Figure 4

Co-activation of TLR4 and TLR2 causes enhanced microglia-dependent neuronal injury in vitro. (A) Primary cortical neurons were supplemented with purified microglia. Co-cultures were stimulated with 100 ng/mL LPS, 100 ng/mL Pam3CysSK4 (Pam), 1 mM loxoribine (lox), or 0.1 μm CpG ODN (CpG) alone or with pairwise combinations of the ligands, as indicated. PBS served as control. After 72 h co-cultures were immunostained with NeuN Ab (neurons, red) and IB4 (microglia, green). Scale bar, 100 μm. (B) Cortical neurons alone or supplemented with microglia were incubated with the TLR ligands named above, as indicated. After 72 h, NeuN⁺ and IB4⁺ cells were quantified and expressed as relative neuronal viability and relative microglial viability, respectively, as indicated. Each condition was performed in duplicate and averaged. Mean ± SEM from four to five individual experiments with ANOVA followed by Bonferroni post-hoc test of control vs. each treatment and of single vs. pair-wise stimulation (relative neuronal viability: co-cultures: *; neurons: †, relative microglial viability: co-cultures *), as indicated. Two-way ANOVA with Bonferroni post-hoc test between indicated groups testing if the neurotoxic effect is dependent on microglia (#). P* <0.05; P** <0.005; P*** <0.001; P# <0.05; P## <0.005; P### <0.001; P † <0.05; P†† <0.005.