OPTICAL IMAGING OF LUNG CAPILLARIES IN VIVO

描述(由申请人提供)： 目的：我们的总体目标是了解在完整的肺毛细血管中以空间分布、节段性方式发展的促炎反应的调节。在这里，我们将在活体内确定肺胸膜表面不同深度的毛细血管中的这种调节。具体地说，我们将检验线粒体机制调节肺泡间隔毛细血管中白细胞边际的假设。具体目标是首次定量测定不同深度的毛细血管内皮细胞线粒体和胞浆钙离子的调节(特异性目标1)、内皮细胞线粒体活性氧(ROS)的产生(特异性目标2)和线粒体介导的白细胞边际化(特异性目标3)。 操作步骤：(1)形态测量。线粒体和内体钙储存(ER)的活体成像将在大鼠肺的毛细血管中进行，使用双光子显微镜。(2)钙离子定量。钙离子、钙离子细胞和内质网钙离子的变化将使用以适当隔室为靶点的荧光团来确定。(3)ROS量化。EC ROS的产量将使用ROS指示剂二氯荧光素的荧光成像来确定，使用我们报告的方案。(4)免疫荧光成像。P-选择素在毛细血管中的表达将通过间接原位免疫荧光成像进行检测。(5)白细胞边集。用罗丹明6G标记的白细胞的双光子显微镜来确定白细胞的边际和迁移。(6)钙离子升高。通过(A)注入激动剂和(B)光刺激细胞内去势，可增加细胞内钙离子浓度。反应将根据增加ROS产生和白细胞边际的线粒体机制来确定。 意义：这项建议提出了一种新的理解，即在与胸膜不同深度的毛细血管中肺部炎症的生物学。该计划是在原位单个内皮细胞的水平上实现对肺毛细血管反应的全球理解。了解线粒体的作用很重要，因为线粒体机制和线粒体ROS可能关键地调节白细胞的边际作用，从而导致肺损伤。线粒体ROS也可能参与信号基因转录，从而参与肺重塑。如果初步数据成立，那么这项研究将首次证明线粒体机制调节特定的促炎反应。

DESCRIPTION (provided by applicant): Aims: Our overall objective is to understand the regulation of pro-inflammatory responses that develop in a spatially distributed, segment specific manner in the intact lung capillaries. Here, we will determine this regulation in capillaries at different depths from the pleural surface of the lung in vivo. Specifically, we will test the hypothesis that mitochondrial mechanisms regulate leukocyte margination in alveolar septal capillaries. The specific aims are to quantify for the first time in capillaries at different depths from the pleura, regulation of endothelial (EC) mitochondrial (Ca2+mit) and cytosolic Ca 2+ (Ca2+cyt) (Specific Aim 1), generation of EC mitochondrial reactive oxygen species (ROS) (Specific Aim 2), and mitochondria-mediated leukocyte margination (Specific Aim 3). Procedures: (1) Morphometric measurements. Intravital imaging of mitochondrial and endosomal Ca 2+ stores (ER) will be conducted in capillaries of the rat lung, using two-photon microscopy. (2) Ca2+quantification. Ca2+mit, Ca2+cyto and ER Ca 2+ changes will be determined using fluorophores that target the appropriate compartment. (3) ROS quantification. EC ROS production will be determined using fluorometric imaging of the ROS indicator dichloro fluorescin using our reported protocols. (4) Immunofluorescence imaging. Expression of P-selectin in capillaries will be determined using indirect in situ immunofluorescence imaging. (5) Leukocyte margination. Leukocyte margination and migration will be determined using two photon microscopy of leukocytes labeled with, rhodamine 6G. (6) Ca 2+ increase. EC Ca 2+will be increased by (a) infusion of agonists and (b) in situ photo-excited intracellular uncaging. Responses will be determined in terms of mitochondrial mechanisms that increase ROS production and leukocyte margination. Significance: This proposal addresses a new understanding of the biology of lung inflammation in capillaries at different depths from the pleura. The plan is to achieve global understanding of lung capillary responses at the level of the single endothelial cell in situ. It is important to understand the role of mitochondria, as mitochondrial mechanisms and mitochondrial ROS may critically regulate leukocyte margination and hence, lung injury. Mitochondrial ROS may also be involved in signaling gene transcription and consequently, lung remodeling. If the preliminary data hold, then this research will prove for the first time that mitochondrial mechanisms regulate specific pro-inflammatory responses.