Name: noninvasive intratracheal lipopolysaccharide instillation in mice
Uploaded: 2023-03-31T13:40:00
Description: Watch this Scientific Journal Video about Noninvasive Intratracheal Lipopolysaccharide Instillation in Mice at JoVE.com

这项工作得到了国家自然科学基金（编号：81903902）、中国博士后科学基金（编号：2019M663457）、四川省科技计划（编号：2020YJ0172）和成都中医药大学杏林学者研究预动项目（编号：QJRC2022053）的支持。

动物实验方案经成都中医药大学管委会审核通过（备案号：2021-11）。雄性C57 / BL小鼠（20-25g，6-8周龄）用于本研究。小鼠被饲养在动物室中，在实验过程中可以自由饮水和进食。
1. 准备
<ol><li>确保插管平台由底座、立管、回形针、两根橡皮筋和一些绳子组成。取一根绳子，将绳子穿过立管顶部的两个孔，并将绳子的两端分别绑在立管顶部的小突起上。 注意：...

<ol>
	<li>Xia, Y., 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=Protective+effect+of+human+amnion+epithelial+cells+through+endotracheal+instillation+against+lipopolysaccharide-induced+acute+lung+injury+in+mice.">Protective effect of human amnion epithelial cells through endotracheal instillation against lipopolysaccharide-induced acute lung injury in mice.</a> Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 33 (1), 7-11 (2017).</li><li>Butt, Y., Kurdowska, A., Allen, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+lung+injury:+A+clinical+and+molecular+review.">Acute lung injury: A clinical and molecular review.</a> Archives of Pathology and Lab Medicine. 140 (4), 345-350 (2016).</li><li>Ware, L. B., Matthay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acute+respiratory+distress+syndrome.">The acute respiratory distress syndrome.</a> The New England Journal of Medicine. 342 (18), 1334-1349 (2000).</li><li>Fan, E., Brodie, D., Slutsky, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+respiratory+distress+syndrome:+Advances+in+diagnosis+and+treatment.">Acute respiratory distress syndrome: Advances in diagnosis and treatment.</a> JAMA. 319 (7), 698-710 (2018).</li><li>Meyer, N. J., Gattinoni, L., Calfee, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+respiratory+distress+syndrome.">Acute respiratory distress syndrome.</a> Lancet. 398 (10300), 622-637 (2021).</li><li>An, N., Yang, T., Zhang, X. X., Xu, M. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bergamottin+alleviates+LPS-induced+acute+lung+injury+by+inducing+SIRT1+and+suppressing+NF-&#954;B.">Bergamottin alleviates LPS-induced acute lung injury by inducing SIRT1 and suppressing NF-&#954;B.</a> Innate Immunity. 27 (7-8), 543-552 (2021).</li><li>Liu, L., 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=Comparative+study+of+trans-oral+and+trans-tracheal+intratracheal+instillations+in+a+murine+model+of+acute+lung+injury.">Comparative study of trans-oral and trans-tracheal intratracheal instillations in a murine model of acute lung injury.</a> The Anatomical Record. 295 (9), 1513-1519 (2012).</li><li>Virag, J. A., Lust, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+artery+ligation+and+intramyocardial+injection+in+a+murine+model+of+infarction.">Coronary artery ligation and intramyocardial injection in a murine model of infarction.</a> Journal of Visualized Experiments. (52), e2581 (2011).</li><li>Li, J., 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=Panaxydol+attenuates+ferroptosis+against+LPS-induced+acute+lung+injury+in+mice+by+Keap1-Nrf2/HO-1+pathway.">Panaxydol attenuates ferroptosis against LPS-induced acute lung injury in mice by Keap1-Nrf2/HO-1 pathway.</a> Journal of Translational Medicine. 19 (1), 96 (2021).</li><li>Zhou, Y., 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=Soluble+epoxide+hydrolase+inhibitor+attenuates+lipopolysaccharide-induced+acute+lung+injury+and+improves+survival+in+mice.">Soluble epoxide hydrolase inhibitor attenuates lipopolysaccharide-induced acute lung injury and improves survival in mice.</a> Shock. 47 (5), 638-645 (2017).</li><li>Nosaka, N., 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=Optimal+tube+length+of+orotracheal+intubation+for+mice.">Optimal tube length of orotracheal intubation for mice.</a> Laboratory Animals. 53 (1), 79-83 (2019).</li><li>Cicero, L., Fazzotta, S., Palumbo, V. D., Cassata, G., Lo Monte, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaesthesia+protocols+in+laboratory+animals+used+for+scientific+purposes.">Anaesthesia protocols in laboratory animals used for scientific purposes.</a> Acta Bio-Medica: Atenei Parmensis. 89 (3), 337-342 (2018).</li><li>Ehrentraut, H., Weisheit, C. K., Frede, S., Hilbert, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducing+acute+lung+injury+in+mice+by+direct+intratracheal+lipopolysaccharide+instillation.">Inducing acute lung injury in mice by direct intratracheal lipopolysaccharide instillation.</a> Journal of Visualized Experiments. (149), e59999 (2019).</li><li>Yang, H., 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=STAT6+inhibits+ferroptosis+and+alleviates+acute+lung+injury+by+regulating+P53/SLC7A11+pathway.">STAT6 inhibits ferroptosis and alleviates acute lung injury by regulating P53/SLC7A11 pathway.</a> Cell Death &amp; Disease. 13 (6), 530 (2022).</li><li>Aramaki, O., 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=Induction+of+operational+tolerance+and+generation+of+regulatory+cells+after+intratracheal+delivery+of+alloantigen+combined+with+nondepleting+anti-CD4+monoclonal+antibody.">Induction of operational tolerance and generation of regulatory cells after intratracheal delivery of alloantigen combined with nondepleting anti-CD4 monoclonal antibody.</a> Transplantation. 76 (9), 1305-1314 (2003).</li><li>Lan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+mammalian+target+of+rapamycin+(mTOR)+in+a+murine+model+of+lipopolysaccharide+(LPS)+-induced+acute+lung+injury+(ALI).">Activation of mammalian target of rapamycin (mTOR) in a murine model of lipopolysaccharide (LPS) -induced acute lung injury (ALI).</a> Peking Union Medical College. , (2010).</li><li>Lv, H., 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=Tenuigenin+ameliorates+acute+lung+injury+by+inhibiting+NF-&#954;B+and+MAPK+signalling+pathways.">Tenuigenin ameliorates acute lung injury by inhibiting NF-&#954;B and MAPK signalling pathways.</a> Respiratory Physiology &amp; Neurobiology. 216, 43-51 (2015).</li><li>Yang, H., Lv, H., Li, H., Ci, X., Peng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oridonin+protects+LPS-induced+acute+lung+injury+by+modulating+Nrf2-mediated+oxidative+stress+and+Nrf2-independent+NLRP3+and+NF-&#954;B+pathways.">Oridonin protects LPS-induced acute lung injury by modulating Nrf2-mediated oxidative stress and Nrf2-independent NLRP3 and NF-&#954;B pathways.</a> Cell Communication and Signaling. 17 (1), 62 (2019).</li><li>Im, G. H., 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=Improvement+of+orthotopic+lung+cancer+mouse+model+via+thoracotomy+and+orotracheal+intubation+enabling+in+vivo+imaging+studies.">Improvement of orthotopic lung cancer mouse model via thoracotomy and orotracheal intubation enabling in vivo imaging studies.</a> Laboratory Animals. 48 (2), 124-131 (2014).</li><li>Hamacher, J., 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=Microscopic+wire+guide-based+orotracheal+mouse+intubation:+description,+evaluation+and+comparison+with+transillumination.">Microscopic wire guide-based orotracheal mouse intubation: description, evaluation and comparison with transillumination.</a> Laboratory Animals. 42 (2), 222-230 (2008).</li><li>Nelson, A. M., Nolan, K. E., Davis, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+orotracheal+intubation+in+mice.">Repeated orotracheal intubation in mice.</a> Journal of Visualized Experiments. (157), e60844 (2020).</li><li>Zhang, S., 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=Microvesicles+packaging+IL-1&#946;+and+TNF-&#945;+enhance+lung+inflammatory+response+to+mechanical+ventilation+in+part+by+induction+of+cofilin+signaling.">Microvesicles packaging IL-1&#946; and TNF-&#945; enhance lung inflammatory response to mechanical ventilation in part by induction of cofilin signaling.</a> International Immunopharmacology. 63, 74-83 (2018).</li><li>Feng, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+different+intratracheal+instillation+in+acute+lung+injury+model+of+mice.">Comparative study of different intratracheal instillation in acute lung injury model of mice.</a> Jilin University. , (2010).</li><li>Chen, J. H., 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=Comparison+of+acute+lung+injury+mice+model+established+by+intranasal+and+intratracheal+instillation+of+lipopolysaccharide.">Comparison of acute lung injury mice model established by intranasal and intratracheal instillation of lipopolysaccharide.</a> Pharmacology and Clinics of Chinese Materia Medica. 38 (02), 222-227 (2022).</li><li>Luckow, B., Lehmann, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+method+for+bronchoalveolar+lavage+in+mice+by+orotracheal+intubation+avoiding+tracheotomy.">A simplified method for bronchoalveolar lavage in mice by orotracheal intubation avoiding tracheotomy.</a> BioTechniques. 71 (4), 534-537 (2021).</li><li>Cai, Y., Kimura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+intratracheal+intubation+to+study+the+pathology+and+physiology+of+mouse+lung.">Noninvasive intratracheal intubation to study the pathology and physiology of mouse lung.</a> Journal of Visualized Experiments. (81), e50601 (2013).</li></ol>

最初，我们查看口腔内部以找到气管的位置 19.然而，在此过程中，我们发现C57 / BL小鼠的气管很窄，这使得在没有内窥镜20等设备的帮助下很难通过这种方法找到正确的位置。经过进一步探索，我们发现插管灯的光线可以穿透身体表面，使操作员能够确定套管21的位置。
为了检查管子是否进入气管，最初，我们尝试使用一?...

急性肺损伤（ALI）是一种常见的临床综合征。在多种致病因素作用下，肺上皮细胞和血管内皮细胞生理屏障的破坏导致肺泡通透性增加，从而引起肺顺应性下降、肺水肿和严重低氧血症1。急性呼吸窘迫综合征（ARDS）是最严重的ALI形式。不受控制的炎症和氧化应激损伤被认为是ALI和更严重的ARDS的主要原因2。当肺泡上皮细胞因创伤直接受损时，肺泡巨噬细胞的炎症反应链被激活，导致肺部炎症3。在全球范围内，每年有超过300万急性ARDS患者，约占重症监护病房入院人数的10%;此外，严重病例的死亡率高达46%4,5,6。因此，有必要建立合适的ALI动物模型来研究其发病机制。小鼠是ALI研究中最常用的实验动物，因为它的呼吸道可以很好地模拟人类呼吸道进行ALI研究。此外，ALI 表现为大量炎性细胞浸润、肺血管通透性增加和肺水肿。血清中炎性细胞因子的变化和肺干湿重比反映了ALI7的程度。
目前，模拟小鼠LPS诱导的ALI的主要方法包括鼻内和手术气管插管8,9。在这里，我们提出了一种通过无创口咽插管将LPS输送到气管中的新方法。该方法使用照明插管器找到小鼠的气管，然后将LPS输送到气管和肺部。这种方法比鼻内输送方法更准确地将LPS输送到肺部。与手术气管插管相比，该方法不需要手术，避免引起伤口，减轻小鼠疼痛10。因此，该方法可用于建立更有说服力的ALI小鼠模型。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Lipopolysaccharide</td>
			<td>MERK</td>
			<td>L4130</td>
			<td>LPS</td>
		</tr>
		<tr>
			<td>Microliter Syringes</td>
			<td>SHANGHAI GAOGE INDUSTRY AND TRADE CO., LTD</td>
			<td>10028505008124</td>
			<td>To deliver LPS</td>
		</tr>
		<tr>
			<td>Mouse cannula</td>
			<td>RWD Life Science</td>
			<td>803-03008-00</td>
			<td>Mouse cannula</td>
		</tr>
		<tr>
			<td>Mouse intubation kit</td>
			<td>RWD Life Science</td>
			<td>903-03027-00</td>
			<td>Including a base, a riser, a intubator, a surgical forceps and some strings</td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>Biosharp life science</td>
			<td>BS-XG-03</td>
			<td>To verify the success of intubation</td>
		</tr>
		<tr>
			<td>Pentobarbital sodium</td>
			<td>Beijing Chemical Co., China</td>
			<td>20220918</td>
			<td>To anesthetize mice</td>
		</tr>
	</tbody>
</table>

noninvasive intratracheal lipopolysaccharide instillation in mice

由脂多糖（LPS）或内毒素诱导的急性肺损伤（ALI）小鼠模型仍然是急性肺损伤或急性炎症动物研究中最常用的模型之一。目前急性肺损伤小鼠模型中最常用的方法是腹膜内注射LPS和气管切开术用于LPS的气管输注。然而，前一种方法缺乏肺部靶向并损害其他器官，后一种方法诱发手术创伤，感染风险大，生存率低。在这里，我们推荐一种无创口咽气管插管方法在小鼠中滴注LPS。在这种方法中，LPS通过口咽腔无创地引入气管，在气管插管装置的帮助下滴入肺部。这种方法不仅可以确保肺部靶向，还可以避免动物的损伤和死亡风险。我们预计这种方法将在急性肺损伤领域得到广泛应用。

通过评估LPS滴注后12 h炎症细胞因子TNF-α的表达和肺干湿重比，验证了所提出的LPS滴注小鼠方法。实验中有四组：空白对照（无任何治疗），手术插管16，鼻内17,18和无创口咽插管（n = 6）。与空白对照组相比，无创口咽插管组血清TNF-α水平显著升高（图8A）。肺干湿重量比也增加（<strong cla...

Watch this Scientific Journal Video about Noninvasive Intratracheal Lipopolysaccharide Instillation in Mice at JoVE.com

Noninvasive Intratracheal Lipopolysaccharide Instillation in Mice

在这里，我们提出了通过无创口咽气管插管 输送 气管内脂多糖（LPS）的方案。这种方法最大限度地减少了动物外科手术的创伤，并准确地将LPS输送到气管，然后输送到肺部。

小鼠无创气管内脂多糖滴注

The acute lung injury (ALI) mouse model induced by lipopolysaccharide (LPS) or endotoxin is still among the most commonly used models in animal studies of ...

我们的协议提出了一种新的无创LPS从气管到肺部的无创输送方法，更准确。第一次尝试这种方法的人应该花很多精力来寻找气管的位置。要准备插管平台，请将绳子穿过两个孔并将其末端绑在立管顶部的小突起上。接下来，将两个橡皮筋连接到回形针的每一端，并将它们粘在立管的背面。最后，将立管以 90 度角固定在底座上。将套管组装到套管笔上，然后打开笔的灯。接下来，使用70%酒精对小型手术钳和巴斯德移液管进行消毒。要制备测试化合物溶液，请在一毫升pH 7.2 PBS中加入三毫克脂多糖或LPS。接下来，将10毫克戊巴比妥钠溶解在1毫升生理盐水中，并使用0.45微米注射器过滤器过滤溶液。将麻醉小鼠放在插管平台上。然后用绳子固定上门牙，用橡皮筋固定两个前脚掌。用镊子，拔出舌头并握住它。将套管沿口腔缓慢推至下颌会厌。用套管笔的灯找到气管，然后慢慢地将套管插入其中。然后取出插管笔，将套管留在里面。将巴斯德移液器插入套管接头并按压头部。气管插管成功后，使用平头微型注射器，通过套管以每公斤三毫克剂量滴注制备的LPS溶液。完成后，取出微量注射器和套管。将鼠标从插管平台上取出，并将其放在单独的笼子中以恢复。观察小鼠的呼吸状态，直到它恢复并恢复意识。在LPS气管滴注后12小时，评估炎性细胞因子TNF-α和肺干湿重量比的表达，并与其他滴注方法进行比较。与空白对照组相比，无创口咽插管组血清TNF-α水平显著升高。肺干湿重量比也增加，达到与外科气管插管组相同的水平。在寻找气管位置的过程中不要划伤气管很重要。该技术还可用于输送需要以非侵入性方式到达肺部的其他药物。

Research

JoVE Journal

Biology

Heterotopic Heart Transplantation in Mice

小鼠异位心脏移植

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable ...

科研

生物学

Small Bowel Transplantation In Mice

小鼠小肠移植

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive ...

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

无创在体内小动物的MRI和MRS：基本实验程序

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the ...

Transverse Aortic Constriction in Mice

在小鼠体内的横向主动脉缩窄

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart ...

Ambulatory ECG Recording in Mice

在小鼠体内的动态心电图记录

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although ...

Direct Tracheal Instillation of Solutes into Mouse Lung

溶质直接气管滴入小鼠肺

Intratracheal instillations deliver solutes directly into the lungs.  This procedure targets the delivery of the instillate into the distal regions of the ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

一种无创性的发采样技术，以获得难以捉摸的小型兽类高质量的DNA

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

体内转染和基因敲除在脊髓通过快速无创腰椎鞘内注射小鼠

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter ...

Intravenous Injections in Neonatal Mice

在新生小鼠静脉注射

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

Activating Autophagy by Aerobic Exercise in Mice

小鼠中自噬激活有氧运动

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced ...