实时数字成像大鼠提睾肌缺血再灌注损伤（IRI）的白细胞 - 内皮细胞相互作用
Summary
数字提睾微循环毛细血管静脉活体荧光显微镜是一种方便的方法获得白细胞 – 内皮细胞相互作用的见解<em>在体内</em在缺血再灌注损伤（IRI）的横纹肌组织。我们在这里提供一份详细的协议，安全地执行技术，并讨论了它的应用和限制。
Abstract
在病理条件下，如脑卒中，心肌梗死，肠缺血，以及移植和心血管手术。1以前缺血组织的再灌注后的大阵，而必要的预防不可逆转的缺血再灌注损伤（IRI）有牵连组织损伤，诱发过度炎症反应受影响的组织。毗邻的活性氧，活化补体系统的生产和增加血管通透性，激活白细胞再灌注过程中的病理级联在炎症组织损伤的原则演员之一。2，3白细胞激活是一个多步过程组成轧制，坚定的粘附和轮回之间复杂的相互作用的粘附分子介导的反应，如补体因子，趋化因子，血小板活化因子的趋化因子。
<虽然在毛细血管静脉的白细胞滚动主要选择素5的相互作用介导，与他们的柜台配体，坚定白细胞粘附血管内皮细胞是通过粘附分子（ICAM）与血管细胞结合的选择控制类p =“的jove_content”>粘附分子（VCAM），6，7在体内的白细胞-内皮细胞相互作用观察的金标准是活体显微镜技术，首次在1968年8。
虽然各个器官缺血再灌注损伤（缺血再灌注损伤）的各种型号，9-12只有少数是适合微血管床的白细胞招聘的直接可视化的图像质量高的水平上。
在这里，我们促进提睾微循环的毛细血管后微静脉的活体荧光显微镜的数字作为一种方便的方法大鼠IRI的研究，定性和定量分析白细胞招聘横纹肌组织，并为实现该技术提供了一个详细的手册13。我们进一步说明常见的陷阱，应该使读者能够真正体会，安全地执行方法，并提供有用的提示。
在由步步协议，我们描述如何获得足够的监测，保持时间较长的动物坚决麻醉呼吸控制麻醉下开始。然后，我们描述优秀的光学分辨率为薄平板提睾准备和提供已在我们的实验室建立在IRI白细胞成像的协议。
Protocol
1。麻醉和监测适当的国家和机构伦理应该在之前进行动物实验。继批准从伦理委员会麻醉男性只SD大鼠的体重从120 – 180克。提供2 – 3％异氟醚，异氟醚蒸发器和一个有机玻璃箱，通过放置在大鼠内。 尽快达到适当的麻醉水平（缺乏反应脚趾或尾巴捏）大鼠加权和腹侧颈区剃光。 放在一个加热垫在背卧鼠体温保持在37°C和异氟醚适用于2卷用硅胶面具％。 <p class="jove_co…
Discussion
白细胞-内皮细胞相互作用，生产活性氧和激活补体系统是IRI的诱导组织功能障碍的主要特点。26受影响组织的微循环被视为不可分割的炎症发病网站。除了 如流室检测27，28的 体外实验，它是强制性的，以提供行之有效的活体成像模型，以进一步评估在体内相关。虽然已在不同的器官系统有牵连IRI的，我们在这里描述的方法系统地研究白细胞与内皮细胞相互作用…
Disclosures
The authors have nothing to disclose.
Acknowledgements
授予的“德意志研究联合会”（EI 866/1-1）苏艾森哈特支持这项工作。
Materials
| Name of the equipment: | Company: | Catalogue No.: | Comments: |
| Forene 100% (V/V) | Abbot | B506 | API isoflurane |
| Terylene Suture | Serag Weissner | OC108000 | |
| Portex Fine Bore Polythene Tubing | Smiths Medical | 800/100/100 | 0.28 mm inner Diameter |
| 0,9% saline solution | Fresinus Kabi | 808771 | |
| Change-A-tip deluxe cautery kit | Bovie Medical | DEL1 | |
| Abbocath -T 14G | Venisystems | G713 – A01 | used as lens tube |
| Servo Ventilator 900C | Maquet | used as animal ventialtor | |
| Logical pressure transducer | Smiths Medical | MX1960 | |
| Sirecust 404 Monitor | Siemens | ||
| ABL 700 Benchtop Analyzer | Radiometer | for blood gas measurement | |
| Heating pad | Effenberger | 8319 | |
| Aluminum stage | Alfun | AW7022 | |
| Surgical microscope OPMI 6-SDFC | Carl Zeiss | ||
| Microsurgical instruments lab set | S&T | 767 | |
| Biemer vessel clip | Diener | 64.562 | |
| Applying forceps | Diener | 64.568 | for Biemer vessel clip |
| Rhodamine 6G | Sigma-Aldrich | R4127 | |
| Vaseline white DAB | Winthrop | 2726853 | |
| Cover glasses | 32×32 mm | ||
| Intravital setup | |||
| Zeis Axio Scope A-1 MAT | Carl Zeis | 490036 | epifluorescence microscope |
| 470 nm LED | Carl Zeis | 423052 | fluorescence light source |
| Colibri 2 System | Carl Zeis | 423052 | |
| W Plan-Apochromat 20x/1,0 DIC | Carl Zeis | 421452 | water immersion objective |
| AxioCam MRm Rev. 3 FireWire | Carl Zeis | 426509 | high resolution digital camera |
| Axio vision LE software | Carl Zeis | 410130 | use for offline analysis |
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Tags
Real-timeDigital ImagingLeukocyte-endothelial InteractionIschemia-reperfusion InjuryIRIRat Cremaster MusclePathological ConditionsCerebral StrokeMyocardial InfarctionIntestinal IschemiaTransplantCardiovascular SurgeryReactive Oxygen SpeciesComplement SystemMicrovascular PermeabilityLeukocyte ActivationAdhesion MoleculesChemoattractantsSelectinsCounter LigandsIntercellular Adhesion MoleculesVascular Cellular Adhesion MoleculesIntravital Microscopy
Cite This Article
Thiele, J. R., Goerendt, K., Stark, G. B., Eisenhardt, S. U. Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle. J. Vis. Exp. (66), e3973, doi:10.3791/3973 (2012).