Diabetic retinopathy (DR) is a sight-threatening complication of diabetes mellitus and a leading cause of preventable vision loss worldwide. Classically regarded as a vascular disease, the clinically observable lesions and hallmark histopathological findings are found in the vascular compartment. The metabolic insults affecting retinal cells in DR are multifactorial and complex; however, hyperglycemia, dyslipidemia, and chronic inflammation are thought to be major contributors. Diabetic dyslipidemia affects systemic and local lipid metabolism driving the pro-inflammatory and pro-apoptotic retinal cell changes.Sphingolipids are known to play a key role in cell functioning. Ceramides, the central bioactive sphingolipid species, control cellular responses to cytotoxic stressors. Ceramides can be generated de novo or through sphingomyelinase pathways. Important in vivo and in vitro results have demonstrated that acid sphingomyelinase (ASMase)-induced ceramide generation is a major contributor to retinal barrier cell apoptosis in diabetes. Results from studies in ASMase knockout models have shown that these animals are resistant to an array of cytotoxic insults, confirming that ASMase-dependent ceramide generation is important for apoptosis execution. Recent reports have demonstrated mitochondrial ceramide accumulation in response to cytotoxic insults in animal and in vitro models. Limited observations of oxidative metabolism are reported in the literature as limited biological material is an impediment for comprehensive metabolic characterization of retinal cells in the context of DR. Removal of such obstacles lays the foundation for this work.Comprehensive metabolic examination of DR model systems requires highly sensitive, flexible, and accurate measurements of cell or organelle oxygen consumption, a functional measure of oxidative metabolism. The instruments which perform such a measurement are called respirometers. Though commercially available options exist they each present unique limitations which can be remediated by rational design of a dedicated microfluidics-based microrespirometer.The first part of this work focuses on the development of a sensitive and customizable method of measuring O2 consumption rates by a variety of biological samples in microliter volumes without interference from the aerobic environment. The work demonstrates use of 3D printing utilizing photopolymer (VeroClear) to reproducibly form micron-scale microchannels. The photopolymer demonstrated low oxygen permeability, optical clarity, and, in combination with optode-based O2 sensing, produced a microrespirometer showing > 100x dynamic range for O2 consumption rates. Measurements are demonstrated with solution-based, suspension-based, and adherent samples.The role of ASM-dependent mitochondrial ceramide accumulation in diabetes-induced retinal pigment ephithelial cell damage is described next. Mitochondria isolated from diabetic rat retinas (7 weeks duration) showed an increase in the ceramide-to-sphingomyelin ratio compared to controls whereas, the ceramide-to-sphingomyelin ratio was decreased in mitochondria isolated from ASM-knockout mouse retinas compared to wild-type littermates. Cellular ceramide was elevated in RPE cells derived from diabetic donors compared to control donors, with a corresponding increase in IL-103B2, IL-6, and ASM expression. RPE from diabetic donors showed fragmented mitochondria and a decreased respiratory control ratio (RCR). Treatment of ARPE-19 cells with high glucose resulted in a decrease in citrate synthase activity at 72 h. Inhibition of ASM with desipramine (15 03BCM, 1 h daily) abolished the decreases in metabolic functional parameters. These results are consistent with diabetes-induced increase in mitochondrial ceramide through an ASM-dependent pathway leading to impaired mitochondrial function in RPE cells.
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