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Summary

Abstract

Introduction

Protocol

Representative Results

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Materials

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Biology

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

Published: January 7th, 2019

DOI:

10.3791/58626

1Division of Metabolism, Endocrinology and Lipid Research, Department of Medicine, Washington University School of Medicine, 2Department of Biomedical Engineering, Washington University in Saint Louis, 3Department of Ophthalmology and Visual Science, Washington University School of Medicine

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

The retina is among the most energy-demanding tissues in the central nervous system1. Like most tissues, it generates adenosine triphosphate (ATP) via glycolysis in the cytosol or via oxidative phosphorylation in mitochondria. The energetic advantage of oxidative phosphorylation over glycolysis to produce ATP from one molecule of glucose is clear: 36 molecules of ATP generated from the former vs. 2 molecules of ATP generated from the latter. Accordingly, retinal neurons primarily depend on mitochondrial respiration for energy supply and this is reflected by their high density of mitochondria2. ....

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Protocols followed the Association for Research in Vision and Ophthalmology Statement for the Use of Animals and were approved by Washington University.

1. Animal Preparation

  1. Keep animals in standard housing with a 12 hours dark to 12 hours light cycle. Begin experiments at standardized times to avoid circadian effects, typically in the morning shortly after lights are turned on.

2. Solution Preparation

  1. Prepare base media by dissolving.......

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Using the described techniques (summarized in Figure 1), retinal explants from 8 week-old wild type (WT) mice were compared to age- and background-matched transducin null mice (Gnat1-/-). Because Gnat1-/- animals lack the machinery to close cyclic-nucleotide gated ion channels in response to light stimuli, their rod photoreceptors remain depolarized even in light14. The subsequent n.......

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OCR and ECAR are readily measured in explanted retinal tissue using a bioanalyzer using the described techniques. This method departs from those of other groups in several critical steps. Retinal tissues are isolated through a large corneal incision without enucleating the globe, as originally described by Winkler15. This method of retinal isolation allows for a rapid transfer from the living eye into the tissue capture plate (often within 5 minutes). Tissues are kept at 37 °C throughout the .......

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We thank Dr. Alexander Kolesnikov and Dr. Vladimir Kefalov for providing Gnat1-/- mice, for helpful feedback and advice, and for reading the manuscript.

This work was supported by NIH EY025269 (RR), the Diabetes Research Center at Washington University - NIH DK020579 (JRM and RR), a Career Development Award from Research to Prevent Blindness (RR), the Horncrest Foundation (RR), a Career Development Award from JDRF (JRM), NIH DK101392 (CFS), DK020579 (CFS), DK056341 (CFS), and DK114233 (JRM).

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Name Company Catalog Number Comments
Seahorse XF24 Extracellular Flux Analyzer Agilent, Santa Clara, CA
Seahorse XF24 Islet Capture FluxPak (includes: Islet Capture Microplate, Sensor Cartridge and Calibrant Solution) Agilent, Santa Clara, CA 101174-100 Includes islet capture microplate, sensor cartridge and calibrant solution
RPMI 1640 Media (Powdered medium) Millipore-Sigma R1383 RPMI 1640 Media with L-Glutamine and without glucose or sodium bicarbonate
D-Glucose Millipore-Sigma G8270 1M D-Glucose filtered, for media preparation
Sodium pyruvate Corning 25000CI 100 mM sodium pyruvate
Antimycin-A Millipore-Sigma A8674 Mitochondrial stress protocol component
FCCP Millipore-Sigma C2920 Mitochondrial stress protocol component
Rotenone Millipore-Sigma R8875 Mitochondrial stress protocol component
2-deoxyglucose Millipore-Sigma D6134 Glycolysis protocol component
1 mm skin biopsy punches with plunger Integra-Miltex 33-31AA-P/25 Explanting retinal tissue tool
Dumont Mini-Forceps Straight Fine Science Tools 11200-10 Explanting retinal tissue tool
Dumont Medical #5/45 Forceps- Angled 45 degrees Fine Science Tools 11253-25 Explanting retinal tissue tool
Dumont #7 Forceps - Curved Fine Science Tools 11271-30 Explanting retinal tissue tool
Quant-iT Picogreen dsDNA Assay Kit Fisher Scientific P7589 Loading normalization assay
Trizma base (Tris base) Millipore-Sigma T6066 Component of lysis buffer
Triton X-100 (polyethylene glycol tert-octylphenyl ether) Millipore-Sigma X100 Component of lysis buffer
0.5M EDTA pH 8.0 Ambion AM9262 Component of lysis buffer
C57BL/6J mice  Jackson Laboratories  Strain 000664 Animals
Gnat1-/- and background-matched Gnat1+/+  Vladimir Kefalov, PhD; Washington University School of Medicine Animals

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