FTY720 prevents excitotoxic neuronal death in cerebrocortical primary and organotypic cultures. (A) Neuronal primary cultures 9 DIV were pre-treated with increased concentration of FTY720 (10 to 1,000 nM) for 24 h and then stimulated with N-methyl-D-aspartic acid (NMDA) (25 to 100 μM) in the same conditioned medium. After 24 h of NMDA treatment, lactate dehydrogenase (LDH) released in the medium was measured and mean ± SEM were reported as per cent of non-treated neurons (n = 6, except for FTY720 1 μM, n = 2; two-way ANOVA: * or #
P < 0.05; ** or ##
P < 0.01; #
vs control). (B) Organotypic cultures were pre-treated 24 h with FTY720 (10 and 100 nM) and then incubated with NMDA (50 μM) for 30 min, in the presence or in the absence of FTY720 or MK801 (50 μM, 10 min pre-incubation). FTY720 was added for additional 24 h. The damage was detected by PI uptake 24 h after treatment with NMDA and propidium iodide (PI) intensity was measured as corrected total fluorescence (CTF). Data are expressed as the mean ± SEM (n ≥ 4 independent experiments; in each experiment, at least two slices for condition were used) and referred to percent of NMDA-treated neurons (dotted line). Student’s t test: *P < 0.05 and **P < 0.01 vs NMDA-treated cells. (C) Representative photographs showing PI uptake in cortical slices treated as in (B). Scale bar: 1 mm.