CELLULAR MODULATION OF MICROVESSEL PERMEABILITY IN VIVO

描述（由申请人提供）：白细胞粘附已被认为是炎症期间导致器官功能障碍的关键事件。然而，实验支持白细胞粘附和微血管通透性增加之间的联系一直不一致。我们的初步研究表明，白细胞作为单个细胞的粘附不会导致微血管通透性增加。相反，我们发现白细胞-血小板聚集和聚集性粘附产生了长期的渗透性增加。该项目的总体目的是阐明白细胞募集和通透性增加之间的相互作用。我们建议使用一种新开发的方法，将单个微血管灌注与自体血液灌注相结合，来确定急性炎症期间通透性增加的启动步骤和关键因素。当内皮与血细胞元素直接相互作用时，这种方法可以研究微血管通透性的调节机制，并且在控制良好的实验条件下，还可以保留单血管灌注的能力，以精确测量血管通透性。拟议的研究是为了检验两个假设。1)血小板/白细胞聚集体的形成和聚集体释放的药物对于白细胞依赖性微血管通透性的增加至关重要，而活化的血小板起着核心作用；2)白细胞/血小板/内皮细胞相互作用或炎症介质诱导的微血管通透性增加是通过细胞cAMP水平降低的机制发生的。全身和局部应用细胞因子和/或炎症介质与自体血液灌注会诱导白细胞粘附和迁移以及白细胞/血小板/内皮相互作用。渗透性的变化将通过对同一微血管中白细胞或白细胞/血小板聚集粘附前后的水力电导率或溶质渗透系数的成对测量来确定。为了将功能研究与形态学变化联系起来，在研究渗透性的相同实验条件下，将使用体内银染色、共聚焦显微镜免疫荧光染色和电子显微镜相结合来检查内皮细胞之间的连接变化、粘附和迁移白细胞的位置以及微血管壁中粘附分子的相应空间分布。我们新开发的实验方法克服了以前体内方法的某些局限性，并将推进该领域的知识。

DESCRIPTION (provided by applicant): Leukocyte adhesion has been implicated as the critical event leading to organ dysfunction during inflammation. However, the experimental support for the linkage between leukocyte adhesion and increased microvessel permeability has been inconsistent. Our preliminary studies suggest that leukocyte adhesion as single cells does not result in increased microvessel permeability. Instead, we found that leukocyte-platelet aggregation and aggregate adhesion produced a prolonged permeability increase. The overall aim of this project is to clarify the interaction between leukocyte recruitment and permeability increase. We propose to identify the initiating steps and critical factors contributing to increased permeability during acute inflammation using a newly developed method that combines single microvessel perfusion with autologous blood perfusion. This approach allows the mechanisms that regulate microvessel permeability to be studied when endothelium interacts directly with blood cell elements, and also retains the capability of single vessel perfusion for precise measurements of vascular permeability under well-controlled experimental conditions. The proposed research is to test two hypotheses. 1) The formation of platelet/leukocyte aggregates and agents released from the aggregates are critical for leukocyte-dependent increases in microvessel permeability and the activated platelets play a central role, and 2) the increased microvessel permeability induced by either leukocyte/platelet/endothelial cell interactions or inflammatory mediators occurs through a mechanism that involves decreased cellular cAMP levels. Leukocyte adhesion and migration and leukocyte/platelet/endothelium interactions will be induced by systemic and local application of cytokines and/or inflammatory mediators with autologous blood perfusion. Changes in permeability will be determined by paired measurements of hydraulic conductivity, or solute permeability coefficient before and after leukocyte or leukocyte/platelet aggregate adhesion in the same microvessel. To correlate functional studies with morphological changes, a combination of in vivo silver staining, immunofluorescence staining with confocal microscopy, and electron microscopy will be used to examine the junction changes between endothelial cells, the location of adherent and transmigrating leukocytes and the corresponding spatial distribution of adhesion molecules in microvessel walls under the same experimental conditions whereby permeability is studied. Our newly developed experimental approach overcomes certain limitations of previous in vivo methods and will advance the knowledge in the field.