Mitochondrial Ceramide Effects on the Retinal Pigment Epithelium in Diabetes.

Mitochondrial damage in the cells comprising inner (retinal endothelial cells) and outer (retinal pigment epithelium (RPE)) blood-retinal barriers (BRB) is known to precede the initial BRB breakdown and further histopathological abnormalities in diabetic retinopathy (DR). We previously demonstrated that activation of acid sphingomyelinase (ASM) is an important early event in the pathogenesis of DR, and recent studies have demonstrated that there is an intricate connection between ceramide and mitochondrial function. This study aimed to determine the role of ASM-dependent mitochondrial ceramide accumulation in diabetes-induced RPE cell damage.

Micro-respirometry of whole cells and isolated mitochondria.

Oxygen consumption is a key metric of metabolism in aerobic organisms. Current respirometric methods led to seminal discoveries despite limitations such as high sample demand, exchange with atmospheric O, and cumulative titration protocols leading to limited choice of useable tissue, complex data interpretation, and restricted experimental design. We developed a sensitive and customizable method of measuring O consumption rates by a variety of biological samples in microliter volumes without interference from the aerobic environment.