Described here is a detailed protocol for performing mitochondrial stress assay and glycolytic rate assay in ex vivo retinal tissue samples using a commercial bioanalyzer.
Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. In addition, photoreceptors also exhibit high aerobic glycolysis like cancer cells. Precise measurements of these metabolic activities can provide valuable insights into cellular homeostasis under physiological conditions and in disease states. High throughput microplate-based assays have been developed to measure mitochondrial respiration and various metabolic activities in live cells. However, a vast majority of these are developed for cultured cells and have not been optimized for intact tissue samples and for application ex vivo. Described here is a detailed step-by-step protocol, using microplate-based fluorescence technology, to directly measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration, as well as extracellular acidification rate (ECAR) as an indicator of glycolysis, in intact ex vivo retinal tissue. This method has been used to successfully assess metabolic activities in adult mouse retina and demonstrate its application in investigating cellular mechanisms of aging and disease.
Mitochondria are essential organelle that regulates cellular metabolism, signaling, homeostasis, and apoptosis by coordinating multiple crucial physiological processes1. Mitochondria serve as the powerhouse in the cell to generate adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS) and provide energy that supports almost all cellular events. The majority of cellular oxygen is metabolized in mitochondria, where it serves as the final electron acceptor in the electron transport chain (ETC) during aerobic respiration. Low amounts of ATP can also be produced from glycolysis in the cytosol, where glucose is converted to pyruvate, which can be further converted to lactate or be transported into mitochondria and oxidized to acetyl-CoA, a substrate in the tricarboxylic acid cycle (TCA cycle).
The retina is one of the most metabolically active tissues in mammals2, displaying high levels of mitochondrial respiration and extremely high oxygen consumption3. The rod and cone photoreceptors contain a high density of mitochondria4, and OXPHOS generates most ATP in the retina5. In addition, the retina also relies heavily on aerobic glycolysis6,7 by converting glucose to lactate5. Mitochondrial defects are associated with various neurodegenerative diseases8,9; and with its unique high energy demands, the retina is especially vulnerable to metabolic defects, including those affecting mitochondrial OXPHOS4 and glycolysis10. Mitochondrial dysfunction and defects in glycolysis are implicated in retinal11,12 and macular13 degenerative diseases, age-related macular degeneration10,14,15,16, and diabetic retinopathy17,18. Therefore, accurate measurements of mitochondrial respiration and glycolysis can provide important parameters for assessing the integrity and health of the retina.
Mitochondrial respiration can be measured through the determination of oxygen consumption rate (OCR). Given that the conversion of glucose to pyruvate and subsequently to lactate results in extrusion of protons into and acidification of the extracellular environment, measurements of the extracellular acidification rate (ECAR) provide an indication of glycolysis flux. As the retina is composed of multiple cell types with intimate relationships and active synergy, including the exchange of substrates6, it is imperative to analyze mitochondrial function and metabolism in the context of whole retinal tissue with intact lamination and circuitry. For the past several decades, the Clark type O2 electrodes and other oxygen microelectrodes have been used to measure oxygen consumption in the retina19,20,21. These oxygen electrodes have major limitations in sensitivity, requirement of a large sample volume, and the need for continuous stirring of suspending sample, which usually leads to the disruption of cellular and tissue context. The protocol described here was developed using a microplate-based, fluorescence technique to measure mitochondrial energy metabolism in freshly dissected ex vivo mouse retina tissue. It allows mid-throughput real-time measurements of both OCR and ECAR simultaneously using a small sample (1 mm punch) of ex vivo retinal tissue while avoiding the need for suspension and continuous stirring.
Demonstrated here is the experimental procedure for mitochondrial stress assay and glycolytic rate assay on freshly dissected retinal punch disks. This protocol allows the measurement of mitochondria-related metabolic activities in an ex vivo tissue context. Different from the assays performed using cultured cells, the readings obtained here reflect combined energy metabolism at the tissue level and are influenced by interactions between the different cell types within the tissue. The protocol is modified from a previously published version22,23 to adapt to the new generation of the Agilent Seahorse extracellular flux 24-wells (XFe24) analyzer with Islet Capture plate. The assay medium, injection compound concentrations, and number/duration of assay cycles have also been optimized for retinal tissue. A detailed step-by-step protocol is given for the preparation of retinal punch disks. More information on the program setup and data analysis can be obtained from the manufacturer's user guide24,25,26.
All mouse protocols were approved by the Animal Care and Use Committee of the National Eye Institute (NEI ASP# 650). Mice were housed in 12 h light-dark conditions and cared for by following the recommendations of the Guide for the Care and Use of Laboratory Animals, the Institute of Laboratory Animal Resources, and the Public Health Service Policy on Humane Care and Use of Laboratory Animals.
1. Hydrating sensor cartridge and preparation of the assay medium
2. Coating mesh inserts of islet capture microplate
3. Preparing injection compounds
4. Retinal dissection and retinal punch preparation
5. Loading the sensor cartridge injection ports and calibration
6. Loading the islet capture plate and start assay run
7. Run termination and data storage
8. Saving the retinal punch sample
9. Data analysis
The data reported here are representative mitochondrial stress assay showing OCR trace (Figure 1) and glycolytic rate assay showing OCR trace and ECAR trace (Figure 2), which were performed using freshly dissected 1 mm retinal punch disks from 4 months old transgenic Nrl-L-EGFP mice36 (C57B/L6 background). These mice express GFP specifically in rod photoreceptors without altering normal retinal development, histology, and physiology and have been widely used as wild-type controls in retinal research. Two Nrl-L-GFP littermate mice were used were used in the assays presented here. GFP expressed in the Nrl-L-GFP mice does not interfere with the measurements of OCR and ECAR in this protocol. Five retinal punches were taken from each retina. Ten of the retinal punches were used for mitochondrial stress assay and the other 10 were used for glycolytic rate assay. Seahorse XF DMEM medium, pH 7.4 (constituted with 6 mM glucose, 0.12 mM pyruvate, and 0.5 mM glutamine) and Seahorse XFe24 Islet Capture plates were used in the experiments. The representative data presented here were obtained using the same 1 mm diameter puncher but was not normalized with respect to the DNA/protein content.
In mitochondrial stress assay, the uncoupler Bam1537 was injected after establishing the OCR baseline, leading to enhanced OCR to the maximal level. Rotenone and Antimycin A were injected to inhibit mitochondria respiration at complex I and complex III, respectively, resulting in OCR to drop to the minimal level (Figure 1). The difference between the maximal level of OCR and the last measurement of the basal OCR level reflects mitochondrial reserve capacity (MRC). The MRC is calculated to be 19.2%±3.4% using Eq. 2, consistent with previously measured MRC values in retinas of ~3 months old Nrl-L-EGFP mice using the previous generation Seahorse XF24 analyzer22,38.
In the glycolytic rate assay, Rotenone and Antimycin A were injected after establishing the baseline for the total ECAR. With the production of ATP from OXPHOS halted, the tissue is forced to rely on glycolysis for energy, and an increase in the extracellular release of lactate drives ECAR to the maximal level. Glycolysis is ceased by injection of 2-DG, which competes with glucose for hexokinase binding, causing ECAR to drop to the minimal level (Figure 2). Mitochondria contributed ECAR (mitoECAR) can be calculated from the mitoOCR value (Eq. 4). Glycolysis contributed ECAR glycoECAR is calculated and plotted by subtracting mitoECAR from totalECAR. The difference between maximal level of glycoECAR and the last measurement of glycoECAR basal level reflects the glycolysis reserve capacity (GRC). Here, the GRC is calculated to be 35.7% ± 3.4% using Eq. 5.
As a highly glycolytic tissue, lactate production from the retina accounts for a major source of extracellular acidification, as revealed by the small difference of glycoECAR from the totalECAR. Interestingly, ECAR measurement does not plateau immediately following the Rot/AA injection but drops after the second measurement. The retinal punch disk is an intact ex vivo system composed of different cell types, including the Müller glia cells, which are known to receive lactate (glycolysis end product) released from the photoreceptors6. Hence, a drop in ECAR measurement following the Rot/AA injection is likely explained by increased removal of lactate from the intercellular space, slowing down/preventing its release into the medium.
Figure 1: Mitochondrial stress assay. The plotted graph shows OCR trace from 1 mm retinal punch disks in Seahorse XF DMEM buffer, supplemented with 6 mM of glucose, 0.12 mM of pyruvate and 0.5 mM of glutamine. Each data point represents the average of measurements from 10 wells. Error bar = standard error. MRC is calculated to be 19.2%±3.4%. Please click here to view a larger version of this figure.
Figure 2: Glycolytic rate assay. The plotted graph shows the measured OCR trace, ECAR trace (totalECAR), and the calculated glycolysis contributed ECAR (glycoECAR) from 1 mm retinal punch disks in Seahorse XF DMEM buffer supplemented with 6 mM of glucose, 0.12 mM of pyruvate, and 0.5 mM of glutamine. Each data point represents the average of measurements from 10 wells. Error bar = standard error. GRC is calculated to be 35.7%±3.4% Please click here to view a larger version of this figure.
Provided here are detailed instructions for performing microplate-based assays of mitochondrial respiration and glycolysis activity using ex vivo, freshly dissected retinal punch disks. The protocol has been optimized to: 1) ensure the use of a suitable assay medium for ex vivo retinal tissue; 2) employ proper size of retinal punch disks to obtain OCR and ECAR readings that fall within the machine's optimal detecting range; 3) coating mesh inserts to enhance the adhesiveness of retinal punch for stable reading during the measuring cycle; 4) use of optimal concentration of each injected drug compounds; and 5) ensure altered cycle length to reach a plateau of mitochondrial states at each step. The reagents and protocol have been modified from a previously published version23 to adapt to the new generation Seahorse XFe24 machine. Instead of the Ames' buffer used in the previous protocol23, a basic Seahorse DMEM medium is used here to allow the custom constitution of fuel source by adding glucose, glutamine, and pyruvate separately. This also makes it possible to perform various assays where a specific fuel substrate is supplied or deprived from the medium. In the assays presented here, the medium was constituted to the same concentration of glucose (6 mM), glutamine (0.5 mM), and pyruvate (0.12 mM) as in Ames' buffer, which are proven suitable for retinal tissue. Another advantage of this medium (with 5 mM HEPES) over the Ames' buffer (with 22.6 mM NaHCO3) is its low buffer capacity, which ensures sensitive and accurate measurement of ECAR28.
Both mitochondrial stress and glycolytic rate assays can be performed following the protocol described here with high precision, as evidenced by the tight standard error values between replicating wells. However, it is worthwhile to note the factors that can contribute to data variability. Avoid cell death in retinal tissue. The entire dissection process should be performed in ice-cold 1x PBS, and the process from enucleation of eyes to putting islet capture plate containing the retinal punch into the machine should not exceed 2 hours. Caution should be taken during the dissection of the retina cup to avoid any damage to the retinal tissue, and punches should not be taken from areas damaged by dissection. New, sharp biopsy puncher should be used in each experiment, and change the puncher when the edge is dull or bent to ensure consistency and accuracy in cutting retinal punches at 1 mm diameter. Try to get the retinal disks punched at equidistant from the optic nerve head to avoid regional variations (center versus peripheral). After the assay, check each well for any sign of the retinal punch being detached from the mesh insert. When a retinal punch has poor adhesion on mesh insert or detaches during measurement, the distance from senser probe to the tissue will change, affecting the readings. Omit the data from such wells with detached retinal punch.
Measurement of the real-time mitochondrial metabolism in intact retinal tissue has broad applications and can provide useful information for various studies. These assays have been used to measure mitochondrial respiration in retinal tissues from mice of different genetic backgrounds to reveal their intrinsic difference in mitochondrial activity39,40. It was also used to study changes in mitochondrial energy metabolism during aging of the retina38. By providing different fuel substrates and utilizing various inhibitors targeting different metabolic pathways, it provides insights to the preference of the cell/tissue on certain fuel sources22,38. Furthermore, comparison on OCR and MRC between wild-type mouse and mouse models of inherited retinal degeneration can provide evidence of mitochondrial defects in degenerating retina22.
There are limitations of this technique. The islet capture plate used in these assays only contains 24 wells; hence, it is only able to provide mid-throughput analysis. The data quality from this method is contingent upon the quality of retinal punch disks and viability of cells. Also, retinal dissection and retinal punch disks preparation is a time-consuming process, rendering it less feasible to high-throughput analysis on live ex vivo retinal tissues even when 96-well plates are available. Compared to a monolayer of cultured cells, penetration of drug compound into the retinal tissue also affects data readout. In addition, the measured OCR and ECAR values represent the total performance of the entire tissue, which is composed by many different cell types; hence, one needs to consider the relationship and interactions among different neuronal and glial cells in the retina while interpreting the data. Specific experimental designs should be implemented by tailoring to each project. It is recommended that one includes 3 to 5 retinal punches (from same eye or same mouse) as technical replicates and use samples from 3 or more mice as biological replicates.
The authors have nothing to disclose.
This work is supported by the Intramural Research Program of the National Eye Institute (ZIAEY000450 and ZIAEY000546).
1X PBS | Thermo Fisher | 14190-144 | |
2-Deoxy glucose (2-DG), 500 mM stock solution | Sigma | D6134 | Dissolve in Seahorse XF DMEM medium, prepare ahead of time |
30-gauge needle | BD Precision Glide | 305106 | |
Antimycin A, 10 mM stock solution | Sigma | A8674 | Dissolve in DMSO, prepare ahead of time |
Bam15, 10 mM stock solution | TimTec | ST056388 | Dissolve in DMSO, prepare ahead of time |
Biopsy puncher, 1 mm | Integra Miltex | 33-31AA | |
Cell-Tak | Corning Life Sciences | CB40240 | |
CO2 asphyxiation chamber | |||
Dissection forceps-Dumont #5 | Fine Science Tools | 11251-10 | Stright tip |
Dissection forceps-Dumont #7 | Fine Science Tools | 11274-20 | Curved tip |
Dissection microscope | |||
DMSO | Sigma | D2438 | |
Graefe forceps | Fine Science Tools | 11051-10 | Curved, Serrated tip |
Microscissors | Fine Science Tools | 15004-08 | Curved tip |
NaOH solution, 1 M | Sigma-Aldrich | S8263 | Aqueous solution, prepare ahead of time |
Rotenone, 10 mM stock solution | Sigma | R8875 | Dissolve in DMSO, prepare ahead of time |
Seahorse calibration medium | Agilent | 100840-000 | |
Seahorse XF 1.0 M glucose | Agilent | 103577-100 | |
Seahorse XF 100 mM pyruvate | Agilent | 103578-100 | |
Seahorse XF 200 mM glutamine | Agilent | 103579-100 | |
Seahorse XF DMEM medium | Agilent | 103575-100 | pH 7.4, with 5 mM HEPES |
Seahorse XFe24 Islet Capture FluxPak | Agilent | 103518-100 | Containing Sensor Cartridge and Islet Capture microplate |
Seahorse XFe24, Extra Cellular Flux Analyzer | Agilent | ||
Sodium bicarbonate solution, 0.1 M | Sigma-Aldrich | S5761 | Aqueous solution, prepare ahead of time |
Superfine eyelash brush | Ted Pella | 113 |