The gut microbiota influences blood-brain barrier permeability in mice

Abstract

Pivotal to brain development and function is an intact blood-brain barrier (BBB), which acts as a gatekeeper to control the passage and exchange of molecules and nutrients between the circulatory system and the brain parenchyma. The BBB also ensures homeostasis of the central nervous system (CNS). We report that germ-free mice, beginning with intrauterine life, displayed increased BBB permeability compared to pathogen-free mice with a normal gut flora. The increased BBB permeability was maintained in germ-free mice after birth and during adulthood and was associated with reduced expression of the tight junction proteins occludin and claudin-5, which are known to regulate barrier function in endothelial tissues. Exposure of germ-free adult mice to a pathogen-free gut microbiota decreased BBB permeability and up-regulated the expression of tight junction proteins. Our results suggest that gut microbiota-BBB communication is initiated during gestation and propagated throughout life.

Fig. 1. BBB integrity in fetal mice with germ- or pathogen-free mothers

(A) Representative lateral images of the brains of E13.5 to E17.5 mouse embryos and adult mice (ventral) 1 hour after infrared-labeled antibody was injected into pregnant pathogen-free (PF) mothers. Scale bar, 1 mm. (B) Quantitative analysis of antibody penetration into the fetal brain of mice with pathogen-free mothers. Data are expressed as means ± SEM (7 to 12 embryos per group). ***P < 0.0001 between E17.5 group versus the rest of the groups by one-way analysis of variance (ANOVA). (C) Representative images from the infrared-labeled antibody assay in E16.5 mouse embryos with germ-free (GF) mothers. Scale bar, 1 mm. (D) Sagittal brain sections from each of three E16.5 mouse embryos with germ- or pathogen-free mothers after injecting the dam with IgG. IgG (top row of each pair, Alexa 594), CD31 [platelet endothelial cell adhesion molecule (PECAM); bottom row of each pair, Alexa 488]. Scale bar, 500 µm. (E) Maternal IgG in comparable regions of the brain of E16.5 mouse embryos. Left column: IgG (Alexa 594). Middle column: CD31 (PECAM; Alexa 488). Right column: Merged images. Scale bar, 20 µm. (F and G) Western blots of brain lysates from E18.5 mouse fetuses with germ- or pathogen-free mothers probed for ZO-1, occludin, claudin-5, and glyceraldehyde phosphate dehydrogenase (GAPDH) (control). (F) Representative blots and (G) quantification. Black bars, PF. White bars, GF. Data were normalized for GAPDH expression and expressed as fold change, control fold (c.f.) PF. Data are means ± SEM (four to six mice per group). *P < 0.05 by Student’s t test. ns, not significant.

Fig. 2. Increased BBB permeability in adult germ-free versus pathogen-free mice

(A) In vivo PET imaging of [¹¹C]raclopride. Average coronal, sagittal, and horizontal PET summation images (brain area encircled in purple ellipse) in pathogen-free (PF) or germ-free (GF) adult mice 2 to 3 min after [¹¹C]raclopride injection. (B) Average whole-brain time-activity curves of [¹¹C]raclopride uptake expressed as % standardized uptake value (% SUV) in the two groups. *P < 0.05 and **P < 0.05 by one-way ANOVA. (C) Values (% SUV) obtained at 1-min intervals during the first 5 min. Data are expressed as means ± SEM (five to six mice per group). (D) Representative images showing Evans blue dye extravasation (red) in three brain regions (cortex - upper row, striatum - middle row, and hippocampus - lower row) of germ- and pathogen-free mice and pathogen-free mice treated with TNFα (n = 3 mice per group). Blue, 4′,6-diamidino-2-phenylindole (DAPI) (nuclear staining). Scale bar, 50 µm. (E and F) Neuronal loss in the hippocampus of germ-free adult mice receiving R4A antibody. (E) Photomicrographs of the CA1 region of the hippocampus (middorsal, matched sections) from PBS-treated germ-free adult mice (left) and germ-free mice treated with different concentrations of R4A antibody (100 and 250 µg) (right). Yellow arrows in the right panel indicate neuronal loss and dying neurons in R4A-treated germ-free mice. Scale bar, 10 µm. (F) Quantitative analysis. Percent neuronal survival in the PBS-treated germ-free mouse group was set at 100%. **P < 0.01 by one-way ANOVA compared with the PBS-treated germ-free mouse group (n = 3 mice per group).

Fig. 3. Brain blood vessel and pericyte coverage in germ- and pathogen-free adult mice

(A to D) Two-photon imaging of brain blood vessels in germ-free (GF) and pathogen-free (PF) adult mice. Tetramethyl rhodamine isothiocyanate (TRITC)–dextran was applied retro-orbitally to highlight the brain blood vessels. (A) Representative images of brain vasculature 20 to 80 µm below the dura mater in germfree (left panel) and pathogen-free (right panel) mice reveal mainly large vessels (average diameter, ~40 µm). (B) Representative images of the brain vasculature 120 to 180 µm below the dura mater in germ-free (left panel) and pathogen-free (right panel) mice showing mainly capillaries. (C) Quantitative analysis of blood vessel density 20 to 80 µm below the dura mater. (D) Quantitative analysis of blood vessel density 120 to 180 µm below the dura mater. Scale bars, 100 µm. Data are representative of n = 3 independent experiments. (E) Representative images of pericyte coverage (CD13, green) in the cerebral cortex of pathogen- and germ-free mice (n = 4 mice per group). Laminin (red) was used as an endothelial cell marker. Scale bars, 50 µm.

Fig. 4. Disrupted BBB tight junctions in the brains of germ- and pathogen-free adult mice

(A to C) Representative Western blots showing the expression of ZO-1, occludin, and claudin-5 in the (A) frontal cortex, (B) striatum, and (C) hippocampus of germ-free (GF) and pathogen-free (PF) adult mice. (D to F) Densitometric analysis of Western blots from protein samples of the (D) frontal cortex, (E) striatum, and (F) hippocampus of germ-free mice (white bars) compared with pathogen-free mice (black bars). Data were normalized for GAPDH expression and expressed as fold change, control fold (c.f.) PF. Values represent means ± SEM (6 to 10 mice per group). *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test compared with the corresponding pathogen-free mouse control. (G and H) Representative images of germ- and pathogen-free mouse cerebral motor cortex stained for endothelial cells with (G and H) anti-laminin, (G) anti-occludin, and (H) anti–claudin-5 antibodies. Scale bars, 20 µm. (I) Electron micrographs showing the disorganized tight junction structure between two endothelial cells in striatum (right panel, white arrows) of germ-free adult mice compared with striatum of pathogen-free mice (left panel). (J) Quantitative data indicate a decreased number of organized tight junctions in the striatum of germ-free mice compared with pathogen-free mice. Values represent means ± SEM (seven mice per group). ***P < 0.001 by Student’s t test.

Fig. 5. Microbial colonization of the gut changes BBB integrity

(A) Representative images from three mouse brain regions (frontal cortex, striatum, and hippocampus) showing Evans blue dye (red) in pathogen-free (PF) mice, germ-free (GF)mice, and germ-free mice colonized with pathogen-free flora for 14days (CONV). Blue, DAPI (nuclear staining). Scale bars, 50 µm. (B to G) Quantitative analysis of ZO-1, occludin, and claudin-5 expression in the frontal cortex, striatum, and hippocampus of germ-free and CONV mice. Data were normalized for GAPDH expression and expressed as fold change, fold control (c.f.) GF. Values represent means ± SEM (four to six mice per group). *P < 0.05 by Student’s t test compared to the germ-free control.

Fig. 6. The effect of SCFAs on BBB permeability

(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.