Over-expression of MIP-2γ makes neurons more sensitive to glutamate toxicity. Cortical astrocytes left untreated (control) or transfected with the plasmid pAAV-IRES-hrGFP (mock) or with pAAV-MIP-2γ-hrGFP (MIP-2γ), or together with siRNA-1 (MIP-2γ +siRNA-1) or with pBS/U6 (MIP-2γ + BS/U6), or treated with LPS (LPS) or together transfected with siRNA-1 (LPS + siRNA-1) or with pBS/U6 (LPS+ BS/U6). Neurons were co-cultured with untreated or treated astrocytes for 4 days; after this time, glutamate (50 μM) was added and, 4 hours later, the neuronal survival was determined using an LDH release assay (a) and an MTT assay (b) and normalized to normal N/A co-cultures. (c,d) In parallel experiments, cell viability was assessed by immunocytochemistry using a MAP-2 antibody to detect neurons. (e) Astrocyte conditioned media (AM) derived from different groups was applied to neuronal cell cultures and the neuronal survival was determined using an MTT assay. Results are expressed as the percentage of surviving neurons compared with control cultures (control). Mock–AM, AM derived from pAAV-IRES-hrGFP transfected astrocytes. MIP-2γ–AM, AM derived from pAAV-MIP-2γ-hrGFP transfected astrocytes. MIP-2γ + BS/U6-con –AM, AM derived from astrocytes co-transfected pAAV-MIP-2γ-hrGFP with pBS/U6-con. MIP-2γ+siRNA-1 –AM, AM derived from astrocytes co-transfected pAAV-MIP-2γ-hrGFP with siRNA-1. Shown are the mean ± SEM for three experiments. *P <0.05; **P <0.01 compared with the control group; §
P <0.05 compared with the MIP-2γ group; ¶
P <0.05 compared with the LPS group (ANOVA followed by a Bonferroni test). ANOVA, analysis of variance; LPS, lipopolysaccharide; MAP-2, microtubule-associated protein 2; MIP-2γ, macrophage inflammatory protein-2γ; MTT, 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide; SEM, standard error of the mean; siRNA, small interfering RNA.