OPTICAL IMAGING OF LUNG CAPILLARIES IN VIVO

体内肺毛细血管的光学成像

• 批准号: 7105064
• 负责人: Jahar Bhattacharya
• 金额: $ 31.81万
• 依托单位: ST. LUKE'S-ROOSEVELT INST FOR HLTH SCIS
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-22 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7105064
• 关键词: bioimaging /biomedical imaging; calcium flux; calcium indicator; capillary; cardiovascular imaging /visualization; cell migration; free radical oxygen; immunofluorescence technique; inflammation; laboratory rat; leukocyte adhesion molecules; lung; mitochondria; selectins; technology /technique development; tumor necrosis factor alpha; vascular endothelium

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.

描述（由申请人提供）： 目的：我们的总体目标是了解完整肺毛细血管中以空间分布、节段特异性方式发展的促炎症反应的调节。在这里，我们将在体内确定距离肺胸膜表面不同深度的毛细血管的这种调节。具体来说，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缘化的假设。具体目标是首次量化胸膜不同深度的毛细血管、内皮 (EC) 线粒体 (Ca2+mit) 和胞质 Ca 2+ (Ca2+cyt) 的调节（具体目标 1）、EC 的生成线粒体活性氧 (ROS)（具体目标 2）和线粒体介导的白细胞边缘化（具体目标 3）。 程序：(1)形态测量。将使用双光子显微镜在大鼠肺毛细血管中进行线粒体和内体 Ca 2+ 储存 (ER) 的活体成像。 (2)Ca2+定量。 Ca2+mit、Ca2+细胞和 ER Ca 2+ 变化将使用针对适当区室的荧光团来确定。 (3)ROS定量。 EC ROS 的产生将使用我们报告的方案，使用 ROS 指示剂二氯荧光素的荧光成像来确定。 (4)免疫荧光成像。毛细血管中 P-选择素的表达将使用间接原位免疫荧光成像来确定。 (5)白细胞边缘化。将使用用罗丹明6G标记的白细胞的两个光子显微镜来确定白细胞边缘化和迁移。 (6)Ca 2+ 增加。 EC Ca 2+ 将通过(a) 激动剂的输注和(b) 原位光激发细胞内解笼来增加。反应将根据增加 ROS 产生和白细胞边缘化的线粒体机制来确定。 意义：该提案提出了对胸膜不同深度毛细血管肺部炎症生物学的新认识。该计划是为了在原位单个内皮细胞水平上实现对肺毛细血管反应的全面了解。了解线粒体的作用非常重要，因为线粒体机制和线粒体 ROS 可能关键调节白细胞边缘化，从而调节肺损伤。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症反应。