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/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/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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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 &#38; Rauscher International GmbH &#38; 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&#246;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&#252;hrungsstab, Durchmesser 4.3</td>
			<td>R&#252;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&#160;</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&#160;</td>
			<td>JOZA, M&#252;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 SpO2 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&#160;&#160;&#160;</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 &#38; 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&#252;lke &#38; 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&#252;schelit Super Safety Clear &#62;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&#160;</td>
			<td>ultrasound&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Macintosh Gr&#246;&#223;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&#160;</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&#160;</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/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/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/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/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

826 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

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

在大鼠模型慢性营养输液

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脑Microslices的兴奋性刺激作为<em&gt;在体外</em&gt;中风模型

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality of life when compared to dialysis. Although the last few decades have seen major improvements in patient outcomes following kidney transplantation, the increasing shortage of available organs represents a severe problem worldwide. To expand the donor pool, marginal kidney grafts recovered from extended criteria donors (ECD) or donated after circulatory death (DCD) are now accepted for transplantation. To further improve the postoperative outcome of these marginal grafts, research must focus on new therapeutic approaches such as alternative preservation techniques, immunomodulation, gene transfer, and stem cell administration.
Experimental studies in animal models are the final step before newly developed techniques can be translated into clinical practice. Porcine kidney transplantation is an excellent model of human transplantation and allows investigation of novel approaches. The major advantage of the porcine model is its anatomical and physiological similarity to the human body, which facilitates the rapid translation of new findings to clinical trials. This article offers a surgical step-by-step protocol for an autotransplantation model and highlights key factors to ensure experimental success. Adequate pre- and postoperative housing, attentive anesthesia, and consistent surgical techniques result in favorable postoperative outcomes. Resection of the contralateral native kidney provides the opportunity to assess post-transplant graft function. The placement of venous and urinary catheters and the use of metabolic cages allow further detailed evaluation. For long-term follow-up studies and investigation of alternative graft preservation techniques, autotransplantation models are superior to allotransplantation models, as they avoid the confounding bias posed by rejection and immunosuppressive medication.

Porcine models of organ transplantation provide an important platform to study mechanisms of organ preservation. This article describes a heterotopic porcine renal autotransplantation model, which allows investigating new approaches to improve the outcome of transplantation using marginal kidney grafts.

异位自体肾移植的猪模型:有步骤，分步协议

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Porcine As a Training Module for Head and Neck Microvascular Reconstruction

Live models that resemble surgical conditions of humans are needed for training free-flap harvesting and anastomosis. Animal models for training purposes have been available for years in many surgical fields. We used the female (because they are easy to handle for the procedure) Yorkshire pigs for the head and neck reconstruction by harvesting the deep inferior epigastric artery perforator or the superior epigastric artery perforator flap. The anastomosis site (neck skin defect or tracheal wall defect) was prepared via the dissection of the common carotid artery and the internal jugular vein, in which 3.5&#215; loupe magnification was used for anastomosis as we use on human cases in real life. This procedure demonstrates a new training method using a reliable learning model and provides a detailed anatomy in a live scenario. We focused on the ischemia time, harvesting, vessel anastomosis, and designing the flap to fit the defect site. This model improves tissue handling and with the use of proper instruments can be repeated many times so that the surgeon is fully confident before starting the surgery on humans.

Here we present a protocol for the use of the pig superior epigastric artery perforator flap as a learning module for head and neck microvascular reconstruction.

猪作为头颈部微血管重建训练模块

Live models that resemble surgical conditions of humans are needed for training free-flap harvesting and anastomosis. Animal models for training purposes ...

A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart failure. Multiple small-animal models of heart transplantation have been used to study the acute and long-term effects of novel therapies. However, few are tested and demonstrated success in clinical trials. It is of critical importance to evaluate new therapies in a clinically relevant large-animal model for efficient and reliable translation of basic studies&#39;&#160;findings. Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and previously used to investigate novel cardioprotective strategies. This procedure focuses on acute ischemia-reperfusion injury and is a reliable method to investigate novel interventions which have been tested and validated in smaller experimental models, such as the murine model. We demonstrate its usefulness in assessing cardiac performance during the early post-transplantation period and other potential possibilities enabled by the model.

Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and utilized to investigate novel cardioprotective strategies.

正交心脏移植的临床前猪模型

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart ...

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

使用分裂的角质按钮的孔膜内皮器官培养模型

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. Several rodent models have been developed to study CP, of which the bile duct 2,4,6 -trinitrobenzene sulfonic acid (TNBS) infusion model replicates the features of neuropathic pain seen in CP. However, bile duct drug infusion in mice is technically challenging. This protocol demonstrates the procedure of bile duct TNBS infusion for generation of a CP mouse model. TNBS was infused into the pancreas through the ampulla of Vater in the duodenum. This protocol optimized drug volume, surgical techniques, and drug handling during the procedure. TNBS-treated mice showed features of CP as reflected by bodyweight and pancreas weight reductions, changes in pain-associated behaviors, and abnormal pancreatic morphology. With these improvements, mortality associated with TNBS injection was minimal. This procedure is not only critical in generating pancreatic disease models but is also useful in local pancreatic drug delivery.

Chronic pancreatitis (CP) is a disease characterized by inflammation and fibrosis of the pancreas, often associated with intractable abdominal pain. This article focuses on refining the technique to generate a mouse model of CP via bile duct infusion with 2,4,6 -trinitrobenzene sulfonic acid (TNBS).

通过胆管 TNBS 输液治疗慢性胰腺炎的鼠标模型

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. ...

Establishment and Evaluation of a Porcine Vein Graft Disease Model

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the investigation of disease mechanisms and the development of therapeutic strategies. 
To perform the surgery, we enter the cardiac chamber through the third intercostal space and carefully dissect the internal mammary vein and immerse it in normal saline. The right main coronary artery is then treated for ischemia. The target vessel is incised, a shunt plug is placed, and the distal end of the graft vein is anastomosed. The ascending aorta is partially blocked, and the proximal end of the graft vein is anastomosed after perforation. The graft vein is checked for patency, and the proximal right coronary artery is ligated. 
CABG surgery is performed in minipigs to harvest the left internal mammary vein for its use as a vascular graft. Serum biochemical tests are used to evaluate the physiological status of the animals after surgery. Ultrasound examination shows that the proximal, middle, and distal end of the graft vessel are unobstructed. In the surgical model, turbulent blood flow in the graft is observed upon histological examination after the CABG surgery, and venous graft stenosis associated with intimal hyperplasia is observed in the graft. The study here provides detailed surgical procedures for the establishment of a repeatable CABG-induced VGD model.

In this protocol, novel pig vein bypass grafting was performed through a small incision in the left chest wall without cardiopulmonary bypass. A postoperative pathology study was done, which showed intimal thickening.

脂多糖输注作为猪内毒素血症休克模型

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