Measuring Uptake of the Glucose Analog, 6-(N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Amino)-6-Deoxyglucose, in Intact Murine Neural Retina

Our lab investigates metabolic changes in the retina and throughout the central nervous system that occur with age as well as in neurodegenerative disease. Our overriding goal is to leverage these findings to identify new therapeutic targets to treat age-related neurodegenerative diseases. Detecting glucose uptake in retinal tissue is often expensive and time consuming, requiring complicated imaging modalities or even radioactive substances.

We established this protocol to provide a novel ex vivo technique to measure fluorescent glucose analog uptake in retinal samples. The protocol that we have developed provides a straightforward and cost effective strategy to measure ex vivo uptake of fluorescent glucose analogs into whole intact retina. This method is also advantageous as it allows researchers to measure multiple samples simultaneously.

This protocol will enable researchers to evaluate changes in retinal uptake of fluorescent glucose analogs, which will help us to better understand how the retina utilizes glucose as an energetic substrate both with age and in healthy and disease states. To begin, prepare a stock solution of five millimolar 6-NBDG in a mixture of 50%DMSO and PBS. Store 500 microliter aliquots of the solution at minus 20 degrees Celsius.

Place a 50 milliliter conical tube containing Neurobasal-A Medium without glucose on ice. Label 1.5 milliliter micro centrifuge tubes as glucose starvation, 6-NBDG incubation, sonication, and sample collection for the pierce assay. Add 100 microliters of chilled Neurobasal-A Medium to the pre-labeled sonication tubes on ice.

Power on the plate reader. Set up plate parameters in the plate reader software using the fluorescence endpoint assay feature with excitation set to 483 nanometers, emission set to 550 nanometers, and cutoff set to 530 nanometers. Dilute the five millimolar stock solution of six NBDG in Neurobasal-A Media to prepare a 500 micromolar working solution and cover it with aluminum foil to protect it from light.

Pipette 200 microliters of the working solution into the pre-labeled 6-NBDG incubation tubes and cover as demonstrated. After isolating the retina from a euthanized mouse using a trimmed transfer pipette or tweezers, transfer the retina into a 1.5 milliliter tube containing 200 microliters of Neurobasal-A Medium for glucose starvation. Incubate the tube in a 37 degrees Celsius water bath for 20 minutes.

Transfer the retina into a pre-labeled tube containing 200 microliters of 500 micromolar 6-NBDG in Neurobasal-A Medium. Incubate the tube in a 37 degrees Celsius water bath for 60 minutes. To prepare 6-NBDG standards, label eight 1.5 milliliter micro centrifuge tubes as standards one to eight.

For standard one, pipette 32 microliters of 500 micromolar 6-NBDG stock solution into the labeled tube. Then add 368 microliters of Neurobasal-A Medium to the tube and mix thoroughly by pipetting up and down. Cover all tubes with aluminum foil.

After the 60 minute 6-NBDG incubation, remove the tubes from the 37 degrees Celsius water bath. To wash the retina, remove the 6-NBDG solution using a pipette without disturbing the retinal tissue and add 500 microliters of fresh ice cold Neurobasal-A Medium on ice. Next, using forceps, carefully transfer the retina to the sonication tubes containing Neurobasal-A Medium on ice.

Using small dissection scissors, chop the retina into small pieces. Sonicate each retina for five to 10 seconds at an amplitude of 10, and immediately place the tube back on ice. Centrifuge the samples at 21, 130 G for 15 minutes at four degrees Celsius.

Transfer 100 microliters of supernatant into a 96-well plate. Insert the plate into the plate reader. Select the fluorescence endpoint assay with specified wavelengths.

Run the assay and save the results. After the run, pipette the samples from the 96-well plate into pre-labeled tubes for the pierce assay. Using the pre-diluted pierce assay protein standards, add 10 microliters of each standard in duplicate into a 96-well clear bottom plate.

Then transfer 10 microliters of sample into the plate. Add 150 microliters of pierce reagent to all wells containing standards or samples. Cover the plate and mix on a plate shaker at medium speed for one minute.

Incubate the plate at room temperature for five minutes. Insert the plate into the plate reader and select the absorbance endpoint assay with a wavelength set to 660 nanometers. Measure the absorbance of the standards and samples, save the results and plot a standard curve to determine the total microgram protein in each sample.

Glucose fluorescence measurements showed an increase in 6-NBDG uptake in wild type mouse retina after 30 minutes of incubation, peaking at 60 minutes, followed by a decrease in arbitrary units after 90 minutes. The addition of the GLUT1 inhibitor, bay 876, reduced 6-NBDG uptake by 24%indicating the involvement of GLUT1 independent mechanisms in the wild type mouse retina.