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W. A. Lagreze, P. Grieshaber, J. Lasseck; Staining of Fluorogold-Prelabeled Retinal Ganglion Cells With Calcein-AM: A New Method for Cell Vitality Assessment. Invest. Ophthalmol. Vis. Sci. 2009;50(13):126.
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The number of retinal ganglion cells (RGC) is often used as an outcome measure in neuroprotection experiments. The gold standard is retrograde labeling, e.g. with fluorogold (FG). However, it requires a craniotomy and differentiation of viable and dead cells is not possible, because dying cells evade quantification only after complete microglial phagocytosis. To differentiate between viable and dead but still existent RGC, we additionally stained FG-labeled RGC with calcein-AM.
The left optic nerves of 19 rats were crushed 6 days after stereotactical injection of FG into both superior colliculi. The right eyes served as controls. Retinal flat mounts were prepared after 2, 5, 8 or 11 days after the crush and incubated for 1 hour in culture media containing 0.01% calcein-AM after previous removal of the vitreous. RGC densities were determined in defined areas at different eccentricities under a fluorescence microscope using the appropriate bandpass emission filters (418 nm for FG, 515 nm for calcein-AM). Double positive RGC were counted after merging both filters.
Normalized to controls, 100% of FG-positive RGC were visible on day 2, 79% on day 5, 43% on day 8, and 22% on day 11. The percentages of FG-positive RGC staining with calcein-AM were 83% in controls, 68% on day 2, 48% on day 5, 26% on day 8, and 9% on day 11.
After optic nerve crush, the decay rate of prelabeled RGC appears accelerated and becomes more linear when counting only viable RGC being positive for calcein-AM as well. FG-positive and calcein-AM-negative cells are most probably dying RGC with preserved morphology not yet cleared by phagocytosis. Hence, additional staining with calcein-AM seems to identify vital RGC and may help to characterize neuroprotective agents more precisely.
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