炎症敏感晚期缺氧缺血性脑损伤的Ferret模型
Summary
该方法描述了P17雪铁龙中发炎意识低氧缺血和高氧脑损伤，以模拟一些晚期早产儿经历的长期炎症和氧化性脑损伤之间的复杂相互作用。
Abstract
目前需要临床上相关的围产期感染和缺氧-缺血（HI）模型，以测试早产症患者的治疗干预措施。铁杉是模拟早产儿大脑的理想候选者，因为它们出生时是脑电，产后发育有陀螺脑。出生时，雪铁龙大脑发育类似于13周的人类胎儿，产后（P）17个试剂盒被认为相当于妊娠32-36周的婴儿。我们描述了P17雪铁龙中的损伤模型，其中脂多糖组分后为双边脑缺血、缺氧和高血糖。这模拟了许多出现脑损伤的新生儿经历的长期炎症、缺血、缺氧和氧化应激的复杂相互作用。受伤的动物表现出一系列严重的损伤严重性，大脑中的形态变化包括缩小多个皮质陀螺和相关的硫化物。受伤的动物也表现出缓慢的反射发育，在自动走道中运动速度较慢和变速，在开阔的田野中减少探索。该模型提供了一个平台，用于测试与炎症和HI相关的新生儿脑病婴儿的假定疗法，研究影响皮质发育的损伤机制，并研究在不受影响的动物。
Introduction
目前需要大型动物模型，以反映早产和围产期缺氧-缺血症的病理生理学，在其中可以测试婴儿的治疗干预措施。2017年，在美国出生的382，726名婴儿中，9.93%是早产儿，其中84%的婴儿在妊娠32至36周之间出生。在早产儿中，围产期接触感染或炎症很常见，其中由于病毒或细菌病原体而使产妇免疫活化可以启动早产。产后，早产儿有早或晚患败血症的高风险2。早产儿也经常经历缺氧、低血压和高血糖期，原因是其心肺系统不成熟，大气中的氧气紧张程度高于子宫内的婴儿，以及异体性暴露。此外，在早产儿中，抗氧化防御是不成熟的3，亲凋亡因子自然调节4。氧化应激和细胞死亡导致免疫系统的激活和神经炎症。这些综合因素被认为有助于大脑的发育和生理脆弱性，并导致或加剧与早产儿5、6、7发育不良结果相关的脑病。
由于雪铁龙大脑与人脑在物理和发育上的相似性，雪铁龙是一种有吸引力的物种，可以模拟脑损伤8、9、10、11、12。Ferrets也是模拟早产儿大脑的理想候选者，因为它们出生时是脑电，产后发育有陀螺的大脑，这为发育中的大脑暴露在侮辱中提供了一个窗口，模仿早产儿经历的侮辱。出生时，雪铁龙大脑发育类似于13周的人类胎儿，产后（P）17试剂盒被认为相当于妊娠13周32-36周的婴儿。
我们小组最近发布了一个模型，在P10雪铁龙中，通过结合炎症敏感性与大肠杆菌脂多糖（LPS）与随后接触缺氧和高血糖12的异常早产（<28周妊娠）脑损伤。在下面的协议中，我们现在描述了P17雪铁龙的晚期早产模型，LPS敏化之后是双边脑缺血、缺氧和高血糖。这导致一部分动物的损伤更为严重，并更密切地模拟了一些先期婴儿出现脑损伤的长期炎症、缺血、缺氧和氧化应激的复杂相互作用。
Protocol
程序是按照NIH《实验室动物护理和使用指南》和华盛顿大学机构动物护理和使用委员会批准的协议的一部分进行的。 1. 准备和LPS管理 注：有关过程的时间表，请参阅图 1。 在开始手术之前，密封、消毒和高压灭菌所有手术器械和手术窗帘。在无菌小瓶中准备术前药物。计算在 8–10 分钟内用实验气体替换缺氧/高…
Representative Results
在受伤群体中，来自6个垃圾中的34只（n = 18男，n=16雌性）动物中，有8只动物（24%;n = 4雄，n=4雌性）在第二个缺氧期（n = 5），在温度管理期间（n = 2），或在侮辱后过夜（n = 1）。在受伤群体中，26名幸存者中有9人（35%）有明显的严重伤害。5只动物（n = 5只雄性）有中度损伤，4只动物（n = 2雄，n =2雌性）有严重伤害，定义为病理评分分别为2⁄5和6+9（图2A）。因?…
Discussion
由于雪铁龙大脑和人脑之间的物理和发育相似性，雪铁龙正越来越多地被用来模拟成人和发育性脑损伤。8，9，10，11，12。然而，迄今为止的研究表明，雪铁龙大脑既对初始损伤有抵抗力，而且具有高度可塑性，行为缺陷会随着时间而减少，即使在可见的病理损…
Disclosures
The authors have nothing to disclose.
Acknowledgements
该模型的开发由比尔和梅林达·盖茨基金会以及NIH赠款5R21NS093154-02（NICHD）资助。
Materials
| 80% Oxygen | Praxair | ||
| 9% Oxygen | Praxair | ||
| Absorbent benchtop protector | Kimtech | 7546 | |
| Automated catwalk | Noldus | ||
| Betadine surgical scrub | |||
| Bupivacaine | Patterson Veterinary | 07-888-9382 | |
| Buprenorphine | |||
| Calipers | SRA Measurement Products | ME-CAL-FP-200 | 200mm range, .01 mm resolution |
| Cotton Gauze Sponge | Fisher Scientific | 22028556 | |
| Curved fine hemostat | Roboz | RS-7101 | |
| Curved forceps | World Precision Instruments | 501215 | |
| Curved suture-tying hemostat | Roboz | RS-7111 | |
| Ethovision tracking software | Noldus | ||
| Eye Lubricant | Rugby | NDC 0536-1970-72 | |
| Ferrets (Mustela putorius furo) | Marshall Biosciences | Outbred (no specific strain) | |
| Formalin | Fisher Scientific | SF100-4 | 10% (Phosphate Buffer/Certified) |
| Hair Clippers | Conair | GMT175N | |
| Insulin Syringes | BD | 329461 | 0.3 cc 3 mm 31G |
| Isoflurane | Piramal | 66794-017-25 | |
| Lidocaine | Patterson Veterinary | 07-808-8202 | |
| LPS | List Biological | LPS Ultrapure #423 | |
| Oxygen sensor | BW Gas Alert | GAXT-X-DL-2 | |
| Pentobarbital | |||
| Plastic chamber | Tellfresh | 1960 | 10L; 373x270x135mm |
| Saline Solution, 0.9% | Hospira | RL-4492 | |
| Scalpel blade | Integra Miltex | 297 | |
| Scalpel handle | World Precision Instruments | 500236 | #3, 13cm |
| Sterile suture | Fine Science Tools | 18020-50 | Braided Silk, 5/0 |
| Surgical clip applicator | Fine Science Tools | 12020-09 | |
| Surgical clip remover | Fine Science Tools | 12023-00 | |
| Surgical drapes | Medline Unidrape | VET3000 | |
| Surgical gloves | Ansell Perry Inc | 5785004 | |
| Surigical clips | Fine Science Tools | 12022-09 | |
| Thermometer (rectal) | YSI | Precision 4000A | |
| Thermometer (water) | Fisher Scientific | 14-648-26 | |
| Umbilical tape | Grafco | 3031 | Sterile |
| Water bath | Thermo Scientific | TSCOL19 | 19L |
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Cite This Article
Wood, T., Moralejo, D., Corry, K., Fisher, C., Snyder, J. M., Acuna, V., Holden-Hunt, A., Virk, S., White, O., Law, J., Parikh, P., Juul, S. E. A Ferret Model of Inflammation-sensitized Late Preterm Hypoxic-ischemic Brain Injury. J. Vis. Exp. (153), e60131, doi:10.3791/60131 (2019).