Astrocyte-mediated Blood Flow Autoregulation as a Disease Mechanism in Glaucoma

描述（由申请人提供）：血流自动调节受损被认为是导致青光眼BF减少和视网膜神经节细胞损伤的重要病理机制之一。在该范例中，当眼内压（IOP）和/或血压（BP）波动时，眼睛中，特别是视神经乳头中的血流量（BF）不再保持在正常范围内。如在临床和实验室实验中所证明的，这样的血液动力学变化导致眼组织内的血流减少以及通过视网膜神经纤维层厚度和功能丧失测量的相关的神经节细胞损伤。因此，针对导致自动调节功能障碍的特定途径的早期干预可以减轻青光眼的血流动力学变化并保护视网膜神经节细胞免受损伤。然而，目前的知识与疾病的机制相关的眼睛BF自动调节是不够的，以解释许多现象在临床和实验中观察到的。星形胶质细胞在脑和视网膜中以前馈机制将神经元活动与血流耦合（称为神经血管耦合）是必不可少的。一系列研究表明，星形胶质细胞也是参与血流自动调节的重要细胞成分。这具有临床意义，因为星形胶质细胞在BF自动调节中的作用的鉴定可能导致一种新的病理学改变， 血液动力学变化的机制和随后创新的青光眼药物干预。本研究旨在开发新的技术来检测动脉周围星形胶质细胞的生物活性和血管反应与血管内和血管外压力变化的关系。这一举措将导致未来的研究，利用这些技术来检查假设的机制，BF自动调节正常和青光眼的眼睛。具体目标1：（a）开发并验证一种系统，该系统可将血管内压力（模拟BP）和血管外压力（模拟IOP）设置和操纵到不同水平，同时对动脉周围星形胶质细胞中的钙信号（用Fluo-4AM标记）和血管直径（用Dil标记）进行成像。(b)比较动脉周围星形胶质细胞内的钙信号和血管直径变化与离体猪视网膜中血管内和/或血管外压力变化诱导的机械刺激的幅度。具体目标2：建立一种新的在体钙显像技术，监测血压变化时动脉周围星形胶质细胞内钙的变化。这将通过玻璃体内注射钙指示剂（Fluo-4AM）来实现。利用共焦扫描激光检眼镜和光谱域光学相干断层扫描的荧光模式，对大鼠眼底进行连续成像，监测动脉周围星形胶质细胞的钙信号和血管直径， 大鼠血压确定性地升高和降低。证明星形胶质细胞参与BF自动调节，识别治疗干预的特异性靶点可以保护眼组织免受缺血性损伤和随后的人青光眼进行性RGC损伤。

DESCRIPTION (provided by applicant): Compromised blood flow autoregulation has been proposed as one of important pathological mechanisms contributing to decreased BF and retinal ganglion cell damage in glaucoma. Within this paradigm, blood flow (BF) in the eye, particularly in the optic nerve head, can no longer be held within a normal range when intraocular pressure (IOP) and/or blood pressure (BP) fluctuates. As evidenced in clinical and laboratory experiments, such hemodynamic changes result in a blood flow decrease within the ocular tissues and associated ganglion cell damage measured by retinal nerve fiber layer thickness and functional loss. Thus, early intervention targeting specific pathways leading to autoregulation dysfunction could mitigate the hemodynamic changes and protect retinal ganglion cells from damage in glaucoma. However, the current knowledge of ocular BF autoregulation associated with the mechanism of the disease is insufficient to interpret many of the phenomena observed in clinics and in experiments. Astrocytes are essential to couple neuronal activities with blood flow (known as neurovascular coupling) in both brain and retina in a feed forward mechanism. A line of studies have suggested that astrocyte is also an essential cellular element involved in blood flow autoregulation. This has clinical implications because identification of the astrocyte role in BF autoregulation is likely to lead to a novel pathological mechanism of hemodynamic change and subsequently innovative pharmacological interventions for glaucoma. This study proposes to develop new techniques to examine peri-arterial astrocyte bioactivities and vascular response in relation to intra and extravascular pressure alteration. This initiative will lead future studies by utilizing these techniques to examine hypothesized mechanisms of BF autoregulation in normal and in glaucomatous eyes. In Specific Aim 1: (a) To develop and validate a system to set and manipulate both intravascular pressure (simulating BP) and extravascular pressure (simulating IOP) to different level and, simultaneously, image calcium signals (labeled with Fluo-4AM) in peri-arterial astrocytes and vessel diameter (labeled with Dil). (b) Compare the calcium signals within the peri-arterial astrocytes and vessel diameter changes against the magnitude of mechanical stimuli induced by changes in intra- and/ or extravascular pressure in isolated pig retina. In Specific Aim 2: To develop a novel in vivo calcium imaging technique to monitor calcium changes within peri-arterial astrocytes while BP is changed. This will be achieved by intravitreal injection of a calcium indicator (Fluo-4AM). Utilizing fluorescence mode in confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography, the rat fundus is imaged continuously to monitor the calcium signals in peri-arterial astrocytes and vessel diameter, whilst the rat BP is deterministically increased and decreased. Proving the involvement of astrocytes in BF autoregulation, identification of specific targets for therapeutic intervention may protect te ocular tissue from ischemic insult and subsequent progressive RGC damage in human glaucoma.