Quantification of Oxygen Consumption in Retina Ex Vivo Demonstrates Limited Reserve Capacity of Photoreceptor Mitochondria.

Defects in mitochondrial respiration and oxidative phosphorylation are associated with pleiotropic phenotypes, which, at least in part, can be attributed to aberrant generation of reactive oxygen (O2) and nitrogen species.1–4 Mitochondrial dysfunction has been correlated with the accumulation of cellular damage in aging and in late-onset neurodegenerative diseases.5–8 Tissues with a high metabolic requirement, such as the retina, are especially vulnerable to mitochondrial damage, which is implicated as a causal factor in neuronal cell death in retinal and macular degeneration.9,10 Within the retina, rod and cone photoreceptors exhibit high energy metabolism and associated O2 consumption, primarily driven by ion transport and synaptic transmission in the dark and by cGMP turnover in the light.11–14 Quantification of O2 consumption rate (OCR) is generally adopted as a readout of overall mitochondrial function.15 Measurements of respiration have traditionally been conducted with the O2-consuming Clark-type electrode on mitochondria preparations,16 which require sample volumes in the milliliter range and continuous stirring of samples in suspension, with loss of cellular/tissue context. The Clark-type electrode has also been used to measure O2 consumption in the intact rat retina, detecting higher O2 consumption in the outer retina compared to inner retina, with downward or upward changes, respectively, during light adaptation.17 Microelectrodes that are O2-sensitive and intraretinal measurements taken at differential tissue depths18 confirmed that photoreceptors have the highest O2 consumption.18 Finally, fresh mouse retina slices in a flow culture system have been used to determine the metabolic dependence of photoreceptors on glucose as a fuel.19 A limitation of all of the above experimental conditions is that they only permit evaluation of one retina/sample at a time and are not readily amenable for simultaneous and high throughput evaluation of multiple experimental conditions. Microplate-based assays using an extracellular flux (XF) analyzer (Seahorse XF Analyzer; Seahorse Bioscience, Billerica, MA, USA) allow parallel comparison of different samples in small microliter volumes without the need for sample suspension, continuous stirring, or large measuring volumes in the order of ∼2 mL, as is the case with typical Clark-type electrode systems. The XF analyzer (Seahorse Bioscience) has been used to determine the effects of Ca++ and oxidative stress on mitochondrial function in the photoreceptor-like cell line 661W.20 Using the same technology, mitochondrial function has been examined and compared in acutely derived sections from several brain areas of a rat model of migraine.21 Direct comparative OCR determination has not yet been accomplished in the freshly isolated mouse retina. Here, we introduce an improved protocol for microplate-based measurement of O2 consumption in acute retina samples. Photoreceptors account for the majority of O2 consumption in the retina,22 with the entire inner retina consuming at most 17% of the amount used by photoreceptors.13 Thus, the measurement of O2 consumption in the intact retina largely reflects photoreceptor respiration. Our newly optimized method directly evaluates retinal mitochondrial function and should be valuable for assessing alterations associated with retinal dysfunction and/or disease.