Figure 5

Blockade of adenosine A _2A receptors prevents the interleukin (IL)-1β-induced exacerbation of glutamate-mediated excitotoxicity in hippocampal cultured neurons. Rat hippocampal neuronal cultures with 7 days in vitro (DIV) were exposed to vehicle (Krebs buffer) or to 100 ng/ml IL-1β , added 5 minutes before vehicle or 100 μmol/l glutamate (Glu) was added for 25 minutes, in the absence or in the presence of the selective A_2A receptor antagonist, SCH58261 (50 nmol/l), as indicated below each bar. Neurons were washed twice with Krebs buffer (pH 7.4) and fresh Neurobasal medium (with or without SCH58261) was added. Neurons were kept for 24 hours in the incubator (37°C, 95% O₂/ 5% CO₂) until analysis of neuronal viability using SYTO-13 plus propidium iodide (PI). (A) Representative images of SYTO-13 (green fluorescence) plus PI (red fluorescence) staining of nucleic acids for the indicated experimental conditions; (B) average results. (C) The significant findings were further confirmed using the lactate dehydrogenase (LDH) assay. Note that neither IL-1β nor SCH58261 alone had any effect on cell viability but IL-1β exacerbated Glu-induced neurotoxicity, which was abrogated by SCH58261. (D) The p38 inhibitor SB 203580 (10 μmol/l) and the JNK inhibitor SP600125 (10 μmol/l) respectively prevented and attenuated the exacerbation by IL-1β of glutamate-induced neurotoxicity, without affecting neuronal viability. Results are mean ± SEM of three to seven experiments (corresponding to the different cultures); *P < 0.05, **P < 0.01 and ***P < 0.001 compared with control (absence of any added drug), #P < 0.05, ##P < 0.01 and ###P < 0.001 compared between the indicated groups.