Name: measurement of energy metabolism in explanted retinal tissue using extracellular flux analysis
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis at JoVE.com

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).

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
<ol>
	<li>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.</li>
</ol>
2. Solution Preparation
<ol>
	<li>Prepare base media by dissolving...

<ol>
	<li>Wong-Riley, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+metabolism+of+the+visual+system.">Energy metabolism of the visual system.</a> Eye and brain. 2, 99-116 (2010).</li><li>Ames, A., Li, Y. Y., Heher, E. C., Kimble, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+metabolism+of+rabbit+retina+as+related+to+function:+high+cost+of+Na++transport.">Energy metabolism of rabbit retina as related to function: high cost of Na+ transport.</a> The Journal of neuroscience: the official journal of the Society for Neuroscience. 12 (3), 840-853 (1992).</li><li>Warburg, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+origin+of+cancer+cells.">On the origin of cancer cells.</a> Science. 123 (3191), 309-314 (1956).</li><li>Wubben, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoreceptor+metabolic+reprogramming+provides+survival+advantage+in+acute+stress+while+causing+chronic+degeneration.">Photoreceptor metabolic reprogramming provides survival advantage in acute stress while causing chronic degeneration.</a> Scientific reports. 7 (1), 17863 (2017).</li><li>Du, J., Linton, J. D., Hurley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+Metabolism+in+the+Intact+Retina+Using+Stable+Isotope+Tracers.">Probing Metabolism in the Intact Retina Using Stable Isotope Tracers.</a> Methods in enzymology. 561, 149-170 (2015).</li><li>Felder, A. E., Wanek, J., Tan, M. R., Blair, N. P., Shahidi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Method+for+Combined+Retinal+Vascular+and+Tissue+Oxygen+Tension+Imaging.">A Method for Combined Retinal Vascular and Tissue Oxygen Tension Imaging.</a> Scientific reports. 7 (1), 10622 (2017).</li><li>Hurley, J. B., Lindsay, K. J., Du, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose,+lactate,+and+shuttling+of+metabolites+in+vertebrate+retinas.">Glucose, lactate, and shuttling of metabolites in vertebrate retinas.</a> Journal of neuroscience research. 93 (7), 1079-1092 (2015).</li><li>Winkler, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycolytic+and+oxidative+metabolism+in+relation+to+retinal+function.">Glycolytic and oxidative metabolism in relation to retinal function.</a> The Journal of general physiology. 77 (6), 667-692 (1981).</li><li>Pelletier, M., Billingham, L. K., Ramaswamy, M., Siegel, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+flux+analysis+to+monitor+glycolytic+rates+and+mitochondrial+oxygen+consumption.">Extracellular flux analysis to monitor glycolytic rates and mitochondrial oxygen consumption.</a> Methods in enzymology. 542, 125-149 (2014).</li><li>Joyal, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+lipid+and+glucose+metabolism+dictates+angiogenesis+through+the+lipid+sensor+Ffar1.">Retinal lipid and glucose metabolism dictates angiogenesis through the lipid sensor Ffar1.</a> Nature medicine. 22 (4), 439-445 (2016).</li><li>Kooragayala, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Oxygen+Consumption+in+Retina+Ex+Vivo+Demonstrates+Limited+Reserve+Capacity+of+Photoreceptor+Mitochondria.">Quantification of Oxygen Consumption in Retina Ex Vivo Demonstrates Limited Reserve Capacity of Photoreceptor Mitochondria.</a> Investigative ophthalmology &amp; visual science. 56 (13), 8428-8436 (2015).</li><li>Pearsall, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PPARalpha+is+essential+for+retinal+lipid+metabolism+and+neuronal+survival.">PPARalpha is essential for retinal lipid metabolism and neuronal survival.</a> BMC biology. 15 (1), 113 (2017).</li><li>Nicholls, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioenergetic+profile+experiment+using+C2C12+myoblast+cells.">Bioenergetic profile experiment using C2C12 myoblast cells.</a> Journal of visualized experiments. (46), (2010).</li><li>Lobanova, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transducin+translocation+in+rods+is+triggered+by+saturation+of+the+GTPase-activating+complex.">Transducin translocation in rods is triggered by saturation of the GTPase-activating complex.</a> The Journal of neuroscience: the official journal of the Society for Neuroscience. 27 (5), 1151-1160 (2007).</li><li>Winkler, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electroretinogram+of+the+isolated+rat+retina.">The electroretinogram of the isolated rat retina.</a> Vision research. 12 (6), 1183-1198 (1972).</li><li>Jastroch, M., Divakaruni, A. S., Mookerjee, S., Treberg, J. R., Brand, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+proton+and+electron+leaks.">Mitochondrial proton and electron leaks.</a> Essays in biochemistry. 47, 53-67 (2010).</li><li>Divakaruni, A. S., Paradyse, A., Ferrick, D. A., Murphy, A. N., Jastroch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+and+interpretation+of+microplate-based+oxygen+consumption+and+pH+data.">Analysis and interpretation of microplate-based oxygen consumption and pH data.</a> Methods in enzymology. 547, 309-354 (2014).</li><li>Du, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototransduction+Influences+Metabolic+Flux+and+Nucleotide+Metabolism+in+Mouse+Retina.">Phototransduction Influences Metabolic Flux and Nucleotide Metabolism in Mouse Retina.</a> The Journal of biological chemistry. 291 (9), 4698-4710 (2016).</li></ol>

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 &#176;C throughout the ...

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. Yet, the retina also relies heavily on glycolytic machinery even when oxygen is abundant. This process of aerobic glycolysis was originally described in cancer cells by Otto Warburg3, who once noted that the retina was the only post-mitotic tissue capable of this form of metabolism4. Since those initial observations, many post-mitotic tissues have been described to engage in varying degrees of glycolysis in addition to oxidative phosphorylation to meet their ATP demands.
Phototransduction, visual pigment recycling, biosynthesis of photoreceptor outer segments, and synaptic activity are all energy demanding processes in photoreceptors, the predominant neuronal subclass in the retina. But the need to actively transport ions against their electrical and concentration gradients is by far the most energetically consuming process in neurons1. Photoreceptors are peculiar neurons in the sense that they are depolarized in the absence of stimulation (i.e., in the dark), whereas a light stimulus triggers channel closure and subsequent hyperpolarization. Therefore, in the dark, the retina consumes large quantities of ATP to maintain its depolarization or &#34;dark current&#34; as it is commonly called. From an adaptive standpoint, a major challenge in supplying these vast quantities of ATP is the need for organisms to maintain visual clarity through the optical axis. The inverted retinal architecture seen in modern creatures is the dominant solution, as it keeps the dense capillary network supplying photoreceptors away from the path of light. But this marvel of natural bioengineering places the retina at a precipice in terms of metabolic reserve. Even small insults to retina can potentially disrupt the delicate balance of energy supply to demand, and visual dysfunction or frank blindness may ensue quickly.
Given the unique energetic demands of the neural retina, coupled with its tight restriction of vascular supply, accurate measurement of ATP consumption in the retina and its changes during disease could have profound implications in understanding and treating blinding conditions such as retinitis pigmentosa and diabetic retinopathy. Traditionally, these measurements require costly, custom-designed equipment with most studies emerging from a handful of laboratories entirely dedicated to measurements of metabolic activity2,5,6,7,8. Techniques include individual assays for specific metabolites, tracer studies using radio-labeled precursors, oxygen consumption recording using Clark electrodes, and metabolomic profiling9.
With advances in high-throughput technology and increased availability of commercial devices, techniques to record retinal metabolism are increasingly accessible and affordable. The method described here measures both oxidative phosphorylation and glycolysis in retina using explanted tissue and a commercially-available extracellular flux analyzer9,10,11,12. This analyzer separately records oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR), serving as indirect indicators of oxidative phosphorylation and glycolysis, respectively13. These measurements are done by a probe submersed within a microchamber created over the tissue of interest. This adaptation of previously published methods uses a capture plate originally designed for pancreatic Islets of Langerhans to record metabolic activity in small, circular sections of mouse retina. Multiple pharmacologic exposures can be delivered to the tissue during the course of a single recording because the system contains 4 injection ports for each sample well. Using this system with separate protocols optimized for ECAR and OCR recordings, the responses of wild-type retinas can be compared to retinas lacking transducin (Gnat1-/-), a cause of congenital stationary night blindness in humans14.

<table border="0" cellpadding="0" cellspacing="0" style="width:777px;" width="778">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:337px;">Seahorse XF24 Extracellular Flux Analyzer</td>
			<td style="width:155px;">Agilent, Santa Clara, CA</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:337px;">Seahorse XF24 Islet Capture FluxPak (includes: Islet Capture Microplate, Sensor Cartridge and Calibrant Solution)</td>
			<td>Agilent, Santa Clara, CA</td>
			<td>101174-100</td>
			<td style="width:167px;">Includes islet capture microplate, sensor cartridge and calibrant solution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">RPMI 1640 Media (Powdered medium)</td>
			<td>Millipore-Sigma</td>
			<td>R1383</td>
			<td style="width:167px;">RPMI 1640 Media with L-Glutamine and without glucose or sodium bicarbonate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">D-Glucose</td>
			<td>Millipore-Sigma</td>
			<td>G8270</td>
			<td style="width:167px;">1M D-Glucose filtered, for media preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:337px;">Sodium pyruvate</td>
			<td>Corning</td>
			<td>25000CI</td>
			<td style="width:167px;">100 mM sodium pyruvate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:337px;">Antimycin-A</td>
			<td>Millipore-Sigma</td>
			<td>A8674</td>
			<td style="width:167px;">Mitochondrial stress protocol component</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:337px;">FCCP</td>
			<td>Millipore-Sigma</td>
			<td>C2920</td>
			<td style="width:167px;">Mitochondrial stress protocol component</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:337px;">Rotenone</td>
			<td>Millipore-Sigma</td>
			<td>R8875</td>
			<td style="width:167px;">Mitochondrial stress protocol component</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:337px;">2-deoxyglucose</td>
			<td>Millipore-Sigma</td>
			<td>D6134</td>
			<td style="width:167px;">Glycolysis protocol component</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:337px;">1 mm skin biopsy punches with plunger</td>
			<td>Integra-Miltex</td>
			<td>33-31AA-P/25</td>
			<td style="width:167px;">Explanting retinal tissue tool</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:337px;">Dumont Mini-Forceps Straight</td>
			<td>Fine Science Tools</td>
			<td>11200-10</td>
			<td style="width:167px;">Explanting retinal tissue tool</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:337px;">Dumont Medical #5/45 Forceps- Angled 45 degrees</td>
			<td>Fine Science Tools</td>
			<td>11253-25</td>
			<td style="width:167px;">Explanting retinal tissue tool</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:337px;">Dumont #7 Forceps - Curved</td>
			<td>Fine Science Tools</td>
			<td>11271-30</td>
			<td style="width:167px;">Explanting retinal tissue tool</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:337px;">Quant-iT Picogreen dsDNA Assay Kit</td>
			<td>Fisher Scientific</td>
			<td>P7589</td>
			<td style="width:167px;">Loading normalization assay</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trizma base (Tris base)</td>
			<td>Millipore-Sigma</td>
			<td>T6066</td>
			<td style="width:167px;">Component of lysis buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Triton X-100 (polyethylene glycol tert-octylphenyl ether)</td>
			<td>Millipore-Sigma</td>
			<td>X100</td>
			<td style="width:167px;">Component of lysis buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">0.5M EDTA pH 8.0</td>
			<td>Ambion</td>
			<td>AM9262</td>
			<td style="width:167px;">Component of lysis buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">C57BL/6J mice&#160;</td>
			<td>Jackson Laboratories</td>
			<td>&#160;Strain 000664</td>
			<td style="width:167px;">Animals</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Gnat1-/- and background-matched Gnat1+/+&#160;</td>
			<td colspan="2">Vladimir Kefalov, PhD; Washington University School of Medicine</td>
			<td style="width:167px;">Animals</td>
		</tr>
	</tbody>
</table>

measurement of energy metabolism in explanted retinal tissue using extracellular flux analysis

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.

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-/-&#160;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...

Watch this Scientific Journal Video about Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis at JoVE.com

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

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 ...

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