慢性假单位病伤口感染的延迟接种模型
Summary
我们描述了一种延迟的接种方案，用于在免疫能力小鼠中产生慢性伤口感染。
Abstract
假单胞菌（P.aeruginosa）是一种与人类健康和疾病越来越相关的主要肿瘤病原体，特别是在糖尿病和住院患者慢性伤口感染的设置中。迫切需要慢性感染模型，以帮助研究伤口发病机制和开发针对这种病原体的新疗法。在这里，我们描述了一个协议，使用延迟接种24小时后，全厚度外伤。目前存在的临时伤口基质的感染可以阻止感染的快速清除或传播，相反，这种感染将形成持续7-10天的慢性感染，而无需植入异物或免疫抑制。该协议模仿人类术后感染的典型时间过程。使用发光P.aeruginosa菌株（PAO1：lux）允许定量的每日评估P.aeruginosa伤口感染的细菌负担。这一新模型可能是研究细菌发病机制和开发慢性P.aeruginosa伤口感染新疗法的有用工具。
Introduction
伪单抗（P.aeruginosa）是一种与人类健康和疾病密切相关的革兰氏阴性棒状细菌。它负责广泛的发病率和死亡率在不治疗环境，特别是涉及伤口感染的免疫功能低下患者1，2。这种病原体的耐多药菌株的出现，为进一步推动了对导致P.aeruginosa毒性的因素、P.aeruginosa抗生素耐药性机制以及预防和治疗这种致命感染的新方法的进一步推动。因此，对慢性伤口感染的动物模型作为研究这些研究问题的工具的需求从未像现在这样大。
不幸的是，许多动物模型的P.aeruginosa感染往往模拟急性感染与快速解决感染或快速下降由于败血症4，5，这没有充分模拟这些感染的经常慢性性质。为了解决这一缺点，一些模型利用异物的植入，如琼脂珠，硅胶植入物，或藻酸盐凝胶6，7，8。其他模型使用由于年高龄、肥胖或糖尿病而免疫功能低下的小鼠，或通过药理学手段，如环磷酰胺引起的中性粒细胞减少症9，10，11，12。然而，无论是使用异物或免疫受损的宿主可能会改变局部炎症过程，使得很难了解在具有正常免疫系统的宿主慢性伤口感染中涉及的病理生理学。
我们已经开发了一种小鼠P.aeruginosa伤口感染的慢性模型，该模型涉及在外伤后延迟接种细菌。延迟接种允许实验评估细菌负担延长到至少7天。这一模型为研究P.aeruginosa慢性感染的发病机制和新的治疗方法开辟了新的机会。
Protocol
Representative Results
Discussion
我们开发了一种新的延迟接种P.aeruginosa伤口感染模型。在切除伤伤后，将细菌延迟接种到24小时的策略，可以在1周的时限内评估伤口感染。通过使用P.aeruginosa的发光菌株，可以跟踪整个感染过程的感染进展。与其他阿鲁吉诺萨感染模型相比，更长的感染过程将为研究宿主-病原体相互作用和针对P. aeruginosa伤口感染的新疗法提供新的机会。例如，我们已经使用这个模型来?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
pUT-Tn5-EM7-lux-Km1发光构造载体是J.哈代赠送的一份厚礼。原理图是使用BioRender.com创建的。我们感谢G.Gurtner实验室对伤口感染模型的建议。我们还感谢斯坦福Vivo成像创新中心的 T. Doyle 的技术专长。这项工作得到了R21AI133370、R21AI133240、R01AI12492093的赠款的支持，以及斯坦福SPARK、法尔克医学研究信托基金和囊性纤维化基金会（CFF）对P.L.B.C.R.D的赠款，得到了T32AI007502的支持。加比兰·斯坦福科学和工程研究生奖学金和卢伯特·斯特勒生物-X斯坦福跨学科研究生奖学金为J.M.S.
Materials
| 0.9% Sodium Chloride injection | Hospira | 2484457 | |
| 18 G x 1 sterile needle | BD | 305195 | |
| 25 G x 1 1/5 sterile needle | BD | 305127 | |
| Alcohol swab | BD | 326895 | |
| Aura Imaging Software | Spectral Instruments Imaging | n/a | |
| Betadine | Purdue Frederick Company | 19-065534 | |
| Buprenorphine SR LAB | Zoopharm | n/a | |
| C57BL/6J male mice | The Jackson Laboratory | 000664 | |
| Disposable biopsy punch, 6mm | Integra | 33-36 | |
| Fine scissors – Tungsten Carbide | Fine Science Tools | 14568-09 | |
| Glass Bead Dry Sterilizer | Harvard Apparatus | 61-0183 | |
| Granulated Agar | Fisher BioReagents | BP9744 | |
| Heating Pad | Milliard | 804879481218 | |
| Insulin syringe with 28 G needle | BD | 329461 | |
| Lago X Imaging System | Spectral Instruments Imaging | n/a | |
| LB broth | Fisher BioReagents | BP1426 | |
| Leur-Lok 1 mL syringe | BD | 309628 | |
| Mini Arco Animal Trimmer | Wahl Professional | 919152 | |
| Nair Hair Removal Lotion with Baby Oil | Church and Dwight | n/a | Available at any pharmacy |
| Octagon Forceps | Fine Science Tools | 11041-08 | |
| Petri dish | Falcon | 351029 | |
| Phosphate Buffered Saline (PBS) 1x | Corning | 21-040-CV | |
| Press and Seal Cling Wrap | Glad | n/a | |
| SafetyGlide Insulin syringe with 30 G needle | BD | 305934 | |
| Safetyglide Insulin syringe, 1/2 mL, 30 G x 5/16 TW | BD | 305934 | |
| Scale | Ohaus Scout Pro | SP202 | |
| Supplical Nutritional Supplement | Henry Schein Animal Health | 29908 | |
| Tegaderm, 6 cm x 7 cm | 3M | 1624W |
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