活体显微镜及血栓诱导在无毛小鼠的耳垂
Summary
无毛SKH1-HR hr小白鼠的耳朵模型使而不在检查微血管床之前手术准备微循环和光毒性血栓诱导的活体荧光显微镜。因此，无毛小鼠的耳朵是一个极好的体内模型微血管血栓形成，血栓进化，与溶栓期间学习复杂的相互作用。
Abstract
血管疾病的血栓并发症是工业国家的发病率和死亡率的一个主要原因。由于在血栓形成的细胞和非细胞的血液成分之间复杂的相互作用，生理学和血栓形成的病理生理学的研究可靠只能在体内进行。因此，本文提出了在无毛小鼠耳模型，并专注于微循环，血栓形成，血栓和进化的体内分析。通过使用活体荧光显微镜和相应的荧光染料的静脉内（iv）应用程序，能够容易地进行微循环在耳廓的重复分析，而不需要外科手术制备。此外，该模型可以适合于体内的不同问题的研究，包括伤口愈合，再灌注损伤，或血管发生。综上所述，无毛小鼠的耳朵对于在利沃夫的理想模型在生理学或病理生理学条件和它的不同的全身或局部治疗反应的评估皮肤微循环邻研究。
Introduction
本制品的目的是描述施加到无毛小鼠的耳廓用于直接观察和微循环的分析，血栓形成，和血栓进化活体显微镜的技术。随着1在1000的发病率，静脉血栓形成仍是发病的常见原因。虽然诊断，预防策略和治疗已在近几年得到了发展，静脉血栓形成的三分之一表现为肺动脉栓塞1。动脉血栓形成在心血管疾病，这是在工业化国家死亡的最常见原因至关重要的作用。基于粥样硬化斑块的破裂动脉血栓形成涉及心脏发作，肠系膜梗死，中风和。每个手术暴露属下的结构中血液成分，改变血流动力学，并固定了病人。在下肢，器官的T人工关节手术ransplantation和皮瓣手术血栓并发症的常见原因。尤其是微血管血栓形成经常导致不可逆的损害，因缺乏临床症状。同样，微血管栓塞起着至关重要的规则在几种疾病，包括血栓性血小板减少性紫癜，败血症，弥漫性血管内凝血，抗磷脂综合征，慢性静脉功能不全，等等。
用于治疗和预防血栓形成的一些新的药物，近年来被开发，但抗血小板药物和抗凝药物仍然有副作用，缺乏拮抗剂，并配备持续时间长的效果。这些缺陷导致紧急医疗问题。因此，需要更多的研究来发现血栓形成，这很难在体外模拟过程中发生的复杂过程。
无毛SKH1-HR 人力资源小鼠在伦敦动物园里发现了1926年。由于14号染色体上的基因缺陷，动物失去了它的皮毛10日龄后，这使得对血管的活体显微镜访问的良好血管耳廓。耳的平均厚度为300μm。它由真皮的两层，其通过软骨隔开。对软骨的凸背侧，3个维管束进入耳垂。心尖血管弧和基础分流连接三个包。小静脉具有200微米（基础）和10微米（心尖）之间的直径。关闭网状毛细血管周围的空毛囊2。无毛SKH1-HR 人力资源鼠标的剖析，使耳廓血栓形成研究的强大和高性价比机型。
Protocol
所有的体内实验（7221.3-1-006 / 15）在符合有关动物保护和NIH指南实验动物的护理和使用（实验动物资源研究所，国家研究理事会）德国法律进行。 1.动物一般饲养执行与年龄4至6周雄性SKH1-HR 小时小鼠实验。使用动物用20和25克的重量。 保持动物在无病原体的设施和24至26℃下在标准条件和约60％相对湿度下，以稳定获得水和食物随意 。 <l…
Representative Results
在血栓形成大麻素治疗的影响在0.05 ml的FITC-葡聚糖的的注入，光毒性血栓诱导导致内皮损伤和血小板壁层插头的形成（ 图2和3）。在本研究中，血栓感应大麻素的腹膜内注射（5 mg / kg体重）或载体后导致所有小静脉血栓形成的血管闭塞（ 图4）。在载体处理的动物中，时间到血栓形成为430秒?…
Discussion
有对成功血栓诱导SKH1-HR 人力资源小鼠的耳垂几个关键步骤。进行故障排除，协议的各个步骤中括号表示。
考试条件是幼龄动物的理想在4岁 – 6周，与表皮的角化低。在较老的动物中，血管的可视化的质量要差少可比由于皮肤表面和靶血管（步骤1.1）之间的距离更高。
为了防止在考试区域FITC葡聚糖外渗，固定缝线必须尽可能勉强放置越好。在接?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者没有确认。
Materials
| SKH-1/hr mice | Charles River | 477 | can be purchased from other vendors |
| standard laboratory food | ssniff Spezialdiaeten | V1594-0 | can be purchased from other vendors |
| operation stereomicroscope | Leica | M651/M655 | can be purchased from other vendors |
| intravital microscope | Zeiss | Axiotech Vario 100 | can be purchased from other vendors |
| objective (20x/0.95) | Zeiss | 20x/0,50 W; Plan-NEOFLUAR | can be purchased from other vendors |
| objective (63x/0.95) | Zeiss | 63x/0,95 W; ACHROPLAN | can be purchased from other vendors |
| black and white CCD-camera | Pieper | FK 6990 IQ-S | can be purchased from other vendors |
| DVD-recorder | Panasonic | DMR-EX99V | can be purchased from other vendors |
| sodium chloride | Braun | 5/12612055/1011 | can be purchased from other vendors |
| Ketamine 10% | Bela pharm | F3901-6 | can be purchased from other vendors |
| Xylazine 2% | Bayer | 6293841.00.00 | can be purchased from other vendors |
| FITC-dextran 5% | Sigma | 46945-100MG-F | can be purchased from other vendors |
| dexapanthenol 5% eye ointment | Bayer | 6029009.00.00 | can be purchased from other vendors |
| formaldehyde 4% | Sigma | HT501128-4L | can be purchased from other vendors |
| DMSO | Sigma | 472301 | can be purchased from other vendors |
| coverslips 5 x 5 x 1 mm | Menzel | L4339 | can be purchased from other vendors |
| plasters | Leukosilk | 4683400 | can be purchased from other vendors |
| centrifuge | Beckman Coulter | CLGS 15 | can be purchased from other vendors |
| hematology analyzer | Sysmex | KX-21 A6980 | can be purchased from other vendors |
| EDTA-blood tube | Sarstedt | 201,341 | can be purchased from other vendors |
| cotton swabs | Sanyo | 604-A-1 | can be purchased from other vendors |
| infrared light | Beurer | 5/13855 | can be purchased from other vendors |
| single use synringe | Braun | 2020-08 | can be purchased from other vendors |
| insulin syringe | Braun | 9161502 | can be purchased from other vendors |
| disposable hypodermic needles | Braun | 465 7640 | can be purchased from other vendors |
| end-to-end capillary | Sarstedt | 19,447 | can be purchased from other vendors |
| heating plate | Klaus Effenberg | OP-T 185/03 | can be purchased from other vendors |
| scissors 14,5 cm | Aesculap | BC259R | can be purchased from other vendors |
| needle Holder | Aesculap | BM081R | can be purchased from other vendors |
| microforceps | Aesculap | BD331R | can be purchased from other vendors |
| microscissors | Aesculap | OC496R | can be purchased from other vendors |
| scalpel 21 | Dahlhausen | 11.000.00.511 | can be purchased from other vendors |
| Prolene 7-0 | Ethicon | XNEH7470 | can be purchased from other vendors |
| Prolene 6-0 | Ethicon | XN8706.P33 | can be purchased from other vendors |
| electrocautery | Servoprax | H40140 | can be purchased from other vendors |
| acrylglass pad | integrated heating, 0,5 cm high plane |
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Citer Cet Article
Strüder, D., Grambow, E., Klar, E., Mlynski, R., Vollmar, B. Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse. J. Vis. Exp. (122), e55174, doi:10.3791/55174 (2017).