title: Staining of fluorogold-prelabeled retinal ganglion cells with calcein-AM: A new method for assessing cell vitality.
abstract: OBJECTIVE
  The number of retinal ganglion cells (RGC) is often used as an outcome measure in neuroprotection. The gold standard for staining RGC is retrograde labeling, e.g. with fluorogold (FG). However, this method alone does not permit to differentiate between viable and dead cells, because dying cells only avoid being counted once they have undergone complete microglial-phagocytosis. To differentiate between viable and dead but still existent RGC, we additionally stained FG-labeled RGC with calcein-acetoxymethylester (CAM).
  
  
  METHODS
  The left optic nerves of rats were crushed 6 days after stereotactical injection of FG into both superior colliculi. The right eyes served as controls. Retinal whole mounts were prepared 2, 5, 8 or 11 days after optic nerve crush (ONC), and incubated for 30min in culture media containing 0.01% CAM. RGC densities were determined in defined areas at different eccentricities under a fluorescence microscope using the appropriate filters. Twice-positive RGC were counted after merging both filters.
  
  
  RESULTS
  The loss of RGC induced by ONC is identified earlier when these cells are detected by FG+CAM rather than by FG-labeling alone. The percentages of FG-positive RGC stained with CAM were 83% in controls, 68% on day 2, 48% on day 5, 26% on day 8, and 9% on day 11 after ONC. The decay rate of FG-prelabeled RGC appears accelerated and becomes more linear when only viable RGC positive for CAM are counted.
  
  
  CONCLUSIONS
  The staining of FG-prelabeled RGC with CAM permits the discrimination between dead and viable RGC in retinal whole mounts, which enables to quantify RGC degeneration earlier after injury than by using microglial-phagocytosis-dependant retrograde labeling alone.
authors.affiliation: University Eye Hospital Freiburg, Killianstraße 5, 79106 Freiburg im Breisgau, Germany.
authors.forename: Philippe
authors.initials: P
authors.lastname: Grieshaber
authors.forename: Wolf Alexander
authors.initials: WA
authors.lastname: Lagrèze
authors.forename: Christian
authors.initials: C
authors.lastname: Noack
authors.forename: Daniel
authors.initials: D
authors.lastname: Boehringer
authors.forename: Julia
authors.initials: J
authors.lastname: Biermann
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D002452:Cell Count; D002470:Cell Survival / Q000187:drug effects; D005260:Female; D005452:Fluoresceins / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000494:pharmacology*; D008297:Male; D009409:Nerve Crush; D020221:Optic Nerve Injuries; D051381:Rats; D017207:Rats, Sprague-Dawley; D012165:Retinal Ganglion Cells / Q000166:cytology* / Q000187:drug effects*
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; C085925:calcein AM