Microfluidic approach to investigating the role of ceramide in respiratory response of mitochondria in diabetic retina.

Abstract

Purpose : Mitochondrial damage and increased ROS production in diabetes contributes to pro-inflammatory changes and development of diabetic retinopathy. Acid sphingomyelinase (ASM) is upregulated in diabetic retina leading to higher ceramide and glycosilceramide production. Ceramide was shown to affect mitochondrial function at several levels including mitochondrial fission and mitophagy, ROS production, formation of ceramide channels in outer mitochondrial membrane and release of pro-apoptotic proteins. ASM inhibition and reduction in ceramide production protects from retinopathy development. The mechanism of ceramide-induced mitochondrial dysfunction is not well understood 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 retina.

Methods : To access the influence of ceramide on the level of overall mitochondrial activity, we developed a novel microfluidic sensor for functional capacity of respiratory activity. It was applied to RPE cells treated with normal (5.5 mM) and high (25 mM) glucose for 24 hours, with and without ASM inhibition using siRNA.

Results : Hyperglycemia-induced increase in ASM activity lead to increased ceramide and glycosyl ceramide production in RPE cells. This increase was associated with higher ROS, IL-1β and VEGF production and ICAM1 expression. Inhibition of ASM using siRNA prevented these pro-inflammatory changes. Fitness of the microfluidic sensor for studies of the overall mitochondrial activity modulated by ceramide was assessed. This sensor is capable of measuring mitochondrial activity in very small sample (~ 1000 cells in <10 microliters), making is suitable for studies on scarce samples. Glucose oxidase measurements under variety of conditions were used to test reproducibility over wide dynamic range. Efforts are currently under way to integrate catalytic capacity measurements with simultaneous electrochemical manipulations of mitochondria.

Conclusions : Hyperglycemia-induced ASM activation and ceramide production could lead to mitochondrial dysfunction and pro-inflammatory changes in RPE cells. Microfluidic sensor for respiratory and electrochemical activity has a good 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 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.