一种新生儿啮齿动物气管内滴注药物治疗肺部疾病的微创方法
Summary
这种将药物直接滴入新生儿啮齿动物气管的技术对于研究局部施用的药物或生物制剂对新生儿肺部疾病的影响非常重要。此外，该方法还可用于在动物模型中诱导肺损伤。
Abstract
用直接滴入气管的药物治疗新生儿啮齿动物可以作为研究局部施用药物影响的宝贵工具。这具有直接的转化作用，因为表面活性剂和药物是局部施用于肺部的。虽然文献中有许多出版物描述了成年小鼠和大鼠在治疗实验中的微创经口插管，但新生儿大鼠幼崽缺乏这种方法。幼崽中口气管区域/咽部的小尺寸使得喉腔（声带）的可视化变得困难，导致气管内药物递送的成功率参差不齐。我们在此证明对新生大鼠幼崽的有效口服插管 – 一种非创伤性和微创的技术，因此可用于连续给药。我们使用带有照明系统和放大镜的手术耳镜来可视化大鼠新生儿的气管开口。然后使用连接到移液器尖端的1mL注射器滴注药物。使用Evans蓝色染料施用证明了递送方法的准确性。这种方法很容易接受培训，可以作为将药物灌输到气管的有效方法。该方法还可用于施用接种物或药剂以模拟动物的疾病状况，也可用于各种肺部疾病的基于细胞的治疗策略。
Introduction
早产儿的肺发育不良，需要许多介入治疗，如长期通气。这些干预措施使存活的新生儿面临随后后遗症1的高风险。实验动物模型是模拟各种疾病状况，研究疾病的病理生物学和评估治疗干预措施的重要工具。尽管从小鼠，大鼠和兔子到早产羔羊和猪的广泛动物模型可用，但小鼠和大鼠是最常用的。
使用小鼠和大鼠的主要优点是相对较短的妊娠期和降低成本。它们也很容易获得，易于在无病环境中维护，遗传上是同质的，并且伦理问题相对较少2，3。啮齿动物模型的另一个主要优点是，出生时，新生儿幼崽处于肺发育的晚期运河/早期囊骨阶段，这在形态上相当于24周早产新生儿的肺继续发展支气管肺发育不良4。此外，由于他们的肺发育在生命的前4周内迅速进展到完成，因此在合理的时间范围内研究产后肺成熟是可行的4。尽管有这些优点，但小鼠和大鼠幼崽的小尺寸是各种干预措施的关注点，这迫使大多数研究人员使用成年动物而不是幼崽5。新生儿肺处于发育阶段，新生儿对刺激剂的反应与成人不同。这使得使用新生儿动物模型来研究人类新生儿疾病状况变得合适。
有不同的方法可以给肺部施用药物/生物制剂。这包括鼻内6，7 或气管内8，9，10 滴注以及气溶胶吸入11，12。每种方法都有自己的技术挑战、优点和局限性13.气管内给药途径优选治疗剂以研究在绕过全身效应的器官中的直接治疗影响。该途径也可用于研究由刺激剂引起的肺部病理学。有侵入性和微创技术可以做到这一点，并且很容易在成人中执行。然而，在幼崽中，由于动物的体型小，插管过程存在技术挑战。目前的研究提出了一种简单，一致，非手术气管内滴注（ITI）方法，可用于研究各种新生儿治疗干预措施的疗效以及生成模拟新生儿呼吸道疾病的动物模型。
Protocol
所有实验均由凯斯西储大学的机构动物护理和使用委员会（协议#2020-0035）批准。所有动物均按照NIH关于实验室动物护理和使用指南进行治疗。 1. 动物 商业上获得怀孕的Sprague Dawley大鼠。 将动物保持在批准的兽医设施中，光暗循环为14小时/ 10小时，相对湿度为45-60%。 2. 测试化合物的制备 使用Evans蓝染料作为测试化合物来评估气?…
Representative Results
Evans Blue的滴注揭示了涉及所有肺叶的染料的多灶分布（图4A，B）。如图 4 所示的结果证明了分布到所有波瓣的功效。这张照片是在染料进入气管的ITI后立即拍摄的。将染料滴入气管，然后将其扩散到两侧的所有肺叶中，实现了100%的功效。预计染料将在肺小叶内进一步扩散。通过反复给药，我们已经能够确保100%成功地将其传递到肺叶和所?…
Discussion
气管内滴注是一种极好的方法，与现有的呼吸系统疾病干预方法以及疾病模型开发相比，它具有多种优势。这是一种快速的方法，并且经验丰富，可以以每只动物的平均速度2-3分钟进行。成功插管的关键考虑因素是动物的正确镇静，正确的位置，特别是头部，以及口咽部窥器的准确位置深度/大小。适当的镇静剂将为操作员，特别是初学者提供足够的工作时间。动物以45°角定位对于声带的正确可?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了从NICHD到AH的R01HD090887-01A1的部分支持。作者还承认Peter Mc Farlane博士的实验室提供的设施，如吸入麻醉/加热垫系统。凯瑟琳·梅耶尔女士在建立该系统方面提供的宝贵协助值得赞赏。资助机构在研究、收集、分析和解释数据或撰写手稿方面没有发挥任何作用。
Materials
| Evans Blue dye | Sigma-Aldrich, St Louis, MO, USA | 314-13-6 | Confirmation of drug administration into lungs |
| Ketamine Hydrochloride | Hospira. Inc, Lake Forest, IL, USA | Dispensed from Animal care facility | For sedation |
| Operating Otoscope | Welch Allyn, Hillrom, Chicago, IL, USA | 21770- 3.5V | For visualization of vocal cords |
| Otoscope Rechargeable Handle | Welch Allyn, Hillrom, Chicago, IL, USA | 71050-C | |
| Pipette tip (Gel loading) | Fisherbrand | 02-707-139 | Administering the drug |
| Platform for restraining (inclined plane) | Animal care facility | Dispensed from Animal care facility | Wired roof of mice cage can be used |
| 3M Micropore Surgical White Paper (sticking tape) | 3M, St. Paul, MN, USA | 1530-2 | |
| Luer Lock SyringeSyringes (1 ml) | BD Franklin Lakes, NJ , USA | NBD2515 | Administering the drug |
| Xylazine | Hospira. Inc, Lake Forest, IL, USA | For sedation |
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Cite This Article
Sudhadevi, T., Ha, A. W., Harijith, A. A Minimally Invasive Method for Intratracheal Instillation of Drugs in Neonatal Rodents to Treat Lung Disease. J. Vis. Exp. (174), e61729, doi:10.3791/61729 (2021).