Measurement of energy metabolism in dissociated mouse retinal rod photoreceptors

Abstract

Purpose : Photoreceptors are metabolically active retinal cells. Disturbance in photoreceptor metabolism leads to neuronal degeneration associated with a broad range of diseases such as age-related macular degeneration, and diabetic retinopathy. Understanding the changes in this process is critical for unraveling disease mechanisms and developing new treatments. The goal of this study is to develop an accurate and reliable approach for the measurement of energy metabolism in isolated mouse retinal photoreceptors.

Methods : Rod photoreceptor cells from dark-adapted mouse retinas were isolated using enzymatic digestion and mechanical dissociation. Cell viability was evaluated using Trypan blue and live-dead assays. Neuronal population and preservation of cell structure were examined by immunofluorescence staining. Cellular bioenergetics were measured by extracellular flux analysis using a Seahorse extracellular flux analyzer. To maintain a high number of metabolically active cells, dark conditions were used throughout the process of dissociation, cell seeding, and metabolic analysis.

Results : Successful isolation of retinal photoreceptors was achieved using as few as one single mouse retina. Immunostaining for multiple retinal neuronal markers revealed that 90% of the dissociated cell population were rod photoreceptors. The majority of photoreceptor cells preserved an intact structure and 93% of them were viable. Reproducible functional data were obtained in oxygen consumption rate (OCR) for mitochondrial respiration and extracellular acidification rate (ECAR) for glycolysis with adjustment of cell densities and titration of substrate concentrations. Compared to analysis using retinal explants, dissociated retinal photoreceptors demonstrated an enhanced response to inhibitors for mitochondrial respiration in metabolic assays.

Conclusions : Our study provided a novel approach for reproducible and accurate measurement of energy metabolism in dissociated mouse retinal rod photoreceptors, which may have a broad application in future studies on retinal degenerative disease.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.