In vivo two-photon imaging of brain vascular endothelial cell calcium dynamics

脑血管内皮细胞钙动力学的体内双光子成像

• 批准号: 8312965
• 负责人: Tyler Clark Brown
• 金额: $ 5.57万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8312965
• 关键词: Acetylcholine; Alzheimer' s Disease; Animals; Arteries; Astrocytes; Biological Models; Biological Neural Networks; Blood - brain barrier anatomy; Blood Circulation; Blood Vessels; Blood Volume; Blood flow; Brain; Brain imaging; Brain region; Calcium; Cell physiology; Cells; Communication; Data; Development; Diabetes Mellitus; Distal; Elements; Endothelial Cells; Epilepsy; Event; Exhibits; Failure; Functional disorder; Future; Health; Human; Hyperemia; Image; Imagery; In Vitro; Individual; Laboratories; Lead; Link; Measures; Methods; Molecular; Multiple Sclerosis; Mus; Neocortex; Network-based; Neuraxis; Neurons; Neurosciences; Organism; Pathway interactions; Photons; Physiological; Play; Population; Positioning Attribute; Process; Regulation; Relaxation; Research Training; Role; Second Messenger Systems; Sensory; Signal Transduction; Site; Smooth Muscle; Stroke; Testing; Time; Tissues; Training; Travel; Vascular Endothelial Cell; Vascular Endothelium; Vascular blood supply; Vasodilation; Vibrissae; arteriole; awake; barrel cortex; base; cell type; cholinergic; constriction; excitatory neuron; extracellular; hemodynamics; improved; in vivo; inflammatory pain; inhibitory neuron; mouse model; neocortical; nervous system disorder; neurovascular unit; novel therapeutic intervention; optogenetics; relating to nervous system; response; second messenger; selective expression; somatosensory; two-photon

DESCRIPTION (provided by applicant): An intimate relationship exists between brain function and the brain vasculature. In the brain, the vasculature is engaged in active, bidirectional communication with neurons and astrocytes, collectively termed the neuro(glio)vascular unit. When local populations of neurons are active, coordinated activity in the neurovascular unit leads to increased blood flow and volume to the activated brain region, a process known as "functional hyperemia". Endothelial cells are a specialized cell-type located within vessel walls that play a crucial role in regulation of the vasculature. Perhaps most prominent is their role as the primary substrate of the blood-brain barrier (BBB). Disrupting vascular endothelial cell (vEC) physiology can lead to breakdown of the BBB and development of several pathological conditions, including diabetes, multiple sclerosis, inflammatory pain, Alzheimer's disease, epilepsy, and stroke. Calcium (Ca2+) is generally recognized as a universal second messenger that contributes to essential cellular signaling events in a broad range of tissues and organisms. Depletion of extracellular Ca2+ levels or conditions that lead to abnormally high or low intracellular Ca2+ concentrations in vECs can lead to alterations in vascular responses and eventually to a breakdown in the BBB. Despite the clear importance of vEC Ca2+ signaling to vascular dynamics, to our knowledge there has never been a study of vEC Ca2+ dynamics in the brain in vivo, nor any direct examination of the natural processes or specific cell types that drive vEC dynamics. The aims of this proposal are to test the hypothesis that Ca2+ dynamics are spontaneously expressed in vECs, driven by natural (sensory) input, and driven by local and distal neuromodulatory activation in vivo. To this end, we optimized two new methods that will allow visualization of vEC Ca2+ dynamics using two- photon imaging of genetically-encoded Ca2+ indicators in neocortical vECs in awake animals. Preliminary data using this approach has led to the characterization of ongoing basal vEC Ca2+ oscillations in the brain. Further, for the first time, we have found that vEC Ca2+ dynamics can be evoked by physiological sensory input to the mouse barrel cortex. We propose to extend these preliminary findings and use cell-type specific optogenetic activation to functionally dissect the precise cellular components within local somatosensory and distal neural networks that contribute to sensory-evoked vEC Ca2+ dynamics in the brain. PUBLIC HEALTH RELEVANCE: The blood-brain barrier is a crucial component of the central nervous system that restricts the passage of potentially harmful substances from the bloodstream into the brain. Breakdown of the blood-brain barrier can lead to the development of several pathological conditions, including diabetes, multiple sclerosis, inflammatory pain, Alzheimer's disease, epilepsy, and stroke. Gaining a better understanding of the individual cells that comprise the blood-brain barrier has the potential to uncover new therapeutic approaches to treating these conditions and improving human health.

描述（由申请人提供）：大脑功能和大脑脉管系统之间存在密切关系。在大脑中，脉管系统与神经元和星形胶质细胞进行主动的双向通信，统称为神经（胶质）血管单元。当局部神经元群活跃时，神经血管单元的协调活动会导致激活的大脑区域的血流量和容量增加，这一过程称为“功能性充血”。内皮细胞是位于血管壁内的一种特殊细胞类型，在脉管系统的调节中发挥着至关重要的作用。也许最突出的是它们作为血脑屏障（BBB）的主要基质的作用。破坏血管内皮细胞 (vEC) 生理机能会导致血脑屏障崩溃和多种病理状况的发展，包括糖尿病、多发性硬化症、炎性疼痛、阿尔茨海默病、癫痫和中风。钙 (Ca2+) 通常被认为是一种通用的第二信使，有助于多种组织和生物体中的重要细胞信号传导事件。细胞外 Ca2+ 水平的耗尽或导致 vEC 中细胞内 Ca2+ 浓度异常高或低的条件可能会导致血管反应的改变，并最终导致 BBB 崩溃。尽管 vEC Ca2+ 信号传导对血管动力学具有明显的重要性，但据我们所知，从未对大脑中的 vEC Ca2+ 动力学进行过体内研究，也没有对驱动 vEC 动力学的自然过程或特定细胞类型进行任何直接检查。该提案的目的是检验以下假设：Ca2+ 动态在 vEC 中自发表达，由自然（感觉）输入驱动，并由体内局部和远端神经调节激活驱动。为此，我们优化了两种新方法，利用清醒动物新皮质 vEC 中基因编码 Ca2+ 指标的双光子成像，实现 vEC Ca2+ 动力学可视化。使用这种方法的初步数据已经确定了大脑中持续的基础 vEC Ca2+ 振荡的特征。此外，我们首次发现 vEC Ca2+ 动力学可以通过小鼠桶状皮层的生理感觉输入引起。我们建议扩展这些初步发现，并使用细胞类型特异性光遗传学激活来功能性地剖析局部体感和远端神经网络中的精确细胞成分，这些成分有助于大脑中感觉诱发的 vEC Ca2+ 动力学。 公共卫生相关性：血脑屏障是中枢神经系统的重要组成部分，可限制潜在有害物质从血液进入大脑。血脑屏障的破坏可导致多种病理状况的发生，包括糖尿病、多发性硬化症、炎性疼痛、阿尔茨海默病、癫痫和中风。更好地了解构成血脑屏障的单个细胞有可能发现治疗这些疾病和改善人类健康的新治疗方法。