Figure 1.
Glial activation and resulting neurotoxicity in neuron-BV2 co-cultures.
Neuronal viability (MAP2-ABTS-ELISA assay) in neuron-BV2 co-cultures 24 h or 48 h after treatment with 100 ng/mL LPS +0.5 ng/mL IFN-γ (A) or 100 ng/mL LPS +5 ng/mL IFN-γ (B). Results are presented as % of MAP2 immunostaining vs each control. Bars are means + SEM of three to five independent experiments. *p<0.05 and ***p<0.001 vs each control, unpaired Student’s t-test. (C-H) MAP-2 immunostaining in control neuron-BV2 co-cultures (C, D) and co-cultures treated with LPS 100 ng/mL + IFN-γ 0.5 ng/mL for 24 h (E) or 48 h (G) and with LPS 100 ng/mL + IFN-γ 5 ng/mL for 24 h (F) or 48 h (H). Bar = 100 µm.
Figure 2.
Increased extracellular K+ concentration enhanced reactive glia-induced.
neurotoxicity in neuron-BV2 co-cultures. (A) Neuronal viability (MAP2-ABTS-ELISA assay) in neuron-BV2 co-cultures grown in 5 mM and 25 mM KCl 24 h after 100 ng/mL LPS +0.5 ng/mL IFN-γ treatment. Results are presented as % of MAP2 immunostaining in control co-cultures grown in 5 mM KCl. Bars are means + SEM of four independent experiments. ***p<0.001 vs respective control; two-way ANOVA (repeated measures) and Bonferroni post-test. (B) MAP2 immunostaining in control (B) and 100 ng/mL LPS +0.5 ng/mL IFN-γ treated co-cultures grown in high K+. Bar = 100 µm. (D) Increasing BV2:neuron ratio results in neurotoxicity in control neuron-BV2 co-cultures. (E) Increasing BV2:neuron ratio does not result in increased neurotoxicity in 100 ng/mL LPS +0.5 ng/mL IFN-γ treated co-cultures.
Figure 3.
Glial activation and resulting neurotoxicity in neuron-primary microglia co-cultures.
(A) Neuronal viability (MAP2-ABTS-ELISA assay) in neuronal cultures and neuron-primary microglia co-cultures 48 h after treatment with 100 ng/mL LPS and increasing concentrations of IFN-γ (15 and 30 ng/mL). (B) The addition of microglial cells at a microglia:neuron ratio of 1∶2 did not result in neurotoxicity after 48 h. Results are presented as % of MAP2 immunostaining vs each control. Bars are means + SEM of 3–4 independent experiments. *p<0.05 vs control; one-way ANOVA and Newman-Keuls post-test. MAP-2 immunocytochemistry in neuronal cultures (C-D) and neuron-primary microglia co-cultures (E-F) in control conditions (C and E) and 48 h after 100 ng/mL LPS +30 ng/mL IFN-γ (D and F).
Figure 4.
High extracellular K+ concentration did not potentiate reactive glia-induced neurotoxicity in neuron-primary microglia co-cultures.
Neuronal viability (MAP2-ABTS-ELISA assay) in neuron-primary microglia co-cultures grown in 5 mM and 25 mM KCl and treated with 100 ng/mL LPS +30 ng/mL IFN-γ for 24 h. Results are presented as % of MAP2 immunostaining in control co-cultures grown in 5 mM KCl. Bars are means + SEM of four independent experiments. **p<0.01 vs each control; two-way ANOVA (repeated measures) and Bonferroni post-test.
Figure 5.
Pro-inflammatory response in BV2 and primary microglia cell cultures in response to LPS/IFN-γ.
BV2 cells were treated 100 ng/mL of LPS +0.5 ng/mL of IFN-γ and primary microglial cells with 100 ng/mL LPS +30 ng/mL IFN-γ, and different pro-inflammatory parameters were measured. TNF-α (A) and IL-6 (B) levels were determined in the culture medium 24 h after treatment. COX-2 (C) and iNOS protein expression (D) were measured by western blot 12 h after treatment, and data normalized by β-actin. NO production (E) was determined in the culture medium 24 h after treatment. Bars are means + SEM of four to six independent experiments. *p<0.05 and **p<0.01 vs control; paired Student’s t test. (F) shows representative western blots.
Figure 6.
IL-10 pre-treatment did not inhibit the neurotoxicity induced by reactive microglia in neuron-primary microglia co-cultures.
Neuron-primary microglia co-cultures were treated with 100 ng/mL LPS +30 ng/mL IFN-γ for 48 h in the presence or absence of 50 ng/mL of IL-10 administered 1 h prior to LPS/IFN-γ. Evaluation of neuronal viability by MAP2-ABTS-ELISA assay. Results are presented as % of MAP2 immunostaining in control co-cultures. Bars are means + SEM of four independent experiments. **p<0.01 vs control; one-way ANOVA (repeated measures) and Newman-Keuls post-test.
Figure 7.
IL-10 pre-treatment inhibited the pro-inflammatory response induced by LPS/IFN-γ in primary microglia cultures.
Primary microglial cultures were treated with 100 ng/mL LPS +30 ng/mL IFN-γ for 24 h in the presence or absence of 50 ng/mL IL-10 administered 1 h prior to LPS/IFN-γ. TNF-α (A) and IL-6 (B) levels were determined in the culture medium 24 h after treatment. (C) COX-2 and (D) iNOS protein expression were measured by western blot 24 h after treatment, and data normalized by β-actin. (E) NO production was determined in the culture medium 24 h after treatment. Bars are means + SEM of three to four independent experiments. *p<0.05, **p<0.01 and ***p<0.001 vs control; ###p<0.001 vs LPS/IFN-γ; one-way ANOVA (repeated measures) and Newman-Keuls post-test. (F) shows a representative western blot.
Figure 8.
The iNOS inhibitor 1400 W prevented the neurotoxicity induced by reactive microglia in neuron-primary microglia co-cultures.
Neuron-primary microglia co-cultures were treated with 100 ng/mL LPS +30 ng/mL IFN-γ for 48 h in the presence or absence of 1400 W (10 µM) co-administered with LPS/IFN-γ. (A) NO production 48 h after LPS/IFN-γ treatment. (B) Evaluation of neuronal viability by MAP2-ABTS-ELISA assay. Results are presented as % of MAP2 immunostaining in control co-cultures. Bars are means + SEM of three independent experiments. **p<0.01 vs control; ##p<0.01 vs LPS/IFN-γ; one-way ANOVA (repeated measures) and Newman-Keuls post-test.