A glial-endothelial model to examine collective regulation of transport across the retina

负责将光转换为大脑信号的视网膜神经元需要一个由血液-视网膜屏障调节的严格控制的生化环境。这种屏障的功能障碍导致成人视网膜的进行性损伤，并且是视力丧失的主要原因。屏障功能在很大程度上归因于排列在视网膜血管内部的血管内皮细胞（EC）。然而，Müller胶质细胞（MG）是视网膜特有的细胞，它们包裹在这些血管周围，影响屏障的维持以及对压力源的反应。EC和MG之间的相互作用知之甚少，在很大程度上研究不足。该项目开发了一种微流体系统，其中EC和MG层在彼此之上培养，以创建一个定义明确的仿生（生物学模拟）环境。将在正常条件下和存在导致屏障渗漏和改变葡萄糖转运的生化试剂的情况下研究这些培养物的屏障特性，这两种试剂均导致神经变性和视力丧失。拟议的研究将增加我们对MG在调节血液-视网膜屏障中的血源性因子的作用的理解。该项目还将为研究生和本科生提供机会，研究神经血管健康的差异及其对神经胶质介导的治疗和公共卫生政策的发展的社会影响。研究人员假设，MG与同源EC双边相互作用，以保持完整性和调节血液视网膜内屏障（iBRB）的渗透性，以应对生化变化。 为了验证这一假设，该项目研究了EC如何在体外系统中与MG共同形成转运屏障，该系统模拟成人iBRB的关键方面。 研究计划有三个目标。第一个目标将研究集体EC和MG反应如何改变细胞行为和从血管到神经视网膜的分子运输。实验将测量细胞生长和形态的变化，粘附和紧密连接蛋白的表达，以及细胞屏障的电阻率和渗透性。第二个目标将开发一个分层的微流体系统，通过模拟适当的视网膜接近与间质流来测量从EC到MG的血液传播因子的运输。测试将使用不同大小和已知反应性的分子检查跨细胞和细胞旁性质，以及探测跨每个细胞层的运输途径。第三个目标将评估晚期糖基化终产物（AGEs）的环境如何机械地改变EC和MG的电阻率和渗透性。这项研究将集中在AGEs介导的活性氧（ROS），这增加了葡萄糖转运蛋白GLUT-1和血管内皮生长因子α（VEGF）的表达，该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查进行评估，被认为值得支持的搜索.

The retinal neurons responsible for converting light into brain signals require a tightly-controlled biochemical environment that is regulated by the blood-retinal barrier. Dysfunction of this barrier leads to progressive damage in the adult retina and is a major cause of vision loss. Barrier function has been attributed largely to the vascular endothelial cells (EC) that line the inside of retinal blood vessels. However, Müller glia (MG) are cells unique to the retina that wrap around these blood vessels to impact barrier maintenance and how it responds to stressors. The interaction between EC and MG is poorly understood and largely understudied. This project develops a microfluidic system wherein layers of EC and MG are cultured atop one another to create a well-defined biomimetic (biology mimicking) environment. The barrier properties of these cultures will be investigated under normal conditions and in presence of biochemical agents that cause barrier leakiness and alter glucose transport, both of which result in neural degeneration and vision loss. The proposed studies will increase our understanding of the role of MG in the regulation of bloodborne factors across the blood-retinal barrier. The project will also provide opportunities for both graduate and undergraduate students to examine disparities in neurovascular health and its societal implications for development of glial-mediated therapies and public health policy.The investigators hypothesize that MG interact bilaterally with cognate EC to preserve the integrity and modulate permeability of the inner blood retinal barrier (iBRB) in response to biochemical changes. To test this hypothesis, the project investigates how EC collectively form a transport barrier with MG in an in vitro system that mimics critical aspects of the adult iBRB. The Research Plan is organized under three Aims. The First Aim will examine how collective EC and MG responses alter cellular behaviors and molecular transport from blood vessels into neuroretina. Experiments will measure changes in cell growth and morphology, expression of adhesion and tight junction proteins, and the resistivity and permeability of cell barriers. The Second Aim will develop a layered, microfluidic system to measure transport of bloodborne factors from EC to MG by modeling appropriate retinal proximity with interstitial flows. Tests will examine trans- and paracellular properties using molecules of different sizes and known reactivity, as well as probe the route(s) of transport across each cell layer. The Third Aim will assess how environments of advanced glycation end products (AGEs) mechanistically alter resistivity and permeability across EC and MG. The study will focus on AGEs-mediated, reactive oxygen species (ROS), which increase the expression of both the glucose transporter GLUT-1 and vascular endothelial growth factor alpha (VEGF), which in turn causes abnormal angiogenesis and irreversible retinal damage in adults.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.