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Gut microbiota‐derived bEVs do not trigger detectable neuroinflammation. (A) Representative confocal microscopy microphotographs of microglial cells labelled for the Iba‐1 marker (green) and the pro‐inflammatory activation marker <t>NF‐κB‐p65</t> (red) to evaluate activated microglia (white arrows) in Prefrontal Cortex (PFC) and (B) Nucleus Accumbens (NAc). Nuclei were stained with DAPI (blue). Microglia activation was assessed as the percentage of Iba1 + cells showing nuclear staining for NF‐κB‐p65. Data indicated no significant differences between the bEV‐injected groups and the vehicle‐injected group. Data are shown as mean ± SEM. One‐way ANOVA and Dunnett's test, p > 0.05 n = 5 per group. Scale bar = 30 µm. (C) RNA levels of pro‐inflammatory cytokines IL‐1β, IL‐6 and TNF‐α in the PFC and NAc show that bEVs treatment had no significant impact on classical pro‐inflammatory cytokine production in both brain regions. One‐way ANOVA and Dunnett's test, p > 0.05. Data are presented as mean ± SEM; n = 5 per experimental group. (D) Representative dot plots of CD11b/c + CD68 + cells determined from brain cortices of bEV and vehicle‐treated animals. (E) Comparison of the percentage of CD68 + cells of total CD11b/c + cells between brain cells of different experimental groups. Data indicated no significant differences between the bEV‐injected groups and the vehicle‐injected group. Data are shown as mean ± SEM. One‐way ANOVA and Dunnett's test, p > 0.05 n = 5 per group.
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Gut microbiota‐derived bEVs do not trigger detectable neuroinflammation. (A) Representative confocal microscopy microphotographs of microglial cells labelled for the Iba‐1 marker (green) and the pro‐inflammatory activation marker <t>NF‐κB‐p65</t> (red) to evaluate activated microglia (white arrows) in Prefrontal Cortex (PFC) and (B) Nucleus Accumbens (NAc). Nuclei were stained with DAPI (blue). Microglia activation was assessed as the percentage of Iba1 + cells showing nuclear staining for NF‐κB‐p65. Data indicated no significant differences between the bEV‐injected groups and the vehicle‐injected group. Data are shown as mean ± SEM. One‐way ANOVA and Dunnett's test, p > 0.05 n = 5 per group. Scale bar = 30 µm. (C) RNA levels of pro‐inflammatory cytokines IL‐1β, IL‐6 and TNF‐α in the PFC and NAc show that bEVs treatment had no significant impact on classical pro‐inflammatory cytokine production in both brain regions. One‐way ANOVA and Dunnett's test, p > 0.05. Data are presented as mean ± SEM; n = 5 per experimental group. (D) Representative dot plots of CD11b/c + CD68 + cells determined from brain cortices of bEV and vehicle‐treated animals. (E) Comparison of the percentage of CD68 + cells of total CD11b/c + cells between brain cells of different experimental groups. Data indicated no significant differences between the bEV‐injected groups and the vehicle‐injected group. Data are shown as mean ± SEM. One‐way ANOVA and Dunnett's test, p > 0.05 n = 5 per group.
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Gut microbiota‐derived bEVs do not trigger detectable neuroinflammation. (A) Representative confocal microscopy microphotographs of microglial cells labelled for the Iba‐1 marker (green) and the pro‐inflammatory activation marker <t>NF‐κB‐p65</t> (red) to evaluate activated microglia (white arrows) in Prefrontal Cortex (PFC) and (B) Nucleus Accumbens (NAc). Nuclei were stained with DAPI (blue). Microglia activation was assessed as the percentage of Iba1 + cells showing nuclear staining for NF‐κB‐p65. Data indicated no significant differences between the bEV‐injected groups and the vehicle‐injected group. Data are shown as mean ± SEM. One‐way ANOVA and Dunnett's test, p > 0.05 n = 5 per group. Scale bar = 30 µm. (C) RNA levels of pro‐inflammatory cytokines IL‐1β, IL‐6 and TNF‐α in the PFC and NAc show that bEVs treatment had no significant impact on classical pro‐inflammatory cytokine production in both brain regions. One‐way ANOVA and Dunnett's test, p > 0.05. Data are presented as mean ± SEM; n = 5 per experimental group. (D) Representative dot plots of CD11b/c + CD68 + cells determined from brain cortices of bEV and vehicle‐treated animals. (E) Comparison of the percentage of CD68 + cells of total CD11b/c + cells between brain cells of different experimental groups. Data indicated no significant differences between the bEV‐injected groups and the vehicle‐injected group. Data are shown as mean ± SEM. One‐way ANOVA and Dunnett's test, p > 0.05 n = 5 per group.
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Gut microbiota‐derived bEVs do not trigger detectable neuroinflammation. (A) Representative confocal microscopy microphotographs of microglial cells labelled for the Iba‐1 marker (green) and the pro‐inflammatory activation marker <t>NF‐κB‐p65</t> (red) to evaluate activated microglia (white arrows) in Prefrontal Cortex (PFC) and (B) Nucleus Accumbens (NAc). Nuclei were stained with DAPI (blue). Microglia activation was assessed as the percentage of Iba1 + cells showing nuclear staining for NF‐κB‐p65. Data indicated no significant differences between the bEV‐injected groups and the vehicle‐injected group. Data are shown as mean ± SEM. One‐way ANOVA and Dunnett's test, p > 0.05 n = 5 per group. Scale bar = 30 µm. (C) RNA levels of pro‐inflammatory cytokines IL‐1β, IL‐6 and TNF‐α in the PFC and NAc show that bEVs treatment had no significant impact on classical pro‐inflammatory cytokine production in both brain regions. One‐way ANOVA and Dunnett's test, p > 0.05. Data are presented as mean ± SEM; n = 5 per experimental group. (D) Representative dot plots of CD11b/c + CD68 + cells determined from brain cortices of bEV and vehicle‐treated animals. (E) Comparison of the percentage of CD68 + cells of total CD11b/c + cells between brain cells of different experimental groups. Data indicated no significant differences between the bEV‐injected groups and the vehicle‐injected group. Data are shown as mean ± SEM. One‐way ANOVA and Dunnett's test, p > 0.05 n = 5 per group.
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Gut microbiota‐derived bEVs do not trigger detectable neuroinflammation. (A) Representative confocal microscopy microphotographs of microglial cells labelled for the Iba‐1 marker (green) and the pro‐inflammatory activation marker <t>NF‐κB‐p65</t> (red) to evaluate activated microglia (white arrows) in Prefrontal Cortex (PFC) and (B) Nucleus Accumbens (NAc). Nuclei were stained with DAPI (blue). Microglia activation was assessed as the percentage of Iba1 + cells showing nuclear staining for NF‐κB‐p65. Data indicated no significant differences between the bEV‐injected groups and the vehicle‐injected group. Data are shown as mean ± SEM. One‐way ANOVA and Dunnett's test, p > 0.05 n = 5 per group. Scale bar = 30 µm. (C) RNA levels of pro‐inflammatory cytokines IL‐1β, IL‐6 and TNF‐α in the PFC and NAc show that bEVs treatment had no significant impact on classical pro‐inflammatory cytokine production in both brain regions. One‐way ANOVA and Dunnett's test, p > 0.05. Data are presented as mean ± SEM; n = 5 per experimental group. (D) Representative dot plots of CD11b/c + CD68 + cells determined from brain cortices of bEV and vehicle‐treated animals. (E) Comparison of the percentage of CD68 + cells of total CD11b/c + cells between brain cells of different experimental groups. Data indicated no significant differences between the bEV‐injected groups and the vehicle‐injected group. Data are shown as mean ± SEM. One‐way ANOVA and Dunnett's test, p > 0.05 n = 5 per group.
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Gut microbiota‐derived bEVs do not trigger detectable neuroinflammation. (A) Representative confocal microscopy microphotographs of microglial cells labelled for the Iba‐1 marker (green) and the pro‐inflammatory activation marker <t>NF‐κB‐p65</t> (red) to evaluate activated microglia (white arrows) in Prefrontal Cortex (PFC) and (B) Nucleus Accumbens (NAc). Nuclei were stained with DAPI (blue). Microglia activation was assessed as the percentage of Iba1 + cells showing nuclear staining for NF‐κB‐p65. Data indicated no significant differences between the bEV‐injected groups and the vehicle‐injected group. Data are shown as mean ± SEM. One‐way ANOVA and Dunnett's test, p > 0.05 n = 5 per group. Scale bar = 30 µm. (C) RNA levels of pro‐inflammatory cytokines IL‐1β, IL‐6 and TNF‐α in the PFC and NAc show that bEVs treatment had no significant impact on classical pro‐inflammatory cytokine production in both brain regions. One‐way ANOVA and Dunnett's test, p > 0.05. Data are presented as mean ± SEM; n = 5 per experimental group. (D) Representative dot plots of CD11b/c + CD68 + cells determined from brain cortices of bEV and vehicle‐treated animals. (E) Comparison of the percentage of CD68 + cells of total CD11b/c + cells between brain cells of different experimental groups. Data indicated no significant differences between the bEV‐injected groups and the vehicle‐injected group. Data are shown as mean ± SEM. One‐way ANOVA and Dunnett's test, p > 0.05 n = 5 per group.
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Gut microbiota‐derived bEVs do not trigger detectable neuroinflammation. (A) Representative confocal microscopy microphotographs of microglial cells labelled for the Iba‐1 marker (green) and the pro‐inflammatory activation marker NF‐κB‐p65 (red) to evaluate activated microglia (white arrows) in Prefrontal Cortex (PFC) and (B) Nucleus Accumbens (NAc). Nuclei were stained with DAPI (blue). Microglia activation was assessed as the percentage of Iba1 + cells showing nuclear staining for NF‐κB‐p65. Data indicated no significant differences between the bEV‐injected groups and the vehicle‐injected group. Data are shown as mean ± SEM. One‐way ANOVA and Dunnett's test, p > 0.05 n = 5 per group. Scale bar = 30 µm. (C) RNA levels of pro‐inflammatory cytokines IL‐1β, IL‐6 and TNF‐α in the PFC and NAc show that bEVs treatment had no significant impact on classical pro‐inflammatory cytokine production in both brain regions. One‐way ANOVA and Dunnett's test, p > 0.05. Data are presented as mean ± SEM; n = 5 per experimental group. (D) Representative dot plots of CD11b/c + CD68 + cells determined from brain cortices of bEV and vehicle‐treated animals. (E) Comparison of the percentage of CD68 + cells of total CD11b/c + cells between brain cells of different experimental groups. Data indicated no significant differences between the bEV‐injected groups and the vehicle‐injected group. Data are shown as mean ± SEM. One‐way ANOVA and Dunnett's test, p > 0.05 n = 5 per group.

Journal: Journal of Extracellular Vesicles

Article Title: Gut Microbiota‐Derived Extracellular Vesicles Influence Alcohol Intake Preferences in Rats

doi: 10.1002/jev2.70059

Figure Lengend Snippet: Gut microbiota‐derived bEVs do not trigger detectable neuroinflammation. (A) Representative confocal microscopy microphotographs of microglial cells labelled for the Iba‐1 marker (green) and the pro‐inflammatory activation marker NF‐κB‐p65 (red) to evaluate activated microglia (white arrows) in Prefrontal Cortex (PFC) and (B) Nucleus Accumbens (NAc). Nuclei were stained with DAPI (blue). Microglia activation was assessed as the percentage of Iba1 + cells showing nuclear staining for NF‐κB‐p65. Data indicated no significant differences between the bEV‐injected groups and the vehicle‐injected group. Data are shown as mean ± SEM. One‐way ANOVA and Dunnett's test, p > 0.05 n = 5 per group. Scale bar = 30 µm. (C) RNA levels of pro‐inflammatory cytokines IL‐1β, IL‐6 and TNF‐α in the PFC and NAc show that bEVs treatment had no significant impact on classical pro‐inflammatory cytokine production in both brain regions. One‐way ANOVA and Dunnett's test, p > 0.05. Data are presented as mean ± SEM; n = 5 per experimental group. (D) Representative dot plots of CD11b/c + CD68 + cells determined from brain cortices of bEV and vehicle‐treated animals. (E) Comparison of the percentage of CD68 + cells of total CD11b/c + cells between brain cells of different experimental groups. Data indicated no significant differences between the bEV‐injected groups and the vehicle‐injected group. Data are shown as mean ± SEM. One‐way ANOVA and Dunnett's test, p > 0.05 n = 5 per group.

Article Snippet: Then, sections were incubated overnight at 4°C with a primary rabbit monoclonal anti‐Iba‐1 antibody (Cat# 019–19741, Wako, 1:500 dilution) and with a primary mouse monoclonal anti‐NFκB‐p65 antibody (Cat# sc‐136548, Santa Cruz Biotechnology, 1:200 dilution) in Signal Stain diluent (Cat# 8112L, Cell Signalling).

Techniques: Derivative Assay, Confocal Microscopy, Marker, Activation Assay, Staining, Injection, Comparison