呼吸机相关和医院获得性肺炎的小鼠口咽吸入模型
Summary
传染性肺炎是人类最常见的感染之一。适当的体内模型对于了解疾病发病机制和检测新疗法的疗效至关重要。与这个小鼠口咽吸入肺炎模型, 你可能审查发病机制和新的治疗反对这些致命的传染。
Abstract
小鼠感染模型对于了解疾病发病机制和测试旨在对抗致病病原体的新疗法的功效至关重要。感染性肺炎是临床病人最常见的感染之一, 因此需要适当的体内模型。典型的肺炎模型使用鼻腔接种, 将过量的生物体沉积在肺部外, 造成非靶向并发症和症状, 如鼻窦炎、胃炎、肠炎、物理创伤或微粒喷雾模拟气溶胶。传播更典型的病毒, 结核, 或真菌性肺炎。这些模型不能准确地反映典型的社区或医疗保健获得的细菌性肺炎的发病机制。与此相反, 这种小鼠口咽吸入性肺炎模型模仿了医疗获得性肺炎的雾滴路径。接种50µL 的细菌悬浮液进入麻醉小鼠的咽, 引起反射性吸入, 导致肺炎。通过这个模型, 我们可以研究肺炎引起的病原体的发病机制和新的治疗方法来对抗这些疾病。
Introduction
下呼吸道感染是世界上最致命的传染性疾病, 也是1发展中国家最常见的死亡原因。在全球范围内, 这些感染占320万以上死亡人数的1。此外, 医院内的肺炎是最常见和致命的医疗形式的感染, 并由最耐药性的病原体2,3造成。在社区获得和医院肺炎中获得细菌性肺炎的典型途径是口咽内容进入肺泡的愿望。用于研究这些疾病的小鼠模型经常使用鼻腔接种4, 将大部分细菌存放在肺部外, 造成非靶向并发症和症状, 如鼻窦炎和身体创伤, 这些都是不一致的疾病。在人类的进步, 模型设计来效仿。其他模型可能使用吸入室和 micromisting 设备, 更准确地模仿病毒, 结核, 和真菌肺炎, 但不准确地重述正常的采集路线的典型细菌肺炎。
小鼠口咽吸入性肺炎模型可用于模拟细菌性肺炎的自然途径和发病机制。通过用吸管将细菌悬浮剂的50µL 接种到麻醉小鼠的咽中, 随后会产生自反性的吸入, 导致感染性肺炎。利用这个模型, 我们可以研究肺炎引起的病原体的发病机制和新的治疗方法, 以更高的保真度模型来对抗这些疾病, 更类似于人类所观察到的吸入性肺炎感染。此外, 不同于通过口腔5,6感染的相似模型, 这个模型确保完全接种到达肺部而不是肠道, 在那里它可以引起非现场炎症和感染, 如胃炎和肠炎。最后, 不像另一个发布的模型, 需要喉镜和 inoculates 通过气管7, 这个模型不会阻碍呼吸道与饲针, 不需要注射接种分娩。相反, 接种依赖于老鼠的自然吸入反射。
Protocol
所有涉及动物的程序必须由研究员的机构动物护理和使用委员会 (IACUC) 批准。 1. 细菌接种的制备 分离细菌菌落。 在适当的无菌琼脂培养基上 (例如, 胰蛋白酶大豆琼脂) 上 HUMC1 细菌菌株 (例如,鲍曼), 小心产生孤立的菌落。 在适当的条件下孵化 (例如, 在37摄氏度过夜)。 发展隔夜文化。 从琼脂板中选择一?…
Representative Results
通过仔细遵循该协议, 可以很容易地获得可重现和健壮的数据。严格遵守自己的定制接种准备协议是至关重要的, 因为实验要相互比较。在感染过程中适当处理小鼠也很重要。一定要把老鼠放进一个没有异氟烷的麻醉室里。老鼠会恐慌, 如果他们被放置在一个房间, 已预先填充异氟醚, 可能会遇到过剩的压力, 这可能会损害实验结果。在固定容器盖后, 缓慢地引入异氟醚, 逐步…
Discussion
可以肯定的是, 老鼠不是微型人类。从老鼠模型得到的结果必须考虑在上下文和随后解释为适用性对人, 根据区别和相似性在两个种类6之间。选择合适的小鼠菌株也很重要, 因为某些感染比其他的更容易发生;同样适用于选择16的病原体菌株。
必须以严格和高度可重复的方式进行感染。菌对老鼠的致命作用是一个价值, 但即使是90% 的价值也无?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到国家卫生研究院的国家过敏和传染性疾病研究所的支持 [R01 AI117211、R01 AI130060、R21 AI127954 和 R42 AI106375 到 BS] 和美国食品药品监督管理局 [合同HHSF223201710199C 到 BML]。
Materials
| Agar | BD | 214530 | Combine with TSB to make TSA |
| Beads, Borosilicate Glass | Kimble | 135003 | Sterilize by baking or autoclaving before each use |
| Beaker, 250 mL | Pyrex | 1003 | Used during precise aliquoting of concentrated bacterial inocula |
| Centrifuge | Sorvall | ST 40R | Capable of 4,000×g at 4°C |
| Chamber for Anesthesia | Kent Scientific Corporation | VetFlo-0720 | Accommodates up to 5 mice |
| Cryomold, Intermediate Size | Sakura Tissue-Tek | 4566 | Disposable vinyl specimen molds, 15×15×5 mm |
| Dental Floss | Oral-B | 37000469537 | Tie to stable post approx. 6" above table height |
| Forceps | VWR | 82027-440 | Used to gently pull tongue out of mouse's mouth |
| Homogenizer for Lung Tissue | Omni International | TM125-115 | Autoclave before first use; rinse between samples |
| Isoflurane for Anesthesia | Abbott | 10015516 | Alternative drug can be used; modify procedure accordingly |
| iSTAT Cartridge | Abbott | 03P79-25 | Various cartridges are available to suit your needs |
| Ketamine, 100 mg/mL | Western Medical Supply | 4165 | Dilute 1:10 in PBS to 1 mg/mL and combine with Xylazine at 1 mg/mL |
| Ointment for Eyes | Akorn | Tears Renewed | Avoid touching eye with tip of dispenser |
| Optimal Cutting Temperature (O.C.T.) Compound | Fisher Scientific | 23-730-571 | Used to freeze lung samples at -80 °C to prepare for pathology sectioning |
| Petri Dish | VWR | 25384-302 | Polystyrene, disposable, sterilized, 100×15 mm |
| Phosphate-Buffered Saline (PBS) | Corning | 21-031-CM | Dulbecco's PBS without calcium and magnesium |
| Pipette Tips, 200-μL | VWR | 10017-044 | Autoclave before use |
| Pipetter, 200-μL | Gilson | Pipetman P200 | Autoclave and calibrate before use |
| Spreader, Bacterial Cell | Bel-Art | F377360006 | Sterilize by baking or autoclaving before each use |
| Stir Bar, Magnetic, 7.9 mm Diameter × 38.1 mm Length | VWR | 58948-150 | Used for stiring concentrated bacterial inocula during aliquoting |
| Stir Plate, Magnetic | Corning | PC-620D | Used for stiring concentrated bacterial inocula during aliquoting |
| Tryptic Soy Broth (TSB) | BD | 211822 | Combine with Agar to make TSA |
| Vial, Conical, Sterile, 50 mL | Corning | 431720 | Used for preparing bacterial inocula |
| Vial, Conical, Sterile, 500 mL | Corning | 431123 | Used to concentrate inocula for preparing frozen inocula |
| Vial, Cryogenic, 2.0 mL | Corning | 430659 | Used for cryogenic storage of concentrated bacterial inocula |
| Xylazine, 20 mg/mL | Akorn | AnaSed Injection | Dilute 1:20 in PBS to 1 mg/mL and combine with Ketamine at 1 mg/mL |
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Cite This Article
Nielsen, T. B., Yan, J., Luna, B., Spellberg, B. Murine Oropharyngeal Aspiration Model of Ventilator-associated and Hospital-acquired Bacterial Pneumonia. J. Vis. Exp. (136), e57672, doi:10.3791/57672 (2018).