All animal handling was performed according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Primary retinal and pure hippocampal cultures were obtained from Wistar rat embryos at gestational day 18 as already described
18,19 and were used at 12–14 day in vitro (DIV). Each cell culture, obtained from the embryos of one pregnant rat, was considered as a single experiment. Retinal cultures were obtained from retinas dissociated in trypsin; cells were seeded onto poly-
l-lysine-coated cell culture plates or glass coverslips and grown in synthetic cell culture medium (MEM; Sigma-Aldrich, St. Louis, MO) with 10% fetal calf serum (FCS, Life Technologies, Milan, Italy), giving rise to a mixed glial–neuronal cell population. Hippocampal cultures were obtained from dissected hippocampi. After dissociation in trypsin, cells were seeded on poly-
l-lysine-coated 24-well plates in MEM/FCS. To obtain pure neuronal cultures, the medium was replaced after 2 h with neurobasal medium supplemented with B27 (NBM/B27, Life Technologies); at 1 DIV, 5 μM arabinosyl-cytosine (Sigma-Aldrich) was added to prevent glial proliferation. In these conditions, astrocytes were < 1%.
18 Curcumin (Sigma-Aldrich) was diluted in ethanol as 10 mM stock solution. Cell cultures were pretreated with 5 or 15 μM curcumin for 20 minutes, 2, 4, 6, 24 h, or treated after the agonists. Excitotoxic neuronal damage was examined in ”delayed” toxicity protocols, which characteristically triggers an apoptotic pathway of cell death.
20 Cell cultures were treated with NMDA (1 mM) or AMPA (0.5 mM) or Kainate (0.5 mM) for 20 minutes. Medium was then replaced with fresh NBM/B27 and the cells fixed after 24 h. To evaluate protective effects of curcumin against AMPA- and Kainate-induced apoptosis, we treated cells in the presence of the NMDAr inhibitor MK-801 (5 μM). The protein synthesis inhibitor cycloheximide (CHX) was prepared as a stock solution in DMSO (100 mg/mL). {(
R)-[(
S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydro-quinoxalin-5-yl)-methyl}-phosphonic acid (NVP-AAM077, a kind gift of Yves Auberson and Sandra Kirchhoff of the Novartis Institutes for Biomedical Research, Basel, Switzerland), a specific NR2A antagonist, was prepared as a stock solution in 0.1 N NaOH. Ifenprodil (IFN, Sigma-Aldrich), an NMDA receptor subunit type 2B (NR2B) antagonist, was diluted in cell culture medium.