小鼠无创气管内脂多糖滴注
1State Key Laboratory of Southwestern Chinese Medicine Resources, College of Pharmacy, Innovative Institute of Chinese Medicine and Pharmacy,Chengdu University of Traditional Chinese Medicine, 2Department of Pharmacy,The Second Affiliated Hospital of Hainan Medical University, 3The School of Preclinical Medicine,Chengdu University
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 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：…
Representative Results
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…
Discussion
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?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Lipopolysaccharide | MERK | L4130 | LPS |
| Microliter Syringes | SHANGHAI GAOGE INDUSTRY AND TRADE CO., LTD | 10028505008124 | To deliver LPS |
| Mouse cannula | RWD Life Science | 803-03008-00 | Mouse cannula |
| Mouse intubation kit | RWD Life Science | 903-03027-00 | Including a base, a riser, a intubator, a surgical forceps and some strings |
| Pasteur pipette | Biosharp life science | BS-XG-03 | To verify the success of intubation |
| Pentobarbital sodium | Beijing Chemical Co., China | 20220918 | To anesthetize mice |
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Citazione di questo articolo
Yu, P., Lin, B., Li, J., Luo, Y., Zhang, D., Sun, J., Meng, X., Hu, Y., Xiang, L. Noninvasive Intratracheal Lipopolysaccharide Instillation in Mice. J. Vis. Exp. (193), e65151, doi:10.3791/65151 (2023).