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Michelle M Giarmarco, Ken J. Lindsay, Jianhai Du, Whitney Marie Cleghorn, Susan E Brockerhoff, James B Hurley; Imaging Real-time Glucose Uptake in Retina Reveals Distinct Transport Among Cell Types. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5503.
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© ARVO (1962-2015); The Authors (2016-present)
Glucose transport is central to retinal metabolism and health, particularly for highly glycolytic photoreceptors, and many retinal cell types make limited or no contacts with the blood supply. How glucose and nutrients are delivered to the different cell types in the retina and how this contributes to retinal health is unknown. We evaluated the distribution of the glucose transporter GLUT1 by immunohistochemistry (IHC), and visualized glucose uptake in real time by in situ live imaging mouse and zebrafish retinas with fluorescent glucose analogs.
For IHC, mouse retinas were fixed in 4% paraformaldehyde and cut into 20-µm sections. Sections were incubated overnight with primary antibodies against CRALBP, a marker for Müller glia, and GLUT1, followed by incubation with fluorescent-tagged secondary antibodies. Sections were imaged via confocal microscopy. For live glucose uptake imaging experiments, mouse and zebrafish retinas from freshly enucleated eyes were dissected, flat-mounted onto filter paper, incubated in BODIPY to stain membranes, and sliced into 400-µm sections. Slices were rotated 900 and the filter paper edges buried in strips of wax on a coverslip, then transferred to a temperature-controlled perfusion chamber for confocal imaging experiments. The resulting retinal slices were perfused with NBD-tagged fluorescent glucose analogs (NBDG) to monitor glucose uptake in real time.
IHC demonstrated differential expression of GLUT1 among the layers of mouse retina. The described in situ preparation yields clear transverse sections of retina, which remain viable for several hours. NBDG imaging revealed that glucose uptake is very rapid and occurs at different rates between specific retinal cell types.
GLUT1 localization in the mouse retina coincides with in situ glucose uptake, although rates of glucose uptake vary between retinal cell layers, indicating potential metabolic cooperation. Further investigation with transgenic markers for Müller glia and photoreceptors will clarify the roles of these cells in nutrient distribution throughout the retina.
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