Changes in mitochondria respiration in degenerating mouse retina identified by a novel ex vivo assay

Abstract

Purpose: Oxidative stress and mitochondrial dysfunction play an important role in retinal disease. We hypothesized that the quantification of changes in mitochondrial respiration can be utilized as a biomarker of retinal distress preceding overt degeneration. The goal of this project was to directly assay oxygen consumption in ex vivo retinal tissue from degenerating mouse models and healthy controls.<br />

Methods: We measured oxygen consumption rate (OCR), an indicator of mitochondrial oxidative phosphorylation, with the Seahorse XF24 Bioanalyzer. Punches of retinal tissue (1 mm diameter) were placed on mesh inserts treated with Cell-Tak adhesive and were secured in a XF24 Islet FluxPak microplate containing Ames medium. A mitochondrial uncoupler, either carbonyl cyanide trifluoromethoxyphenylhydrazone (FCCP) or (2-fluorophenyl){6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)}amine (Bam15), was injected during the assay to assess the maximal respiratory capacity. Rotenone was added to determine the background contribution of non-mitochondrial respiration. OCR values were background subtracted and normalized to mitochondrial cytochrome a content.

Results: A steady baseline OCR was maintained for the duration of the experiment (125 minutes) using one-month-old wild type retinal punches. Both FCCP and Bam15 uncouplers induced a 15-20% increase in OCR. Retina isolated from one-month-old mice with retinal disease (Rpgrip1^-/-, rds, Nrl^-/-) displayed basal OCR similar to wild type but exhibited elevated maximal OCR. One-month-old retina from rd1 and rd10 mice had lower basal OCR but exhibited maximal respiratory capacity of almost 200% from baseline.

Conclusions: We have optimized an ex vivo assay that uses freshly isolated retinal punches to measure oxygen consumption by microplate-based respirometry. We detected an unexpected low induction of maximal OCR upon uncoupling in young, healthy retina, suggesting that the retina operates near its peak metabolic capacity. However, maximal respiration appeared to be elevated in the presence of significant retinal degeneration. Changes in maximal respiration were correlated with the degree of degeneration. We propose that OCR measurements in retinal punches offer a convenient and reliable method to study mitochondrial function in the retina and can serve as a marker of disease onset and progression.