Thiele Jan R, Goerendt Kurt, Stark G Bjoern, Eisenhardt Steffen U
Department of Plastic and Hand Surgery, University of Freiburg Medical Centre, USA.
J Vis Exp. 2012 Aug 5(66):e3973. doi: 10.3791/3973.
Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery. Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion. Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor. While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM). Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968. Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality. We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method. In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.
缺血再灌注损伤（IRI）与一系列病理状况有关，如脑卒 中、心肌梗死、肠道缺血以及移植和心血管手术后的情况。对先前缺血组织进行再灌注虽然对于预防不可逆组织损伤至关重要，但会引发受影响组织的过度炎症。除了产生活性氧、补体系统激活和微血管通透性增加外，白细胞的激活是再灌注期间炎症组织损伤病理级联反应的主要因素之一。白细胞激活是一个多步骤过程，包括滚动、牢固黏附和迁移，由黏附分子之间的复杂相互作用介导，以响应补体因子、趋化因子或血小板活化因子等化学引诱剂。虽然毛细血管后微静脉中的白细胞滚动主要由选择素与其相应配体的相互作用介导，但白细胞与内皮的牢固黏附是通过与细胞间黏附分子（ICAM）和血管细胞黏附分子（VCAM）结合而由选择素控制的。体内观察白细胞与内皮相互作用的金标准是活体显微镜技术，该技术于1968年首次描述。尽管已经针对各种器官描述了多种IRI（缺血再灌注损伤）模型，但只有少数模型适合在高质量图像水平上直接观察微血管床中的白细胞募集。我们在此推广大鼠提睾肌微循环中毛细血管后微静脉的数字活体落射荧光显微镜检查，作为定性和定量分析横纹肌组织中IRI研究中白细胞募集的便捷方法，并提供完成该技术的详细手册。我们进一步说明了常见的陷阱并提供了有用的提示，使读者能够真正理解并安全地执行该方法。在一个逐步的方案中，我们描述了如何在充分监测下开始进行呼吸控制麻醉，以使动物长时间保持深度麻醉。然后，我们将提睾肌制备成薄平板以获得出色的光学分辨率，并提供我们实验室中已建立的IRI中白细胞成像方案。
