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Published in final edited form as:

Sci Transl Med. 2014 November 19; 6(263): 263ra158. doi:10.1126/scitranslmed.3009759.

The gut microbiota influences blood-brain barrier permeability in

mice
Viorica Braniste1,†,*, Maha Al-Asmakh1,*, Czeslawa Kowal2,*, Farhana Anuar1, Afrouz
Abbaspour1, Miklós Tóth3, Agata Korecka1, Nadja Bakocevic4, Lai Guan Ng4, Parag
Kundu5, Balázs Gulyás3,5, Christer Halldin3,5, Kjell Hultenby6, Harriet Nilsson7, Hans
Hebert7, Bruce T. Volpe8, Betty Diamond2,‡, and Sven Pettersson1,5,9,†,‡
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1Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, 17177 Stockholm,
Sweden.
2Centerfor Autoimmune and Musculoskeletal Disease, The Feinstein Institute for Medical
Research, North Shore-LIJ Health System, Manhasset, NY 11030, USA.
3PsychiatrySection, Department of Clinical Neuroscience, Karolinska Institutet, 17176
Stockholm, Sweden.
4Singapore Immunology Network, Agency for Science, Technology and Research, Singapore
138648, Singapore.
5Lee Kong Chian School of Medicine, Nanyang Technological University, 60 Nanyang Drive,
Singapore 637551, Singapore.
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6Department of Laboratory Medicine, Karolinska Institutet, 14186 Stockholm, Sweden.

7Department of Biosciences and Nutrition, Karolinska Institutet, and School of Technology and
Health, KTH Royal Institute of Technology, Novum, SE-141 57 Huddinge, Sweden.
8Laboratoryof Functional Neuroanatomy, The Feinstein Institute for Medical Research, North
Shore-LIJ Health System, Manhasset, NY 11030, USA.

Copyright 2014 by the American Association for the Advancement of Science; all rights reserved.
†
Corresponding author. [email protected] (V.B.); [email protected] (S.P.).
*Co-first authors.
‡Co-senior authors.
SUPPLEMENTARY MATERIALS
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www.sciencetranslationalmedicine.org/cgi/content/full/6/263/263ra158/DC1 Material and Methods

Fig. S1. Expression of tight junction proteins in the brains of germ- and pathogen-free adult female mice.
Fig. S2. Electron micrographs showing different tight junction structure (white arrows) in the brains of germ-free adult mice.
Fig. S3. The effect of oral treatment with the bacterial metabolite sodium butyrate or monocolonization with C. tyrobutyricum on
histone acetylation in extracts of mouse brain frontal cortex.
Author contributions: V.B., F.A., B.D., and S.P. conceived of and designed the project. C.K. and M.A.-A. determined the
permeability of the BBB in the fetal mice; M.T. performed the in vivo PET imaging; V.B., A.A., M.A.-A., and F.A. performed the
Evans blue dye study; V.B. and B.T.V. performed the R4A injection and quantitative analysis; N.B. conducted the two-photon study;
A.A. performed the immunofluorescence studies; V.B. performed the Western blot study for tight junction proteins, and F.A.
performed the Western blot data for histone acetylation; K.H. and H.N. performed electron microscopy; and P.K. and M.A.-A. were
involved in the animal experiments concerning the role of SCFAs. V.B., F.A., M.A.-A., C.K., M.T., B.T.V., N.B., L.G.N., B.D., A.K.,
and S.P. analyzed the data. V.B. and S.P. wrote the manuscript with significant input from B.G., C.H., L.G.N., C.K., H.H., M.A.-A.,
and B.D.
Competing interests: The authors declare that they have no competing interests.
 Braniste et al. Page 2

9Singapore Centre on Environmental Life Sciences Engineering (SCELSE), Nanyang

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Technological University, Singapore 637551, Singapore.

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
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expression of tight junction proteins. Our results suggest that gut microbiota–BBB communication
is initiated during gestation and propagated throughout life.

INTRODUCTION
Our gut microbiota is important for many biological functions in the body, including
intestinal development, barrier integrity and function (1, 2), metabolism (3, 4), the immune
system (5), and the central nervous system (CNS). The effects of the gut microbiota on brain
physiology include synaptogenesis, regulation of neurotransmitters and neurotrophic factors
such as brain-derived neurotrophic factor and nerve growth factor-A1 (6). However, the
development of the CNS also includes the formation of an intact blood-brain barrier (BBB)
that ensures an optimal microenvironment for neuronal growth and specification (7). An
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intact and tightly regulated BBB is also required to protect against colonizing microbiota in
neonates during the critical period of brain development (8, 9). It also protects against
exposure to “new” molecules and bacterial metabolites due to the postnatal metabolic switch
from predominant dependence on carbohydrates during fetal life to a greater dependence on
fatty acid catabolism after birth.

The BBB begins to develop during the early period of intrauterine life (10, 11) and is formed
by capillary endothelial cells sealed by tight junctions, astrocytes, and pericytes. Tight
junction proteins restricting paracellular diffusion of water-soluble substances from blood to
the brain (12) consist mainly of transmembrane proteins such as claudins, tricellulin, and
occludin, which are connected to the actin cytoskeleton by the zona occludens (ZO-1) (13).
Tight junction proteins are dynamic structures and are subject to changes in expression,
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subcellular location, posttranslational modification, and protein-protein interactions under

both physiological and pathophysiological conditions (12). Disruption of tight junctions due
to disease or drugs can lead to impaired BBB function, compromising the CNS. Therefore,
understanding how BBB tight junction proteins are affected by various factors is important
for elucidating how to prevent and treat neurological diseases.

Here, we report that the intestinal microbiota affects BBB permeability in both the fetal and
adult mouse brain. Using as a model system germfree mice that have never encountered a

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live bacterium and pathogen-free mice that were reared in an environment free of monitored
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mouse pathogens, we demonstrated that lack of gut microbiota is associated with increased
BBB permeability and altered expression of tight junction proteins. Fecal transfer from mice
with pathogen-free gut flora into germ-free mice or treatment of germ-free mice with
bacteria that produce short chain fatty acids (SCFA) decreased the permeability of the BBB.

RESULTS
The maternal gut microbiota can influence prenatal development of the BBB
First, we characterized BBB permeability of mouse embryos with pathogen-free mothers by
administering infrared-labeled immunoglobulin G2b (IgG2b) antibody to dams during timed
pregnancies to see whether the antibody was excluded by the BBB or was able to cross the
BBB into the brain parenchyma. The qualitative analysis of mouse embryos with pathogen-
free mothers showed a shift from a diffuse infrared-labeled antibody signal present within
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the embryonic brain at E13.5 and E14.5 to a signal confined to the developing vasculature
starting at E15.5 to E17.5 (Fig. 1A). This signal was most pronounced in adult offspring of
pathogen-free dams (Fig. 1A). The quantitative analysis of the penetration into the fetal
brain of infrared-labeled IgG2b antibody injected intravenously into pathogen-free dams
supported the qualitative data, showing a decrease at E15.5 to E17.5 (Fig. 1B). In contrast,
the analysis of E16.5 brains from fetal mice of germ-free dams showed a diffuse signal from
the infrared-labeled IgG2b antibody (Fig. 1C) present in the brain parenchyma (Fig. 1, D
and E). Higher-magnification images of the brain showed that the IgG2b antibody was
limited only to the vessels in E16.5 fetal mice of pathogen-free dams in contrast to age-
matched fetal mice of germ-free dams (Fig. 1, D and E). Because BBB integrity is
controlled in part by sealing of the endothelial cells via tight junctions, we determined
expression of the main tight junction proteins in brain lysates from E18.5 fetal mice of
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pathogen-free versus germ-free dams. Expression of the brain endothelial tight junction
proteins claudin-5 and ZO-1 was similar between the two groups, whereas the expression of
occludin was significantly lower in the brain lysates from E18.5 fetal mice of germ-free
dams compared to that in age-matched fetal mice of pathogen-free dams (P < 0.05) (Fig. 1,
F and G).

Lack of gut microbiota is associated with increased BBB permeability in adult mice
Three techniques were used to determine whether the BBB was more permeable in germ-
free adult mice: (i) in vivo positron emission tomography (PET) imaging with
[11C]raclopride (Fig. 2, A to C); (ii) extravasation of Evans blue tracer from the circulation
(Fig. 2D); and (iii) the capacity of an anti–N-methyl-D-aspartate receptor reactive antibody
(R4A) to induce neuronal death after intravenous administration (Fig. 2, E and F).
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In germ-free adult mice, [11C]raclopride uptake was increased compared with that for
pathogen-free adult mice (Fig. 2A), but only during the first 4 min after injection (Fig. 2, B
and C). This period of time represents the “flow phase” (that is, the presence of the
radioligand in the whole brain due to BBB permeability). Because the radioligand was given
in tracer doses, it does not exert any pharmacological effects on the brain (or body)
vasculature or heart rate. These differences were present only in the initial flow phase and

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not in the later phase of the time activity curves, indicating no differences in radioligand
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binding to dopamine D2 receptors between the groups.

Fluorescence microscopy images of different brain regions (cortex, striatum, and

hippocampus) of pathogen-free adult mice showed the presence of Evans blue dye (bright
red) only in the blood vessels, whereas Evans blue staining in germ-free mice was detected
not only in the blood vessels but also in the brain parenchyma, demonstrating leakage of the
dye across the BBB (Fig. 2D). A group of mice receiving an intravenous injection of tumor
necrosis factor–α (TNFα) 15 hours before the experiment served as a positive control for
BBB leakiness (Fig. 2D).

In germ-free adult mice, intravenous administration of the monoclonal antibody R4A (250
µg) was associated with abnormal neurons, marked by condensed cytoplasm and shrunken
cell bodies in the CA1 region of the hippocampus (Fig. 2E, right panel). Abnormal neurons
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were not present or were rare in the CA1 region of the hippocampus in the control group
[phosphate-buffered saline (PBS)–treated germ-free group; Fig. 2E, left panel]. Furthermore,
the R4A injection (at low and high doses) in germ-free adult mice was associated with a
significant reduction in neuron numbers in the CA1 region of the hippocampus, −38% for
low-dose R4A (100 µg) and −42% for high-dose (250 µg) R4A compared with the PBS-
treated germ-free control group (P < 0.01) (Fig. 2F). Intravenous administration of R4A in
pathogen-free adult mice did not induce any changes, indicating that the R4A monoclonal
antibody did not penetrate the BBB (Fig. 2F).

Vascular density and pericyte coverage show no difference in germ- and pathogen-free
adult mice
We used intravital two-photon microscopy to exclude the possibility that high BBB
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permeability in germ-free adult mice was caused by higher vascular density in the brain.
Using the second harmonic generation signals from collagen fibers of dura mater as a
reference point, we observed that the subdural region, which is 20 to 80 µm below the dura
mater, contains mainly large vessels (average diameter, ~40 µm) (Fig. 3, A and C). In
contrast, deeper regions of the brain 120 to 180 µm below the dura mater consist mostly of
capillaries (Fig. 3, B and D). Quantitative analysis of vasculature density in the brain
revealed no significant gross differences between germ- and pathogen-free adult mice (Fig.
3, C and D).

Pericytes play an important role in regulating BBB properties, and decreased pericyte
coverage has been associated with increased BBB permeability (10, 14).
Immunofluorescence staining using CD13, a cell surface marker for pericytes in different
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brain regions, revealed no difference in pericyte coverage between germ- and pathogen-free
mice (Fig. 3E).

Brain endothelial tight junctions are altered in the absence of a gut microbiota
Permeability of CNS vessels is controlled in part by dynamic opening and closing of the
endothelial junctions (15). Therefore, we assessed the expression of the main tight junction
proteins (ZO-1, occludin, and claudin-5) by Western blot in three regions of an adult mouse

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brain: frontal cortex, striatum, and hippocampus. Significantly lower expression of occludin
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and claudin-5 was observed in male germ-free mice compared with male pathogen-free mice
in all three brain regions of interest (occludin: P < 0.001 in frontal cortex and hippocampus
and P < 0.05 in striatum; claudin-5: P > 0.001 in frontal cortex and P < 0.01 in striatum and
hippocampus) (Fig. 4, A to F). In contrast, no difference in the expression of the cytoplasmic
protein ZO-1 was observed between the two groups (Fig. 4, A to F). Similar patterns of tight
junction protein expression were observed in the brains of female germ-free mice compared
with female pathogen-free mice (fig. S1), suggesting that the effect of gut microbiota on the
integrity of the BBB is independent of sex. In addition, immunofluorescence analysis
confirmed lower expression of occludin and claudin-5 in the brain vessels of adult male
germ-free mice compared with that of adult male pathogen-free mice (Fig. 4, G and H).

The ultrastructure of the tight junctions was investigated by transmission electron

microscopy analysis. In germ-free adult mice, the tight junctions appeared as a diffuse,
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disorganized structure compared with those in the pathogen-free group (Fig. 4I). A scoring
system was used to quantitatively determine the number of intact tight junctions as follows:
perfect tight junctions, 3; patches of blurriness, 2; totally blurred, 1 (fig. S2 shows examples
of the rating scale). In the striatum of germ-free adult mice, the number of intact tight
junctions was significantly lower than that in pathogen-free mice (P < 0.001) (Fig. 4J).

BBB permeability and tight junction protein expression are associated with changes in the
gut microbiota
Colonization of germ-free adult mice with flora from pathogen-free mice [conventionalized
(CONV)] was associated with increased integrity of the BBB as shown by restriction of the
Evans blue tracer to the blood vessels and decreased extravasation of the dye into the brain
parenchyma (Fig. 5A). Quantitative analysis of tight junction proteins in the CONV group
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compared with germ-free mice showed a significant increase in the expression of occludin in
the frontal cortex (P < 0.05) and striatum (P = 0.05) and of claudin-5 in the hippocampus
and striatum (P < 0.05) (Fig. 5, B to G). Increased expression of the intracellular protein
ZO-1 was detected in the striatum and hippocampus of CONV mice compared with germ-
free controls (P < 0.05) (Fig. 5, B to G).

SCFAs or metabolites produced by bacteria affect BBB permeability

SCFAs are known to enhance the integrity of the intestinal epithelial barrier (16, 17) by
facilitating the assembly of tight junctions (18). Hence, we evaluated BBB permeability in
germ-free adult mice monocolonized with a single bacterial strain, Clostridium
tyrobutyricum (CBut), that produces mainly butyrate (19, 20) or with Bacteroides
thetaiotaomicron (BTeta), which produces mainly acetate and propionate (21, 22). We also
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evaluated germ-free adult mice given sodium butyrate by oral gavage for 3 days. Evans blue
perfusion in CBut-, BTeta-, and sodium butyrate–treated mice demonstrated decreased BBB
permeability, compared to that in germ-free adult mice, that was equivalent to that of
pathogen-free adult mice (Fig. 6A). Administration of sodium butyrate to germ-free mice
was associated with increased expression of occludin in the frontal cortex and hippocampus
but had no effect on the expression of claudin-5 (Fig. 6, B to D). Furthermore,
administration of sodium butyrate or monocolonization of germ-free mice with C.

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tyrobutyricum was associated with an increase in histone acetylation in brain lysates (fig.
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S3).

DISCUSSION
The BBB is a physiological barrier that controls the passage of molecules between the brain
parenchyma and the blood and in so doing allows proper functioning of neurons. Our results
highlight the gut microbiota as a potential regulator of BBB integrity. Here, we show that
the lack of a normal gut microbiota in germ-free mice is associated with increased
permeability of the BBB. This result was confirmed using three different techniques: in vivo
PET imaging using radio-labeled ligand, vascular leakage of Evans blue dye, and neuronal
damage after intravenous administration of R4A antibody. Furthermore, our data show that a
more permeable BBB is observed in the fetal mice with germ-free mothers at E16.5 to E18.5
days of embryonic development compared to the fetal mice with pathogen-free mothers at
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the same stages of development. The increased permeability of the BBB in germ-free adult
mice may partly be the consequence of disorganized tight junctions, as shown by electron
microscopy analysis, and low expression of the transmembrane tight junction proteins
occludin and claudin-5. The “conventionalization” of germ-free adult mice through
transplant of the fecal microbiota from pathogen-free adult mice or by administering
bacterial strains that produce SCFAs reinforced the integrity of the mouse BBB.

The BBB matures progressively during intrauterine life and continues to mature during the
early postnatal stages of life (23). Our data confirm previous observations (10, 24) and show
that closure of the BBB to IgG in pathogen-free mice occurs during the later stages of
intrauterine life. A recent study of Mfsd2a knockout mice (lacking the transporter for the
essential omega-3 fatty acid docosahexaenoic acid) showed that the BBB in mice becomes
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functional at E15.5, demonstrating complex regional and temporal differences in maturation

(11). This coincides with our observation of the permeability of the embryonic BBB to
maternal antibodies. In mice, gestational stage E15.5 is a turning point for the restriction of
maternal antibody penetration into the fetal brain. Maternal antibodies or, more precisely,
antibody delivered to the embryo through the placenta was our molecule of choice in our
embryonic BBB studies as a physiological route of delivery. Reduced closure of the BBB
was observed in fetuses from germ-free dams. In humans, marked changes in the
composition of the maternal gut microbiota have been observed between the first and the
third trimesters of pregnancy (25). These observations, together with the present study,
imply that the maternal gut microbiota might contribute to increased nutritional demands in
late pregnancy, which would require more stringent control of BBB permeability in the
growing offspring.
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The BBB is a complex structure formed by capillary endothelial cells, pericytes, and
astrocytes (26). A difference in vascular density might be a confounding factor in assessing
BBB permeability. In our study, increased invasion of circulating substances into the brain
parenchyma appears not to be due to differences in large vascular structures between the two
groups of mice as shown by a comparable equal visualization of the brain vasculature using
140 kD TRITC-dextran. However, we cannot formally exclude that some microcapillary
changes may still exist between the two groups. In germ-free adult mice, pericyte coverage

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was similar to that in pathogen-free mice, suggesting that altering the number of pericytes is
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unlikely to account for the increased BBB permeability observed in germ-free mice. This is
similar to the Mfsd2adeficient mouse model in which leaky vessels are not associated with
changes in the cerebrovascular network or pericyte abnormalities (11). However, in contrast
to the Mfsd2a-deficient mouse where transcytosis across the BBB is affected without
disruption of tight junctions, our model implies that the gut microbiota may regulate the
BBB through modulation of tight junction protein expression.

Tight junctions play a major role in the functional maintenance of the BBB (27, 28). Our
data show disorganized tight junctions in the brains of germ-free adult mice compared with
those of pathogen-free mice, which was associated with lower expression of occludin and
claudin-5. No difference in the expression of ZO-1 was observed. A decrease in occludin
and claudin-5 paralleled by cytoskeletal changes and tight junction protein redistribution
was associated with altered integrity of the BBB (29). Reduced expression of occludin was
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observed in germ-free mice during both intrauterine life and adulthood and was associated
with increased permeability of the BBB. Administration of normal flora from pathogen-free
mice or oral treatment with the bacterial metabolite sodium butyrate to germ-free adult mice
induced an increase in the expression of occludin that was associated with decreased
permeability of the BBB. These observations imply that the expression of occludin by the
brain endothelial cells is sensitive to changes in the intestinal gut microbiota.

Regulation of occludin expression by the intestinal microbiota has been reported in the
intestinal epithelial barrier (30) and blood-testis barrier (31). The expression of claudin-5 by
brain endothelial cells was low in germ-free adult mice but elevated after exposure to the gut
microbiota. However, no difference in the expression of claudin-5 was observed in the
E18.5 cortex of fetal mice with germ- or pathogen-free mothers. This implies that claudin-5
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expression continues to increase during the postnatal period in offspring of pathogen-free

mothers but not in offspring of germ-free mice. Although claudin-5 expression is present
from early intrauterine life in the human brain, it is localized mostly in the endothelial
cytoplasm and shifts toward the endothelial border during development to form tight
junctions (32, 33). Furthermore, claudin-5 knockout mice appear to develop normally during
intrauterine life with morphologically normal blood vessels but have a BBB impairment
associated with increased permeability to small molecules (34). The complete depletion of
claudin-5 is associated with 100% mortality of the newborns within 10 hours of birth (34),
further supporting the role of claudin-5 in the postnatal regulation of the BBB.

Dietary carbohydrates are substrates for fermentation by certain gut bacteria, which produce
SCFAs, primarily acetate, propionate, and butyrate, as end products (35). These SCFAs have
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been shown to regulate intestinal motility (36, 37) and to be involved in central appetite
regulation (38, 39) as well as being taken up directly into the bloodstream and transported to
various organs, including the brain (39, 40), where they modulate tissue development and
function (41, 42). Physiological concentrations of SCFA mixtures or individual SCFAs
regulate intestinal barrier function by increasing the transepithelial electrical resistance and
decreasing paracellular permeability (43). In a rat model of transient focal cerebral ischemia,
intraperitoneally injected sodium butyrate attenuated BBB disruption (6). In our study, we
show that monocolonization of the intestine of germ-free adult mice with either C.

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tyrobutyricum, a bacterial strain producing butyrate (19, 44), or B. thetaiotaomicron, which

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produces mainly acetate and propionate (21), decreased BBB permeability. Oral
administration of the bacterial metabolite sodium butyrate mimicked this effect on the BBB.
The effects of the other metabolites, acetate and propionate, as single substrates may also
have an effect on the permeability of the BBB and should also be explored. Intravenous or
intraperitoneal administration of sodium butyrate has been reported to inhibit histone
deacetylation and facilitate long-term memory consolidation (45), prevent BBB breakdown
(46), and promote angiogenesis and neurogenesis (47, 48). Whether the gut microbiota and
sodium butyrate alter histone acetylation of brain microvascular endothelial cells requires
further study to better understand the effects of SCFAs produced by the gut microbiota on
the CNS. Cumulative and chronic exposure to SCFAs could lead to relatively stable effects
on gene expression in the brain.

Finally, the composition and diversity of the gut microbiota community change over time,
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presumably reflecting different gut microbiota–host interactions. Early in life, there is an

urgent need to support the growing offspring with an almost unlimited amount of energy to
ensure brain development. Germ-free mice show an increase in glucocorticoid production
due to metabolic stress (49). An increase in BBB permeability may, therefore, allow serum
glucocorticoids to enter the growing brain, affecting neurogenesis and impairing production
of brain-derived nerve growth factor in the brain (6). In addition, microbes regulating the
BBB in infants with a growing brain may influence BBB permeability differently compared
to the adult BBB (50).

Although providing a first glimpse into an additional layer of gut-brain communication, the
current study has not revealed the precise signaling mechanisms through which gut
microbiota modulates BBB function. In addition, the consequences of increased BBB
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permeability in germ-free mice on neuronal function throughout life are still not known.
Therefore, the results should be interpreted cautiously until more physiological data are
acquired to corroborate these findings.

MATERIALS AND METHODS

Study design
The objective of this study was to assess the importance of the intestinal microbiota in the
maintenance of BBB integrity in a mouse model. The integrity of the BBB was examined in
germ-free mice and in pathogen-free mice using functional permeability assays and by
determining the status of tight junctions. BBB integrity was also determined in a group of
germ-free adult mice colonized with fecal samples from pathogen-free adult mice or treated
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with bacterial strains that produced SCFAs or the bacterial metabolite sodium butyrate.

Animals
Germ- and pathogen-free NMRI (Naval Medical Research Institute) male mice
andC57BL/6J female and male mice (8 to 10 weeks old) (Core Facility for Germ-free
Research, Karolinska Institutet) were used. C57BL/6J, Balb/c, and NMRI germ- and
pathogen-free female mice undergoing timed pregnancies were used to assess the

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intrauterine development of the BBB. Germ-free mice were raised in special plastic
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isolators. All animals were maintained on autoclaved R36 Lactamin chow (Lactamin), given
sterile drinking water ad libitum, and kept under 12-hour light/dark cycles.

The protocols were approved by the Regional Animal Research Ethical Board, Stockholm,
Sweden, following the proceedings described in the (European Union) EU legislation
(Council Directive 2010/63/EU) and the Institutional Animal Care and Use Committee at
The Feinstein Institute for Medical Research, Manhasset, NY, USA.

Colonization of germ-free mice

For colonization, 8- to 10-week-old C57BL/6J germ-free mice received fecal matter from
the pathogen-free mice through a single gavage and then were left for 14 days (CONV)
before being sacrificed. The control group was gavaged with sterile PBS.
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C. tyrobutyricum (DSM 2637) (a contribution of H. Kozakova, Department of Immunology

and Gnotobiology, Institute of Microbiology, Academy of Sciences of the Czech Republic,
Praha, Czech Republic) and B. thetaiotaomicron (BTeta) were cultured in Bryant Burkey
broth with resazurine (Merck), and 108 bacteria were used to colonize each C57BL/6J germ-
free adult male mouse as previously described (19). Fourteen days later, the mice were
treated with Evans blue dye to assess the BBB permeability. Another group of germ-free
male mice was gavaged for 3 days with sodium butyrate [1 g/kg body weight (BW) per day]
before sacrifice. A group of germ-free male mice and another group of pathogen-free mice
were gavaged with sterile water and used as controls.

Drug treatment
A group of adult male pathogen-free mice receiving a single intravenous injection of TNFα
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(100 µg/kg BW) 15 hours before Evans blue perfusion were used as a positive control for a
“leaky” BBB (51).

Perfusion with Evans blue dye

Evans blue perfusion was performed as previously described (52). Briefly, anesthetized mice
were perfused with PBS followed by Evans blue cocktail. Tissue cryosections were analyzed
by fluorescence microscopy. A detailed description is provided in Supplementary Materials.

In vivo PET imaging of [11C]raclopride

Scans were performed in a micro PET Focus 120 scanner (CTI Concorde Microsystems).
The tracer (maximum volume, 200 µl) was administered by bolus injection via the tail vein
into anesthetized mice. PET data were acquired in full three-dimensional mode, and images
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were reconstructed by standard two-dimensional filtered back projection using a ramp filter.
The % SUV is the regional concentration of tissue radioactivity normalized for injected dose
and BW. A complete description is provided in Supplementary Materials.

R4A antibody injection and quantitative analysis

Germ- and pathogen-free adult male mice received intravenous injections of R4A dissolved
in PBS. Forty-eight hours later, anesthetized mice were given transcardiac perfusion with

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0.9% NaCl followed by 4% paraformaldehyde (PFA) (Histolab). Tissue cryosections stained

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with cresyl violet and neurons were sampled from comparable regions of the anterior dorsal
hippocampus (53, 54). A detailed protocol is provided in Supplementary Materials.

Intravital two-photon laser scanning microscopy

TRITC-dextran (155 kD, Sigma) was injected retro-orbitally before imaging. Two-photon
imaging was performed on a TriM Scope II (LaVision BioTec) equipped with an Olympus
BX51 upright microscope fitted with a 20 × 0.95 numerical aperture water immersion
objective and a Chameleon Ultra-II Tunable, Mode-locked Ti:Sapphire laser (Coherent)
tuned to 880 nm for excitation of TRITC-dextran. Imaris (Bitplane) was used for three-
dimensional image analysis. Volumetric index (VI), a parameter that indicates percentage of
volume occupied by blood vessels within the analyzed Z stack, was calculated according to
the following formula: VI = total blood vessel volume within the Z stack/total volume of the
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Z stack × 100.

Immunofluorescence
Immunofluorescence was performed on 50-µm brain coronal cryotome sections blocked
with 5% bovine serum albumin (BSA) (Sigma) and 0.5% Triton X-100 (Sigma) in PBS,
followed by incubation with primary antibody as follows: rat anti-CD13 (BD Pharmingen,
558744), mouse anti-occludin (1:300, Invitrogen, 33-1500), mouse anti–claudin-5 (1:300,
Invitrogen, 35-2500), and rabbit anti-laminin (1:400, Sigma, L9393). Sections were then
incubated with secondary donkey anti-rat Alexa 488, goat anti-mouse Alexa 488, or goat
anti-rabbit Alexa 594 (1:500, Invitrogen) for 1 hour at room temperature. Z stack images
were visualized and acquired using a Nikon Eclipse TE300 inverted microscope integrated
with a PerkinElmer UltraVIEW spinning disk confocal system. Image processing was done
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using Corel Paint Shop Pro Photo XI software.

Protein extraction and Western blot analysis

Protein expression was determined in brain lysates as previously described (19). Primary
antibodies (occludin: 1:1000, Invitrogen, 331500; claudin-5: 1:1000, Invitrogen, 352500;
and ZO-1: 1:2000, Invitrogen, 617300) diluted in 3% BSA in 0.1% Tween 20 (Sigma)/PBS
were incubated overnight at 4°C with the blot. Protein bands were visualized by
chemiluminescence using Immun-Star WesternC chemiluminescence kit (Bio-Rad). Protein
expression was then quantified using ImageJ software (National Institute of Mental Health).

Transmission electron microscopy

A group of adult pathogen- and germ-free mice were transcardially perfused with 2.5%
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glutaraldehyde (Sigma)/1% PFA and processed for transmission electron microscopy.

Digital images were taken with a Veleta camera (Olympus Soft Imaging Solutions, GmbH).
The full method is described in Supplementary Materials.

Statistical analysis
Statistical significance was determined using one-way ANOVA with Tukey post hoc test for
multiple groups, Bonferroni post hoc analysis for Fig. 1B, or t test when changes were

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 Braniste et al. Page 11

compared between two groups (GraphPad Prism 6). P < 0.05 was considered statistically
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significant unless otherwise stated. Values were expressed as means ± SEM.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Acknowledgments
We thank V. Arulampalam and C. Betsholtz for their valuable comments. We thank A. Samuelsson, J. Aspsäter,
and A. A. Bautista Amoyo for technical assistance.

Funding: This project was supported by grants from Vetenskapsrådet, EU project “Molecular Targets Open for
Regulation by the gut flora—New Avenues for improved Diet to Optimize European health,” Hjärnfonden,
Mérieux Institute, and Singapore Millennium Foundation and funding from Lee Kong Chian School of Medicine,
Nanyang Technological University to S.P. and grants from the NIH and the Simons Foundation to B.D. We also
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thank the Wenner-Gren Foundation for postdoctoral funding of V.B.; the Agency for Science, Technology and
Research for the postdoctoral funding of F.A.; the Higher Education Institute and Qatar University for the Ph.D.
funding of M.A.-A.; Karolinska Institutet doctoral funding for the Ph.D. scholarship to A.A.; and the Center for
Innovative Medicine.

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

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(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

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mm. (D) Sagittal brain sections from each of three E16.5 mouse embryos with germ- or
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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.
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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
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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

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mice. Scale bar, 10 µm. (F) Quantitative analysis. Percent neuronal survival in the PBS-
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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).
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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
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per group). Laminin (red) was used as an endothelial cell marker. Scale bars, 50 µm.

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

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Student’s t test compared with the corresponding pathogen-free mouse control. (G and H)
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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.
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Fig. 5. Microbial colonization of the gut changes BBB integrity

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(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.

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Fig. 6. The effect of SCFAs on BBB permeability

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(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

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(GF) or NaBu for 72 hours. Data were normalized for β-tubulin expression as a loading
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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.
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