因此结果具有高度的可重复性。相反，施用粪便的组成可以显着变化，并且细菌生长受到不可控因素的影响19。
尽管存在某些局限性，但该方案始终诱发内毒素休克，影响多个器官系统。我们观察到肺功能和血流动力学的特征性改变，以及所有LPS治疗动物的乳酸水平升高。由于整个实验过程中持续的深度麻醉，我们无法评估认知功能，认知功能使用人类格拉斯哥昏迷量表纳入 SOFA 评分。然而，我们确实观察到脑氧合减少，这表明LPS诱导的休克对大脑功能有潜在影响。在早期阶段，脓毒症通常与以心输出量升高为特征的高动力阶段有关。由于该模型中症状的快速进展，此处提供的数据并未充分显示这种超动态阶段。如果这个阶段特别感兴趣，则应在实验的早期阶段更定期地进行测量。调整LPS剂量可能有助于减缓症状的发展，并使其更容易观察到高动力阶段。
LPS 和其他细菌内毒素以前已被用于模拟小动物模型中的脓毒症8。然而，与小鼠等小动物模型相比，在这种情况下使用猪会带来一定的挑战。猪的繁殖和维护需要更多的时间和精力，每次实验可以使用的动物更少。然而，大型动物模型，尤其是猪，提供了更真实的人体表现。猪在解剖学、基因组、饮食和免疫系统反应性方面表现出与人类的相似性 9,10。另一个优点是可以重复进行血液样本分析。虽然小动物模型通常需要专门的设备，但人类患者常用的标准医疗设备可以应用于猪，从而类似于临床ICU环境中的仪器和血流动力学监测。总之，该方案通过LPS输注在猪中建立了实验性内毒素血症模型。它提供了一种简单且标准化的方法，可以持续诱导脓毒性休克患者中经常观察到的变化。

脓毒症和脓毒性休克是接受重症监护治疗的患者死亡的主要原因之一1,2,3。当感染引发免疫反应失调导致多器官衰竭时，就会出现脓毒症。它的特征是危及生命的症状，包括血流动力学不稳定、呼吸窘迫、肝肾衰竭以及认知障碍 4,5。脓毒性休克是脓毒症的一个亚型集，症状特别严重，可显著增加死亡率。这些症状包括需要血管加压药治疗的持续性低血压和血清乳酸水平超过 2 mmol∙L-1 4,5。脓毒性休克患者的死亡率估计高达 40%，即使住院治疗也是如此 1,3,5。 
革兰氏阴性菌，如假单胞菌和大肠杆菌，经常引起感染，引发这种失调的免疫反应4。潜在的病理生理机制很复杂，尚未完全了解。一个充分描述的方面涉及病原体相关分子模式 （PAMP） 激活免疫细胞上的 Toll 样受体，导致细胞因子的释放，例如肿瘤坏死因子-α （TNFα） 或白细胞介素 1 （IL 1）4。其中一种 PAMP 是脂多糖 （LPS），它构成革兰氏阴性菌细胞膜的组成部分6。LPS 已在动物模型中用于诱导内毒素血症和内毒素休克 7,8。
动物模型为开发和研究新的治疗策略提供了一个受控和标准化的环境。由于其相似的解剖结构、免疫生理学和相似的血流动力学参数，猪模型特别适合研究内毒素休克的影响 9,10。此外，由于猪的气道和血管大小相似，通常用于人类患者的标准医疗设备可以很容易地应用于猪，从而便于仪器仪表和血流动力学监测。
通过该协议，我们通过静脉输注来自 大肠杆菌的LPS为猪的内毒素休克提供了实验模型。为了监测效果，我们测量了血流动力学和肺参数，包括动脉血压、心率、外周血氧饱和度、肺动脉压和气道压力。为了评估内毒素血症对脑氧供应的影响，我们使用了近红外光谱法（NIRS）。使用这种方法，可以通过施加在前额11上的粘性电极来评估脑血氧饱和度。

<table><tbody><tr><td>Atracurium Hikma 50 mg/5mL</td><td>Hikma Pharma GmbH, Martinsried</td><td></td><td></td></tr><tr><td>Azaperone (Stresnil) 40 mg/mL</td><td>Lilly Deutschland GmbH, Bad Homburg, Germany</td><td></td><td></td></tr><tr><td>BD Discardit II Spritze 2, 5, 10, 20 mL</td><td>Becton Dickinson S.A. Carretera, Mequinenza Fraga, Spain</td><td>syringe</td><td></td></tr><tr><td>BD Luer Connecta&amp;nbsp;</td><td>Becton Dickinson Infusion Therapy, AB Helsingborg, Schweden</td><td>3-way-stopcock</td><td></td></tr><tr><td>Curafix i.v. classics</td><td>Lohmann &amp;amp; Rauscher International GmbH &amp;amp; Co. KG, Rengsdorf, Germany</td><td>Cannula retention dressing</td><td></td></tr><tr><td>Datex Ohmeda S5</td><td>GE Healthcare Finland Oy, Helsinki, Finland</td><td>hemodynamic monitor</td><td></td></tr><tr><td>Engstr&amp;ouml;m Carestation</td><td>GE Heathcare, Madison USA</td><td>ventilator</td><td></td></tr><tr><td>Fentanyl-Janssen 0.05 mg/mL</td><td>Janssen-Cilag GmbH, Neuss</td><td>fentanyl</td><td></td></tr><tr><td>F&amp;uuml;hrungsstab, Durchmesser 4.3</td><td>R&amp;uuml;sch</td><td>endotracheal tube introducer</td><td></td></tr><tr><td>Incetomat-line 150 cm</td><td>Fresenius, Kabi Deutschland, GmbH</td><td>perfusor line</td><td></td></tr><tr><td>Intrafix Primeline</td><td>B. Braun Melsungen AG, Melsungen, Germany</td><td>Infusion line</td><td></td></tr><tr><td>Introducer sheath 5 Fr.</td><td>Terumo Healthcare</td><td>arterial introducer&amp;nbsp;</td><td></td></tr><tr><td>INVOS</td><td>Medtronic, Dublin, Ireland</td><td>near infrared spectrometry</td><td></td></tr><tr><td>JOZA Einmal Nitril Untersuchungshandschuhe&amp;nbsp;</td><td>JOZA, M&amp;uuml;nchen, Germany</td><td>disposable gloves</td><td></td></tr><tr><td>Laryngoscope, 45.48.50, KL 2000</td><td>Medicon</td><td>Laryngoscope handle</td><td></td></tr><tr><td>Littmann Classic III Stethoscope</td><td>3M Deutschland GmbH, Neuss, Germany</td><td>stethoscope</td><td></td></tr><tr><td>LPS (E. coli; Serotype O111:B4)</td><td>Sigma-Aldrich, Switzerland</td><td></td><td></td></tr><tr><td>MAC Two-Lumen Central venous access set</td><td>Arrow international inc. Reading, PA, USA</td><td>venous introducer</td><td></td></tr><tr><td>Maimed Vlieskompresse</td><td>Maimed GmbH, Neuenkirchen, Germany</td><td>Fleece compress to fix the tongue</td><td></td></tr><tr><td>Masimo LNCS Adtx SpO&lt;sub&gt;2 &lt;/sub&gt;sensor</td><td>Masimo Corporation Irvine, Ca 92618 USA</td><td>saturation clip for the tail</td><td></td></tr><tr><td>Masimo LNCS TC-I SpO2 ear clip sensor</td><td>Masimo Corporation Irvine, Ca 92618 USA</td><td>Saturation clip for the ear</td><td></td></tr><tr><td>Masimo Radical 7</td><td>Masimo Corporation Irvine, Ca 92618 USA</td><td>periphereal oxygen saturation&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Midazolam 15 mg/3 mL</td><td>B.Braun Melsungen AG, Germany</td><td></td><td></td></tr><tr><td>Midmark Canine Mask Small Plastic with Diaphragm FRSCM-0005</td><td>Midmark Corp., Dayton, Ohio, USA</td><td>dog ventilation mask</td><td></td></tr><tr><td>Monocryl surgical suture</td><td>Johnson &amp;amp; Johnson, Belgium</td><td></td><td></td></tr><tr><td></td><td>B.Braun Melsungen AG, Germany</td><td>saline solution</td><td></td></tr><tr><td>NaCl 0.9 %</td><td>Sanofi- Aventis, Seutschland GmbH</td><td></td><td></td></tr><tr><td>Octeniderm farblos</td><td>Sch&amp;uuml;lke &amp;amp; Mayr GmbH, Nordenstedt, Germany</td><td>Alcoholic disinfectant</td><td></td></tr><tr><td>Original Perfusor syringe 50 mL</td><td>B.Braun Melsungen AG, Germany</td><td>perfusor syringe</td><td></td></tr><tr><td>PA-Katheter Swan Ganz 7.5 Fr 110 cm</td><td>Edwards Lifesciences LLC, Irvine CA, USA</td><td>Swan-Ganz catheter</td><td></td></tr><tr><td>Perfusor FM Braun</td><td>B.Braun Melsungen AG, Germany</td><td>syringe pump</td><td></td></tr><tr><td>PiCCO catheter</td><td>PULSION Medical Systems SE, Feldkirchen, DE</td><td></td><td></td></tr><tr><td>Potassium chloride 1 M</td><td>Fresenius, Kabi Germany GmbH</td><td></td><td></td></tr><tr><td>Propofol 2% 20 mg/mL (50 mL flasks)</td><td>Fresenius, Kabi Deutschland, GmbH</td><td></td><td></td></tr><tr><td>Pulse-contour continous cardiac output System PiCCO2</td><td>PULSION Medical Systems SE, Feldkirchen, DE</td><td></td><td></td></tr><tr><td>R&amp;uuml;schelit Super Safety Clear &gt;ID 6/6.5 /7.0 mm</td><td>Teleflex Medical Sdn. Bhd, Malaysia</td><td>endotracheal tube</td><td></td></tr><tr><td>Sonosite Micromaxx Ultrasoundsystem</td><td>Sonosite Bothell, WA, USA&amp;nbsp;</td><td>ultrasound&amp;nbsp;</td><td></td></tr><tr><td>Stainless Macintosh Gr&amp;ouml;&amp;szlig;e 4</td><td>Welch Allyn69604</td><td>blade for laryngoscope</td><td></td></tr><tr><td>Sterofundin</td><td>B.Braun Melsungen AG, Melsungen, Germany</td><td>Balanced electrolyte solution</td><td></td></tr><tr><td>Vasco OP sensitive&amp;nbsp;</td><td>B.Braun Melsungen AG, Germany</td><td>sterile gloves</td><td></td></tr><tr><td>Vasofix Safety 22 G-16 G</td><td>B.Braun Melsungen AG, Germany</td><td>venous catheter</td><td></td></tr><tr><td>VBM Cuff Manometer</td><td>VBM Medizintechnik GmbH, Sulz a.N., Germany&amp;nbsp;</td><td>cuff pressure gauge</td><td></td></tr></tbody></table>

lipopolysaccharide infusion as a porcine endotoxemic shock model

脓毒症和脓毒性休克常见于重症监护病房 （ICU） 接受治疗的患者，是这些患者死亡的主要原因之一。它是由对感染的免疫反应失调引起的。即使进行了优化的治疗，死亡率仍然很高，这使得对病理生理学和新的治疗方案有了进一步的了解。脂多糖 （LPS） 是革兰氏阴性菌细胞膜的一种成分，通常是引起败血症和感染性休克的感染的原因。 
脓毒症和脓毒性休克的严重程度和高死亡率使得对人类进行标准化的实验研究变得不可能。因此，需要一个动物模型进行进一步的研究。猪特别适合这个目的，因为它在解剖学、生理学和体型上与人类非常相似。 
该方案为通过LPS输注对猪进行内毒素休克提供了实验模型。我们能够可靠地诱导脓毒性休克患者中经常观察到的变化，包括血流动力学不稳定、呼吸衰竭和酸中毒。这将使研究人员能够获得对这种高度相关疾病的宝贵见解，并在实验环境中评估新的治疗方法。

在这项研究中，对6头年龄为2-3个月、体重30-35公斤的健康公猪进行麻醉，并接受脂多糖（LPS）输注以诱导内毒素血症。为了确定持续诱发休克症状所需的适当LPS剂量，在30分钟内给予猪从100μgkg-1到200μgkg-1的各种诱导剂量的LPS，然后在实验的剩余时间里维持剂量为每小时初始剂量的1/10。所有动物在LPS输注后不久都表现出休克迹象。使用 PiCCO 系统监测血流动力学参数。动物表现出心脏指数下降和心率增加，表明休克状态下的血流动力学不稳定。LPS输注后平均动脉血压下降，但必要时通过液体复苏或去甲肾上腺素输注保持在60mmHg以上（图3）。肺损伤表现为PaO2 FiO 2-1比值降低和肺动脉压升高（图4）。使用近红外光谱 （NIRS） 测量脑氧合，并在诱导休克后降低（图 5）。这些动物还表现出酸中毒和乳酸水平升高（图6）。采用多重比较单因素方差分析确定显著性。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66039/66039fig03.jpg"> 图 3:LPS 输注后血流动力学参数的发展。 （A） 诱导休克后平均动脉血压下降，但必要时使用去甲肾上腺素输注保持在 60 mmHg 以上。（B）输注LPS后心脏指数降低，（C）心率增加。显示均值和标准差。*P < 0.05 与基线测量值相比。缩写:BLH = 基线健康;LPS = 脂多糖。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66039/66039fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66039/66039fig04.jpg"> 图 4:LPS 输注后肺部参数的发展。 （A） LPS 输注后不久 PaO2 FiO2-1 比值降低。（二）激波感应后驱动压力升高。（C）休克期间肺动脉压也升高。显示均值和标准差。*P < 0.05 与基线测量值相比。缩写:BLH = 基线健康;LPS = 脂多糖;FiO2 = 吸气氧分数;PaO2 = 动脉血氧分压。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66039/66039fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66039/66039fig05.jpg"> 图5:LPS输注后的脑氧合。 通过近红外光谱测量的脑氧合在LPS电击诱导后降低。显示均值和标准差。*P < 0.05 与基线测量值相比。缩写:BLH = 基线健康;LPS = 脂多糖;NIRS = 近红外光谱。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66039/66039fig05large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66039/66039fig06.jpg"> 图 6:LPS 诱导的内毒素血症期间的动脉血气分析。 （A）随着时间的推移，动物变得更加酸中毒，（B）LPS输注后乳酸水平增加。显示均值和标准差。*P < 0.05 与基线测量值相比。缩写:BLH = 基线健康;LPS = 脂多糖。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66039/66039fig06large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

Watch this Scientific Journal Video about Lipopolysaccharide Infusion as a Porcine Endotoxemic Shock Model at JoVE.com

作者聚焦:猪实验性内毒素休克的诱导，用于研究血流动力学和呼吸衰竭

我们通过输注脂多糖为猪的实验性内毒素休克模型提供了方案。

脂多糖输注作为猪内毒素血症休克模型

Sepsis and septic shock are frequently encountered in patients treated in intensive care units (ICUs) and are among the leading causes of death in these ...

通过该协议，我们在猪中诱导实验性内毒素休克。我们能够可靠地引起内毒素血症的症状，例如血流动力学不稳定和呼吸衰竭。脂多糖是一种有毒物质。在处理脂多糖时，必须始终戴上手套、护目镜、口罩和实验室外套。该方案提供了一种简单可靠的诱导内毒素血症的方法。由于它们的解剖学和生理学相似，猪特别适合这种研究，因为可以使用标准的医疗设备。使用该方案，我们将能够研究不同通气策略对内毒素休克期间呼吸衰竭和肺损伤的影响。

lipopolysaccharide-infusion-as-a-porcine-endotoxemic-shock-model

Nanyang Technological University

Research

JoVE Journal

Medicine

Lipopolysaccharide Infusion as a Porcine Endotoxemic Shock Model

845 次观看.  University Medical Center of the Johannes Gutenberg-University. 通过该协议，我们在猪中诱导实验性内毒素休克。我们能够可靠地引起内毒素血症的症状，例如血流动力学不稳定和呼吸衰竭。脂多糖是一种有毒物质。在处理脂多糖时，必须始终戴上手套、护目镜、口罩和实验室外套。该方案提供了一种简单可靠的诱导内毒素血症的方法。由于它们的解剖学和生理学相似，猪特别适合这种研究，因为可以使用标准的医疗设备。使...

视频: 作者聚焦:猪实验性内毒素休克的诱导，用于研究血流动力学和呼吸衰竭

Fixed Volume or Fixed Pressure: A Murine Model of Hemorrhagic Shock

It is common knowledge that severe blood loss and traumatic injury can lead to a cascade of detrimental signaling events often resulting in mortality. 1, 2, 3, 4, 5 These signaling events can also lead to sepsis and/or multiple organ dysfunction (MOD). 6, 7, 8, 9 It is critical then to investigate the causes of suppressed immune function and detrimental signaling cascades in order to develop more effective ways to help patients who suffer from traumatic injuries. 10 This fixed pressure Hemorrhagic Shock (HS) procedure, although technically challenging, is an excellent resource for investigation of these pathophysiologic conditions. 11, 12, 13 Advances in the assessment of biological systems, i.e. Systems Biology have enabled the scientific community to further understand complex physiologic networks and cellular communication patterns. 14 Hemorrhagic Shock has proven to be a vital tool for unveiling these cellular communication patterns as they relate to immune function. 15, 16, 17, 18 This procedure can be mastered! This procedure can also be used as either a fixed volume or fixed pressure approach. We adapted this technique in the murine model to enhance research in innate and adaptive immune function. 19, 20, 21 Due to their small size HS in mice presents unique challenges. However due to the many available mouse strains, this species represents an unparalleled resource for the study of the biologic responses. The HS model is an important model for studying cellular communication patterns and the responses of systems such as hormonal and inflammatory mediator systems, and danger signals, i.e. DAMP and PAMP upregulation as it elicits distinct responses that differ from other forms of shock. 22, 23, 24, 25 The development of transgenic murine strains and the induction of biologic agents to inhibit specific signaling have presented valuable opportunities to further elucidate our understanding of the up and down regulation of signal transduction after severe blood loss, i.e. HS and trauma 26, 27, 28, 29, 30. 
There are numerous resuscitation methods (R) in association with HS and trauma. 31, 32, 33, 34 A fixed volume resuscitation method of solely lactated ringer solution (LR), equal to three times the shed blood volume, is used in this model to study endogenous mechanisms such as remote organ injury and systemic inflammation. 35, 36, 38 This method of resuscitation is proven to be effective in evaluating the effects of HS and trauma 38, 39.

The Hemorrhagic Shock model has been a reliable and reproducible resource facilitating the identification and understanding of signaling cascades associated with inflammation and end-organ damage after trauma. This article provides a step-by-step description of surgical and mechanical aspects associated with the Hemorrhagic Shock experimental procedure in mice.

固定体积或固定压力：失血性休克的小鼠模型

It is common knowledge that severe blood loss and traumatic injury can lead to a cascade of detrimental signaling events often resulting in mortality. 1, ...

科研

医学

Oleic Acid-Injection in Pigs As a Model for Acute Respiratory Distress Syndrome

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit patients. Despite treatment advances over the last decades, ARDS patients still suffer mortality rates between 35 and 40%. There is still a need for further research to improve the outcome of patients suffering from ARDS. One problem is that no single animal model can mimic the complex pathomechanism of the acute respiratory distress syndrome, but several models exist to study different parts of it. Oleic acid injection (OAI)-induced lung injury is a well-established model for studying ventilation strategies, lung mechanics and ventilation/perfusion distribution in animals. OAI leads to severely impaired gas exchange, deterioration of lung mechanics and disruption of the alveolo-capillary barrier. The disadvantage of this model is the controversial mechanistic relevance of this model and the necessity for central venous access, which is challenging especially in smaller animal models. In summary, OAI-induced lung injury leads to reproducible results in small and large animals and hence represents a well-suited model for studying ARDS. Nevertheless, further research is necessary to find a model that mimics all parts of ARDS and lacks the problems associated with the different models existing today.

In this article, we present a protocol to induce acute lung injury in pigs by central-venous injection of oleic acid. This is an established animal model for studying the acute respiratory distress syndrome (ARDS).

油酸注射液在猪急性呼吸窘迫综合征模型中的研究

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit ...

Standardized Hemorrhagic Shock Induction Guided by Cerebral Oximetry and Extended Hemodynamic Monitoring in Pigs

Hemorrhagic shock ranks among the main reasons for severe injury-related death. The loss of circulatory volume and oxygen carriers can lead to an insufficient oxygen supply and irreversible organ failure. The brain exerts only limited compensation capacities and is particularly at high risk of severe hypoxic damage.This article demonstrates the reproducible induction of life-threatening hemorrhagic shock in a porcine model by means of calculated blood withdrawal. We titrate shock induction guided by near-infrared spectroscopy&#160;and extended hemodynamic monitoring to display systemic circulatory failure, as well as cerebral microcirculatory oxygen depletion. In comparison to similar models that primarily focus on predefined removal volumes for shock induction, this approach highlights a titration by means of the resulting failure of macro- and microcirculation.

Hemorrhagic shock is a severe complication in seriously injured patients, which leads to life-threatening oxygen undersupply. We present a standardized method to induce hemorrhagic shock via blood withdrawal in pigs that is guided by hemodynamics and microcirculatory cerebral oxygenation.

由脑氧测量和猪期扩展血液动力学监测指导的标准化出血性休克诱导

Hemorrhagic shock ranks among the main reasons for severe injury-related death. The loss of circulatory volume and oxygen carriers can lead to an ...

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

Acute respiratory distress syndrome (ARDS) is a common cause of hypoxemic respiratory failure and death in critically ill patients, and there is an urgent need to find effective therapies. Preclinical studies have shown that inhaled halogenated agents may have beneficial effects in animal models of ARDS. The development of new devices to administer halogenated agents using modern intensive care unit (ICU) ventilators has significantly simplified the dispensing of halogenated agents to ICU patients. Because previous experimental and clinical research suggested potential benefits of halogenated volatiles, such as sevoflurane or isoflurane, for lung alveolar epithelial injury and inflammation, two pathophysiologic landmarks of diffuse alveolar damage during ARDS, we designed an animal model to understand the mechanisms of the effects of halogenated agents on lung injury and repair. After general anesthesia, tracheal intubation, and the initiation of mechanical ventilation, ARDS was induced in piglets via the intratracheal instillation of hydrochloric acid. Then, the piglets were sedated with inhaled sevoflurane or isoflurane using an ICU-type device, and the animals were ventilated with lung-protective mechanical ventilation during a 4 h period. During the study period, blood and alveolar samples were collected to evaluate arterial oxygenation, the permeability of the alveolar-capillary membrane, alveolar fluid clearance, and lung inflammation. Mechanical ventilation parameters were also collected throughout the experiment. Although this model induced a marked decrease in arterial oxygenation with altered alveolar-capillary permeability, it is reproducible and is characterized by a rapid onset, good stability over time, and no fatal complications.
We have developed a piglet model of acid aspiration that reproduces most of the physiological, biological, and pathological features of clinical ARDS, and it will be helpful to further our understanding of the potential lung-protective effects of halogenated agents delivered through devices used for inhaled ICU sedation.

We describe a model of hydrochloric acid-induced acute respiratory distress syndrome (ARDS) in piglets receiving sedation with halogenated agents, isoflurane and sevoflurane, through a device used for inhaled intensive care sedation. This model can be used to investigate the biological mechanisms of halogenated agents on lung injury and repair.

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 ...

A Reproducible Intensive Care Unit-Oriented Endotoxin Model in Rats

Sepsis and septic shock remain the leading cause of death in intensive care units. Despite significant improvements in sepsis management, mortality still ranges between 20 and 30%. Novel treatment approaches in order to reduce sepsis-related multiorgan failure and death are urgently needed. Robust animal models allow for one or multiple treatment approaches as well as for testing their effect on physiological and molecular parameters. In this article, a simple animal model is presented.
First, general anesthesia is induced in animals either with the use of volatile or by intraperitoneal anesthesia. After placement of an intravenous catheter (tail vein), tracheostomy, and insertion of an intraarterial catheter (tail artery), mechanical ventilation is started. Baseline values of mean arterial blood pressure, arterial blood oxygen saturation, and heart rate are recorded.
The injection of lipopolysaccharides (1 milligram/kilogram body weight) dissolved in phosphate-buffered saline induces a strong and reproducible inflammatory response via the toll-like receptor 4. Fluid corrections as well as the application of norepinephrine are performed based on well-established protocols.
The animal model presented in this article is easy to learn and strongly oriented towards clinical sepsis treatment in an intensive care unit with sedation, mechanical ventilation, continuous blood pressure monitoring, and repetitive blood sampling. Also, the model is reliable, allowing for reproducible data with a limited number of animals in accordance with the 3R (reduce, replace, refine) principles of animal research. While animal experiments in sepsis research cannot easily replaced, repetitive measurements allow for a reduction of animals and keeping septic animals anesthetized diminishes suffering.

Here, we present a reproducible intensive care unit-oriented endotoxin model in rats.

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 ...

Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its incidence is still considerably high and therefore, influences patient outcome. Microcirculation plays a key role in tissue perfusion and oxygen supply and is often dissociated from macrohemodynamics. Thus, direct evaluation of spinal cord microcirculation is essential for the development of microcirculation-targeted therapies and the evaluation of existing approaches in regard to spinal cord microcirculation. However, most of the methods do not provide real-time assessment of spinal cord microcirculation. The aim of this study is to describe a standardized protocol for real-time spinal cord microcirculatory evaluation using laser-Doppler needle probes directly inserted in the spinal cord. We used a porcine model of ischemia/reperfusion to induce deterioration of the spinal cord microcirculation. In addition, a fluorescent microsphere injection technique was used. Initially, animals were anesthetized and mechanically ventilated. Thereafter, laser-Doppler needle probe insertion was performed, followed by the placement of cerebrospinal fluid drainage. A median sternotomy was performed for exposure of the descending aorta to perform aortic cross-clamping. Ischemia/reperfusion was induced by supra-celiac aortic cross-clamping for a total of 48 min, followed by reperfusion and hemodynamic stabilization. Laser-Doppler Flux was performed in parallel with macrohemodynamic evaluation. In addition, automated cerebrospinal fluid drainage was used to maintain a stable cerebrospinal pressure. After completion of the protocol, animals were sacrificed, and the spinal cord was harvested for histopathological and microsphere analysis. The protocol reveals the feasibility of spinal cord microperfusion measurements using laser-Doppler probes and shows a marked decrease during ischemia as well as recovery after reperfusion. Results showed comparable behavior to fluorescent microsphere evaluation. In conclusion, this new protocol might provide a useful large animal model for future studies using real-time spinal cord microperfusion assessment in ischemia/reperfusion conditions.

Spinal cord microcirculation plays a pivotal role in spinal cord injury. Most methods do not allow real-time assessment of spinal cord microcirculation, which is essential for the development of microcirculation-targeted therapies. Here, we propose a protocol using Laser-Doppler-Flow Needle probes in a large animal model of ischemia/reperfusion.

猪缺血/再灌注模型中脊髓微灌注的实时评估

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its ...

Description of a Swine Infant Model of Volume-Controlled Hemorrhagic Shock

Hemorrhagic shock is a leading cause of morbidity and mortality in pediatric patients. Interpretation of the clinical indicators validated in adults to guide resuscitation and comparison between different therapies is difficult in children due to the inherent heterogeneity of this population. As a result, compared to adults, appropriate management of pediatric hemorrhagic shock is still not well established. In addition, the scarcity of pediatric patients with hemorrhagic shock precludes the development of clinically relevant studies. For this reason, an experimental pediatric animal model is necessary to study the effects of hemorrhage in children as well as their response to different therapies. We present an infant animal model of volume-controlled hemorrhagic shock in anesthetized young pigs. Hemorrhage is induced by withdrawing a previously calculated blood volume, and the pig is subsequently monitored and resuscitated with different therapies. Here, we describe a precise and highly reproducible model of hemorrhagic shock in immature swine. The model yields hemodynamic data that characterizes compensatory mechanisms that are activated in response to severe hemorrhage.

This article aims to provide researchers with a detailed and accessible guide to set up an infant porcine model of hemorrhagic shock.

容量控制性失血性休克的猪婴儿模型的描述

Hemorrhagic shock is a leading cause of morbidity and mortality in pediatric patients. Interpretation of the clinical indicators validated in adults to ...

<meta charset="utf8"></head><body>研究猪模型中对急性容量超负荷的生理反应

Adult and Pediatric Porcine Model of Acute Volume Overload

This protocol describes an acute volume overload porcine model for adult Yorkshire pigs and piglets. Both swine ages undergo general anesthesia, endotracheal intubation, and mechanical ventilation. A central venous catheter and an arterial catheter are placed via surgical cutdown in the external jugular vein and carotid artery, respectively. A pulmonary artery catheter is placed through an introducer sheath of the central venous catheter. PlasmaLyte crystalloid solution is then administered at a rate of 100 mL/min in adult pigs and at 20 mL/kg boluses over 10 min in piglets. Hypervolemia is achieved either at 15% decrease in cardiac output or at 5 L in adult pigs and at 500 mL in piglets. Hemodynamic data, such as heart rate, respiratory rate, end-tidal carbon dioxide, fraction of oxygen-saturated hemoglobin, arterial blood pressure, central venous pressure, pulmonary artery pressure, pulmonary capillary wedge pressure, partial arterial oxygen pressure, lactate, pH, base excess, and pulmonary artery fraction of oxygen-saturated hemoglobin, are monitored during experimentation. Preliminary data observed with this model has demonstrated statistically significant changes and strong linear regressions between central hemodynamic parameters and acute volume overload in adult pigs. Only pulmonary capillary wedge pressure demonstrated both a linear regression and a statistical significance to acute volume overload in piglets. These models can aid scientists in the discovery of age-appropriate therapeutic and monitoring strategies to understand and prevent acute volume overload.

<meta charset="utf8"></head><body>作者聚焦:探索猪模型中的静脉波形以解决医学中的容量超负荷

The protocol here shows how continuous administration of crystalloids into the central veins of a euvolemic pig/piglet allows for the appropriate investigation of the physiological effects of acute volume overload.

脂多糖输注作为猪内毒素血症休克模型

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此视频中的章节

固定体积或固定压力：失血性休克的小鼠模型

油酸注射液在猪急性呼吸窘迫综合征模型中的研究

由脑氧测量和猪期扩展血液动力学监测指导的标准化出血性休克诱导

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

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

猪缺血/再灌注模型中脊髓微灌注的实时评估

容量控制性失血性休克的猪婴儿模型的描述

成人和儿童猪急性容量超负荷模型

成人和儿童猪急性容量超负荷模型

成人和儿童猪急性容量超负荷模型