Figure 5

Simultaneous activation of TLR4 by HSP60 and of TLR7 by loxoribine causes enhanced neuronal injury . Co-cultures of neurons and microglia as well as neurons alone were stimulated with 1 μg/mL HSP60, 100 ng (A) or 1 μg (B) /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, NeuN⁺ cells (A -C) or after 24 h, cells positive for activated caspase-3 (D) were quantified and expressed as relative neuronal viability or caspase-3⁺ cells/field, respectively. Each condition was performed in duplicate and averaged. Mean ± SEM from (A) three to four and (B, C) four individual experiments with ANOVA followed by Bonferroni post-hoc test of control vs. each treatment and of single vs. pair-wise stimulation (*), as indicated. (A) In addition, data were analyzed by two-way ANOVA with Bonferroni post-hoc test between indicated groups testing if effect is dependent on microglia (#). (D) Mean ± SEM from four individual experiments with ANOVA followed by Bonferroni post-hoc test of control vs. each treatment and of single vs. pairwise stimulation. Scale bar, 100 μm. P* <0.05; P** <0.005; P*** <0.001; P# <0.05; P## <0.005; P### <0.001.