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Anand Rao-Venkata Saripalli, Yan Levitsky, Elahe Crockett, Evangelyn Alocilja, Julia V Busik, Denis Proshlyakov; DEVELOPMENT OF A NOVEL APPROACH FOR ASSESSMENT OF MITOCHONDRIAL RESPIRATION IN DIABETIC RETINA. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2525.
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© ARVO (1962-2015); The Authors (2016-present)
Despite recent success in treatment approaches, diabetic retinopathy (DR) remains a leading cause of progressive vision loss and blindness. It is well accepted that mitochondrial dysfunction contributes to the development of DR, however the mechanism(s) of mitochondrial damage are not well understood. Recent studies demonstrate that there is an intricate connection between ceramide and mitochondrial function. We have previously demonstrated that activation of acid sphingomyelinase (ASM) and ceramide production is an important early event in the pathogenesis of DR, however the role of ceramide-induced mitochondrial dysfunction in DR is unknown due to the lack of sufficiently sensitive tools. This study aims to design the tools for evaluation of the role of mitochondria in ceramide-induced damage in diabetic retinas.
Using differential centrifugation and magnetic nanoparticles (MN) conjugated to anti-translocase of the outer membrane of mitochondria-22 (anti-TOM-22) antibodies, mitochondria were isolated from mouse livers, retinas, and retinal pigment epithelium (RPE) cell cultures in normal (5.5mM) or high (25mM) glucose for 24 hours. Mitochondrial respiration was measured using a NeoFox fluorescent oxygen sensor in both conventional and microfluidic chambers. Glucose oxidase bound MNs were used for calibration of the microfluidic device.
The conventional NeoFox oxygen sensor requires mitochondrial isolation from ~700mg of tissue or about 0.05mg of total protein to achieve the detection limit. The microfluidic device reduced tissue requirements to ~7mg or about 0.0027mg of total protein making it possible to analyze mouse retinal mitochondrial metabolism from a single animal. We have demonstrated that MNs can be used to concentrate mitochondria in the microfluidic chamber. Using this approach we found a 3.6 ± 0.2 fold increase in mitochondrial outer membrane permeability in RPE cells cultured in 25mM glucose compared to control. The increase was corrected by ASM inhibitor desipramine.
MN-based mitochondria isolation coupled with a microfluidic sensor for respiratory and electrochemical activity shows potential for identification of complex-specific changes in the mechanism of ceramide-induced mitochondrial dysfunction in the diabetic retina.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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