Abstract

DO.24.07

Quantification of dead and alive ganglion cells in retinal whole mounts – a methodological comparison

Lasseck J., Grieshaber P., Lagrèze W.
Universitäts-Augenklinik Freiburg

Objective: A reliable quantification of retinal ganglion cells (RGCs) is essential for in vivo analysis in neuroprotection. The gold standard quantification is retrograde labelling (RL) of RGCs. A fluorescent dye (e.g. fluorogold (FG)) has to be injected into the superior colliculus. The axon terminals took up the dye within six days; thereafter it is transferred retrogradely into the cells soma in the retina. This is a highly standardised and specific method, however, it requires an operation of the animal and the experiment is elongated. Furthermore, a differentiation of dead and viable cells is impossible (a dead FG-positive (⁺) cell evades quantification only after complete microglia-phagocytosis). To address this issue, we correlated the gold standard with a cell viability stain using calcein.
Methods: The left optic nerve of rats was crushed six days after stereotactical injection of FG into both superior colliculi. The corresponding right eyes served as controls. Retinal tissue was harvested after five days and further processed for whole mount preparation. After removing the vitreous, retinas were incubated for one hour in culture media containing calcein. Afterwards retinas were analysed in a defined area in all eccentricities under a fluorescence microscope using the appropriate bandpass emission filters. Results were expressed as means and standard deviation.
Results: In control retinas (n=3, 31 analysed squares), 1881±461 cells/mm² stained FG⁺, 1828±444 cells/mm² stained calcein⁺ and 1573±355 cells/mm² (83.7%) stained calcein⁺ and FG⁺. In retinas after optic nerve crush (n=4, 42 analysed squares), 1482±569 cells/mm² stained FG⁺, 1303±438 cells/mm² stained calcein⁺ and 893±334 cells/mm² (60.3%) stained calcein⁺ and FG⁺.
Conclusions: FG⁺/calcein negative (^-) cells are most probable dead RGCs with preserved morphology not yet cleared by phagocytosis. FG^-/calcein⁺ cells are other cells than RGCs. The additional staining with calcein most reliably reflects the number of vital RGCs and may help to characterise neuroprotective agents.

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