COBRE: UNV MED SCH: CORE D: DYNAMIC IMAGING FACILITY

COBRE：UNV MED SCH：核心 D：动态成像设施

• 批准号: 7382024
• 负责人: Terence Keith Smith
• 金额: $ 16.95万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7382024
• 关键词: COBRE; UNV; MED; SCH; CORE

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Core D will support Projects 1, 2 and 4 requiring expertise in fluorescent imaging. This core has purchased two fluorescent high speed imaging systems that can be used for examining activity in both intact tissues and in isolated cells. One imaging set up is based around an upright microscope (Nikon TS100) with water immersion lenses and has a wavelength switcher (DG-5) for ratio-metric (Fura 2-AM) imaging. The latter allows for calibrating calcium activity in pressurized blood vessels, cells in intact tissues and in isolated cells. The second imaging system is based around an inverted microscope and incorporates an electrophysiology set up. This system is used for determining the relationship between voltage and fluorescent calcium signals. Both systems use a highly sensitive back illuminated camera (Cascade 512). These imaging systems will support projects that need to examine the spread of intracellular and intercellular calcium waves that regulate the membrane potential and contractile activity respectively of the smooth muscle layers of the intact but isolated fundus and colon. Project 2 will examine whether these waves are modified PLB knockout mice compared to their wild type littermates. It will also be determined whether CaMKII inhibitors affect the propagation and duration of these waves. Project 4 will use this apparatus to examine the changes in the spread of intracellular and intercellular calcium waves in the smooth muscle layers from hypertrophied regions of bowel. In addition, Project 4 will use confocal calcium imaging to determine if changes occur to the spread of activity through the neurons of the enteric nervous system. This is possible because [Ca 2+]I is a reliable indicator of action potential dependent changes in the activity of enteric neurons. Project 4 will also use this technology in combination with patch clamp studies to determine if there are changes in the ionic conductances underlying spontaneous Ca2+ transients (Sparks and Puffs) in isolated smooth muscle cells taken from different regions of bowel with a partial obstruction. Project 1 will utilize the ratio-metric/pressurized vessel imaging system to determine whether Ca2+ signaling and vascular reactivity are altered in arteries from mdx mice lacking dystrophin compared to their wild type litter mates. In addition, single cell Ca2+ imaging experiments combined with patch clamp techniques will be utilized to provide a detailed understanding of how integrins affect Ca2+ signaling and currents in isolated vascular smooth muscle cells. Ca2+-permeable (L-type and nonselective) channels will be compared in isolated myocytes from mdx and wildtype mice.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。核心D将支持需要荧光成像专业知识的项目1、2和4。该中心购买了两套荧光高速成像系统，可用于检查完整组织和分离细胞的活性。一种成像设置是基于具有水浸透镜的直立显微镜（Nikon TS 100），并且具有用于比率度量（Fura 2-AM）成像的波长切换器（DG-5）。后者允许校准加压血管、完整组织中的细胞和分离细胞中的钙活性。第二个成像系统是基于周围的倒置显微镜，并纳入了电生理设置。该系统用于确定电压和荧光钙信号之间的关系。两种系统都使用高灵敏度背照式摄像机（Cascade 512）。这些成像系统将支持需要检查细胞内和细胞间钙波传播的项目，这些钙波分别调节完整但分离的胃底和结肠平滑肌层的膜电位和收缩活动。项目2将检查这些波是否是修饰的PLB基因敲除小鼠相比，其野生型同窝出生。还将确定CaMKII抑制剂是否影响这些波的传播和持续时间。项目4将使用该仪器来检查肠肥大区域平滑肌层中细胞内和细胞间钙波传播的变化。此外，项目4将使用共聚焦钙成像来确定活动通过肠神经系统的神经元的传播是否发生变化。这是可能的，因为[Ca 2+]I是肠神经元活动中动作电位依赖性变化的可靠指标。项目4还将使用该技术与膜片钳研究相结合，以确定从部分梗阻的肠不同区域采集的分离平滑肌细胞中自发性Ca 2+瞬变（火花和抽吸）的离子电导是否发生变化。项目1将利用比率度量/加压血管成像系统来确定与野生型同窝小鼠相比，缺乏肌营养不良蛋白的mdx小鼠的动脉中Ca 2+信号传导和血管反应性是否发生改变。此外，单细胞钙离子成像实验结合膜片钳技术将被用来提供一个详细的了解整合素如何影响钙离子信号和电流在孤立的血管平滑肌细胞。将在mdx和野生型小鼠的分离肌细胞中比较Ca 2+渗透性（L型和非选择性）通道。