Date of Award

May 2020

Degree Type

Thesis

Degree Name

Master of Science

Department

Biological Sciences

First Advisor

Janis Eells

Second Advisor

Elizabeth Liedhegner

Committee Members

Michael Laiosa

Keywords

Diabetic Retinopathy, Muller Cells, Photobiomodulation, Red Light

Abstract

Diabetic Retinopathy (DR) is the most common complication of diabetes mellitus and a leading cause of blindness. The pathophysiology of DR is complicated, involving mitochondrial dysfunction, oxidative stress, inflammation and vascular degeneration. The most common therapeutic approach for DR includes the use anti-vascular endothelial growth factor (VEGF) drugs to reduce vascular proliferation. These treatments are invasive, frequently ineffective and have numerous adverse effects. A non-invasive more effective therapy is clearly needed. An alternative, non-invasive therapy using far-red light (photobiomodulation, PBM) may be an improvement over current therapy. PBM has documented efficacy in experimental and clinical studies of retinal disease including DR. Müller glial cells play a critical role in retinal metabolism and are among the first cells to demonstrate metabolic changes in response to retinal stress or disease and play an important role in the pathophysiology of DR. High glucose treatment of Müller cells increases the activity of Nuclear Factor Kappa beta (NFB) resulting in DR-associated protein synthesis. Previous studies in our laboratory have shown that that mimicking hyperglycemia in cultured Müller cells activates the NFB signaling pathway resulting in an increase in intracellular adhesion molecule 1 (ICAM-1) and that 670 nm PBM blocks this activation [Fisher, 2015]. However, the upstream mechanisms by which PBM reduces NFB activity remain to be elucidated. We hypothesized that 670 nm photobiomodulation would attenuate hyperglycemia-induced mitochondrial dysfunction and protect against oxidative stress in a rat Müller glial cell model of diabetic retinopathy. We tested this hypothesis in the following two aims: Specific Aim 1: Determine the effects of 670 nm PBM on mitochondrial function in a cultured Müller cell model of DR at 24 hours. Specific Aim 2: Determine the effects of 670 nm PBM on oxidative stress and glutathione status in a cultured Müller cell model of DR at 24 hours. We showed for the first time that as little as 24 hour of exposure to high glucose conditions disrupted mitochondrial metabolic activity, and decreased mitochondrial membrane potential, and ATP production. A single treatment with 670 nm light (4.5 J/cm2) restored metabolic activity, mitochondrial membrane potential and ATP production to values measured under normal glucose conditions within one hour following treatment. We also showed that a 24-hour exposure to high glucose stimulated ROS production. These effects were also reversed by a single treatment of 670 nm light within one hour. We conclude that this in vitro model of diabetic retinopathy in a dish has considerable potential for use in the development and testing of therapeutic agents to treat diabetic retinopathy. Our model can assess disruptions in metabolic activity beginning with ROS generation and mitochondrial redox changes culminating in increased production of angiogenic and inflammatory mediators characteristic of diabetic retinopathy.

Available for download on Friday, November 25, 2022

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