Figure 5

TLR4 modulation affected motor neuron survival. Viability of motor neurons directly (A and B) or indirectly (C) exposed to TLR4 agonists (1 µg/mL LPS or lipid A) with or without an antagonist (20 µg/mL VB3323 or RsLPS), under different coculture conditions, is shown. (A) Motor neuron/glia cocultures were exposed to LPS ± TLR4 antagonists for 24 h. ***p < 0.001; two-way ANOVA and Bonferroni posttest. F_interaction < 0.001. (B) Motor neuron/glia cocultures were exposed to lipid A ± VB3323 for 24 h. **p < 0.01: one-way ANOVA and Tukey test. C: Mixed glial layers were exposed to LPS ± VB3323 for 24 h. After removing the TLR4 ligands, purified motor neurons were added to this conditioned glial layer. These co-cultures were allowed to grow for 1 wk, and motor neuron viability was assessed. ***p < 0.001, *p < 0.05; two-way ANOVA and Bonferroni posttest. Data are mean number ± standard deviation (SD) of viable motor neurons normalized against control. At least nine wells for each condition were analyzed from three independent experiments. (D-I) Representative pictures of SMI32-positive motor neurons maintained in control conditions (D, G) or treated with LPS alone (E, H) or in combination with VB3323 (F, I). Scale bar (D-F), 40 µm; scale bar (G-I), 20 µm.