大鼠烧伤模型研究全层皮肤热烧伤和感染
1UNC Eshelman School of Pharmacy,University of North Carolina at Chapel Hill, 2Department of Biochemistry & Pharmacology, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences,The University of Melbourne, 3Department of Microbiology & Immunology,University of North Carolina School of Medicine, 4Department of Surgery,University of North Carolina at Chapel Hill, 5Curriculum in Toxicology and Environmental Medicine,University of North Carolina at Chapel Hill, 6Department of Microbiology, Monash Biomedicine Discovery Institute,Monash University
Summary
模拟烧伤和感染临床情况的模型对于进一步烧伤研究是必要的。本协议展示了一种与人类相当的简单且可重复的大鼠烧伤感染模型。这有助于研究烧伤后烧伤和感染，以开发新的局部抗生素治疗。
Abstract
烧伤诱导方法在大鼠模型中的描述不一致。代表临床情况的统一烧伤创面模型对于进行可重复的烧伤研究是必要的。本协议描述了一种简单且可重复的方法，可在大鼠中产生~20%的总体表面积（TBSA）全层烧伤。在这里，将22.89cm2 （直径5.4cm）铜棒在水浴中加热97°C以诱导烧伤。具有高导热性的铜棒能够将热量散发到皮肤组织中的更深处，从而产生全层灼伤。组织学分析显示表皮减毒，真皮和皮下组织的全层厚度存在凝血损伤。此外，该模型代表了在烧伤后住院烧伤患者中观察到的临床情况，例如免疫失调和细菌感染。该模型可以概括革兰氏阳性和革兰氏阴性细菌的全身性细菌感染。综上所述，本文提出了一种易于学习且稳健的大鼠烧伤模型，该模型模拟了包括免疫失调和细菌感染在内的临床情况，对于开发用于烧伤创面和感染的新型外用抗生素具有相当大的实用性。
Introduction
烧伤是最具破坏性的创伤形式之一，即使在专门的烧伤中心，死亡率也达到 12%1，2，3。根据最近发表的报告，美国每年有~486，000名烧伤患者需要医疗护理，其中近3，500人死亡1，2，3，4，5，6。烧伤对患者的免疫系统构成重大挑战，并形成明显的开放性伤口，愈合缓慢，使他们容易受到皮肤、肺部和全身性院内机会性细菌的定植。免疫失调与细菌感染相结合与烧伤患者的发病率和死亡率增加有关7。
动物烧伤和感染模型对于研究皮肤损伤和烧伤创伤相关免疫抑制后细菌感染的发病机制至关重要。这些模型能够设计和评估治疗烧伤患者细菌感染的新方法。大鼠和人类具有先前记录的相似的皮肤生理和病理特征8。此外，与较大的动物模型相比，大鼠的体型更小，使它们更容易处理、更实惠、更容易采购和维护。
这些特征使大鼠成为研究烧伤和感染的理想模式动物9。不幸的是，烧伤诱导技术不一致，并且通常描述最少10，11，12，13，14。本协议旨在开发一种简单，具有成本效益和可重复的程序，用于在模拟临床场景的大鼠模型中创建一致的全层烧伤损伤，并可用于评估免疫抑制和细菌感染。
Protocol
所有程序均由北卡罗来纳大学机构动物护理和使用委员会（IACUC）批准，并按照其既定指南进行。使用7-9周龄的雄性和雌性Sprague Dawley大鼠（250-300g）进行实验。所有动物均被饲养在12 h：12 h明暗循环中，可随意获取食物 和水。在研究开始之前，请务必与您的机构兽医合作制定镇痛计划。 1.为大鼠准备烧伤 在烧伤前24小时准备动物烧伤。 在诱?…
Representative Results
这里提出的方案是高度可重复的，并导致大鼠三度全层烧伤。烧伤诱导后烧伤创面呈蜡白色（图2B）。烧伤后72小时烧伤的颜色从白色变为棕色（图2B-E）。 组织学分析证实烧伤后24小时全层烧伤（深度>2.61毫米; 图 3B）。与完整的非烧伤皮肤相比，烧伤动物的皮肤样本在烧伤后24、48和7…
Discussion
已经提出了几种烧伤模型来研究烧伤的病理生理学8，12，16，17。在本研究中，我们采用大鼠模型开发了一种简单且可重复的方案，以诱导全层烧伤，然后是细菌感染，以模拟患者的感染烧伤创伤。选择大鼠作为动物模型来模拟人类条件是基于成本、易用性、可重复性和数据可靠性的平衡。本文中使用…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢北卡罗来纳大学比较医学系提供和照顾动物。我们感谢病理学服务核心的Lauren Ralph和Mia Evangelista在组织病理学/数字病理学方面的专家技术援助，包括组织切片和成像。这项研究得到了国防部的研究资助（奖励号W81XWH-20-1-0500，GR和TV）。
Materials
| 1 mL syringe | BD, USA | 309597 | Used to inject the analgesic |
| 1.7 mL Microtube | Olympus, USA | 24-282 | Used to carry morphine |
| 10% NBF | VWR, USA | 16004-115 | Used to fix the skin piece for staining |
| 30 mL syringe | BD, USA | 302832 | Used to inject the lactate ringer solution |
| 70% ethyl alcohol | Fischer Scientific, USA | BP28184 | |
| Aperio AT2 Digital Pathology Slide Scanner with ImageScope software | Aperio, Technologies Inc., Vista, CA, USA | n/a | Scanning of H & E slides and analysis |
| Cetrimide agar plates | BD, USA | 285420 | Selective media plates for Pseudomonas aeruginosa growth |
| Copper rods | n/a | n/a | Used to induce the burn injury |
| Cotton tipped applicators | OMEGA Surgical supply, USA | 4225-IMC | Used to apply eye ointment |
| Electric shaver | Oster, USA | Golden A5 | Used to remove the dorsal side hairs |
| Eye lube | Dechra, UK | n/a | The eye wetting agent to provide long lasting comfort and avoid eye dryness |
| Fluff filled underpads | Medline, USA | MSC281225 | Used in the burn procedure |
| Forcep | F.S.T. | 11027-12 | Used to hold the skin piece |
| Gauze sponges | Oasis, USA | PK412 | Used to clean the applied nair cream from the dorsal side |
| Heat-resistant gloves | n/a | n/a | Used to hold the heated copper rods |
| Hematology Analyzer | IDEXX laboratories, USA | ProCyte Dx | |
| Induction chamber | Kent Scientific, USA | vetFlo-0730 | Used to anesthesize the animals |
| Insulin syringe | BD, USA | 329461 | |
| Isoflurane | Pivetal, USA | NDC46066-755-04 | Used to anesthesized rats to induce a loss of consciousness |
| Isoflurane vaporiser | n/a | n/a | |
| Lactated ringer's solution | icumedical, USA | NDC0990-7953-09 | Used to resuscitate the rats |
| L-shaped spreader | Fischer Scientific, USA | 14-665-230 | |
| Mannitol Agar | BD, USA | 211407 | Selective media plates for Staphylococcus aureus growth |
| Minicollect tubes (K2EDTA) | greiner bio-one, USA | 450480 | Used to collect the blood |
| Morphine | Mallinckrodt, UK | NDC0406-8003-30 | This analgesia was used to induce the inability to feel burn injury pain |
| Muller Hinton Broth | BD, USA | 275730 | |
| Muller Hinton II Agar | BD, USA | 211438 | |
| Nair hair removal lotion | Nair, USA | n/a | Used to remove the residual hairs on dorsal side |
| Needle 23 G | BD, USA | 305193 | Used to inject the lactate ringer solution |
| Normal saline | n/a | n/a | |
| Spectrophotometer | ThermoScientific, USA | Genesys 30 | |
| Sprague-Dawley rats, male and female | Charles River Labs | n/a | 7-9 weeks old for burn induction |
| Surgical Scissor | F.S.T. | 14501-14 | Used to cut the desired skin piece |
| Tissue collection tubes | Globe Scientific | 220101236 | |
| Tissue Homogenizer | Kinematica, Inc, USA | POLYTRON PT2100 | Used to homogenize the tissue samples |
| Water bath | Fischer Scientific, USA | n/a | Used to induce the burn injury |
| Weighted heating pad | Comfytemp, USA | n/a | Used during the procedure to keep rat's body warm |
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Cite This Article
Sharma, R., Yeshwante, S., Vallé, Q., Hussein, M., Thombare, V., McCann, S. M., Maile, R., Li, J., Velkov, T., Rao, G. Rat Burn Model to Study Full-Thickness Cutaneous Thermal Burn and Infection. J. Vis. Exp. (186), e64345, doi:10.3791/64345 (2022).