(A) Extravasation of Evans blue dye (red) observed in the brain regions (frontal cortex, striatum, and hippocampus) of germ-free (GF) mice. In germ-free mice monocolonized with either C. tyrobutyricum (CBut) or B. thetaiotaomicron (BTeta) for 2 weeks or mice treated with the bacterial metabolite sodium butyrate (NaBu) for 72 hours, Evans blue dye was detected only in the blood vessels, without any leakage into the brain parenchyma. Blue, DAPI (nuclear staining). Scale bars, 50 µm. (B to D) Quantitative analysis of ZO-1, occludin, and claudin-5 expression in brain lysates from germ-free mice gavaged with water (GF) or NaBu for 72 hours. Data were normalized for β-tubulin expression as a loading control and expressed as fold change, control fold (c.f.) GF. Values are expressed as means ± SEM (four to five mice per group). *P < 0.05 by Student’s t test compared to the germ-free control.