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

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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.