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Hui Li, Lin Wang, Grant Cull; A novel method to assess astrocyte calcium in rat retina. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4214.
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© ARVO (1962-2015); The Authors (2016-present)
To validate the specificity of calcium indicator Fluo-4 AM (F4) binding to the retinal astrocytes and a method for in vivo monitoring of retinal astrocyte calcium (Ca2+ ) activity with a confocal scanning laser ophthalmoscopy (cLSO).
(1) Three rat retinas were isolated and loaded with F4 for 90 min in a culture dish. The retinas were imaged with a microscope for F4 positive cells (F4+). Following the imaging, the retinas underwent immunocytochemistry (IHC) staining with glial fibrillary acidic protein (GFAP) antibody, a specific marker for astrocytes. GFAP positive cells (GFAP+) were imaged and overlaid on the images for F4+ cells. The agreement of overlay between the two images was evaluated with Image J.(2) To determine the average time required for F4 fluorescence intensity to reach a stable level after intravitreal (IV) injection, each 5 rat eyes was injected with 5 µl F4 solution (0.1µg/µl). The F4 fluorescence intensity, representing relative astrocytic Ca2+ concentration in the retina, was imaged with the cLSO in fluorescence mode (FM, 488 nm) in vivo. The imaging process began 10 minutes after F4 injection and was repeated every 15 minutes thereafter for the next 2 hours. At each time point, 30-second consecutive images were recorded. The time course change in F4 fluorescence intensity was quantified.
(1) Strong fluorescence appeared in cells across the isolated retina particularly along the sides of both arterioles and venules. Overlay of the F4 images with that of GFAP IHC staining in the same retina showed that majority of the F4+ cells were also GFAP+. However, the F4+ cells showed fewer processes compared with GFAP+ cells. (2) cSLO imaging showed fluorescence appeared ten minutes after the IV injection of F4. The F4 fluorescent intensity increased with time and stabilized at ~90 minutes post-injection for at least 30 minutes. However, unlike the F4+ cells under the microscope, no individual cells could be identified due to the limitation in resolution of cSLO. One way RM-ANOVA showed a significant increase in fluorescent intensity over time (P<0.0001). Post-hoc analysis indicated no significant difference in fluorescent intensity from 90 to 120 minutes post-injection.
F4 appeared to bind Ca2+ selectively within retinal astrocytes. F4 fluorescence could be imaged in vivo with the cLSO after IV injection and this novel method will allow further studies for the role of astrocytes in the retina.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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