Gut microbiota and central nervous system development

Accepted Manuscript Gut microbiota and central nervous system development Nicola Principi, Susanna Esposito PII:

S0163-4453(16)30251-1

DOI:

10.1016/j.jinf.2016.09.010

Reference:

YJINF 3824

To appear in:

Journal of Infection

Received Date: 5 August 2016 Revised Date:

26 September 2016

Accepted Date: 29 September 2016

Please cite this article as: Principi N, Esposito S, Gut microbiota and central nervous system development, Journal of Infection (2016), doi: 10.1016/j.jinf.2016.09.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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GUT MICROBIOTA AND CENTRAL NERVOUS SYSTEM DEVELOPMENT

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Nicola Principi, Susanna Esposito

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Pediatric Highly Intensive Care Unit, Department of Pathophysiology and Transplantation,

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Università degli Studi di Milano, Fondazione IRCCS Ca’ Granda Ospedale Maggiore

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Policlinico, Milan, Italy

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Short title: Gut microbiota and central nervous system

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Abstract: 244 words. Text: 5,520 words.

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Correspondence and requests for reprints should be addressed to:

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Susanna Esposito,

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Pediatric Highly Intensive Care Unit,

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Department of Pathophysiology and Transplantation,

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Università degli Studi di Milano,

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Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico,

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Via Commenda 9,

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20122 Milano,

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Italy.

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Tel.: +39-02-55032498; Fax: +39-02-50320206;

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E-mail: [email protected]

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ABSTRACT

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Objectives: Gut dysbiosis has been associated with several clinically relevant conditions,

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including alterations of central nervous system (CNS) structure and function development.

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This review discussed aspects of the relationship between gut microbiota and the CNS

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during development.

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Methods: PubMed was used to search for all of the studies published over the last 15

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years using the key word “microbiota” and “gut” or “intestinal” and “nervous system”. More

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than 350 articles were found, and only those published in English and providing data on

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aspects related to neurologic diseases were included in the evaluation.

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Results: The data indicate that the gut microbiota influences CNS development and

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function and that gut dysbiosis is associated with significant neurological problems.

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However, most of these data have been collected in experimental animals and cannot be

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transferred to humans. Moreover, it is not definitively established whether neurologic

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diseases depend on a generic modification of the gut microbiota or whether a single

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bacterial phylum or species plays a specific role for any single condition. Furthermore,

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limited information exists regarding protective bacteria.

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Conclusions: Both probiotics and prebiotics can have different impacts on CNS according

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to the microbial species or oligosaccharides that are administered. In humans, particularly

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in children, several factors may be important in conditioning gut microbiota modifications;

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unfortunately, most of these factors act simultaneously. More efforts are required to fully

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define both the array of complex behaviors that are influenced by the gut microbiota at the

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CNS level and the mechanisms involved.

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Key-words: attention deficit hyperactivity disorder; autism spectrum disorder; central

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nervous system; gut microbiota; probiotics; prebiotics. 2

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INTRODUCTION Several trillions of commensal microbes live in the human gut and are collectively

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referred to as the gut microbiota. The gut microbiota performs several functions that are

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considered essential for health and survival. Studies have suggested that the gut

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microbiota serves as a barrier inhibiting the proliferation of pathogenic organisms.1

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Moreover, it contributes to the digestion of food and the breakdown of toxins and drugs,1

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regulates lipid and glucose metabolism,2 plays a fundamental role in the induction,

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training, and function of the host immune system,3 modulates gene expression,4 and

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reduces inflammation.5 In addition, 20-40% of the small molecules in the peripheral blood

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are microbial metabolites, many of which have profound effects on the development and

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function of the central nervous system (CNS).3, 4

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Gut dysbiosis, i.e., a significant modification in the gut microbiota composition, has

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been associated with several clinically relevant conditions. These conditions include

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obesity,6 cardiovascular diseases,7 liver diseases,8 kidney diseases,9 type 1 and type 2

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diabetes,10,

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neurological disorders have been associated with gut dysbiosis.15 Alterations of brain

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structure and function development are among the most relevant problems ascribed to

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modifications of the gut microbial composition because interactions between the CNS and

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the gut microbiota, which form the so-called gut-brain axis, appear to be already

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established during fetal life and continue until old age.16 It has been demonstrated that

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communication between the gastrointestinal tract and the CNS occurs continuously

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through several routes (including hormonal, immune, and neuronal pathways) that are

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mostly conditioned by the microbiota composition.17, 18 Consequently, dysbiosis may affect

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the CNS, particularly during the first years of life when the developing brain is highly

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vulnerable.16

rheumatoid arthritis,12 cancer,13 and allergic diseases.14 Moreover, several

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ACCEPTED MANUSCRIPT In this review, several aspects of the relationship between the gut microbiota and the

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CNS during development are discussed. Particular attention is paid to factors that

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condition the modification of the gut microbiota and the possibility of managing

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neurological diseases by modifying the gut microbial composition. PubMed was used to

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search for all of the studies published over the last 15 years using the key word

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“microbiota” and “gut” or “intestinal” and “nervous system”. More than 350 articles were

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found, and only those published in English and providing data on aspects related to

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neurologic diseases were included in the evaluation.

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CHARACTERISTICS OF THE GUT MICROBIOTA DURING DEVELOPMENT

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The gut microbiota’s definitive composition is mainly based on four major phyla

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covering more than 90% of the total bacterial population (namely Firmicutes,

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Bacteroidetes, Actinobacteria, and Proteobacteria) and include many additional minor

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phyla such as Verrucomicrobia and Fusobacteria.18 The Firmicutes phylum is composed of

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Gram-positive aerobic and anaerobic bacteria. Prominent members are included in the

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genus

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Staphylococcus, Escherichia, and Klebsiella. Bacteroidetes are Gram-negative bacteria

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and include genus Bacteroides and Prevotella. Actinobacteria are Gram-positive bacteria,

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among which genus Bifidobacterium, Corynebacterium, Propionibacterium, Atopobium are

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the most frequently detected. The Proteobacteria phylum contains Gram-negative

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bacteria, most notably the family of Enterobacteriaceae, including Enterobacter species.

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However, this composition of the gut microbiota is achieved only at the end of the third

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year of life. At birth and during the first months of life, the proportion of the different phyla

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can differ markedly from subject to subject due to the influence of several factors that are

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summarized below.18, 19 Table 1 summarizes the factors influencing gut microbiota during

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development.

Enterococcus,

Clostridium,

Ruminococcus,

Streptococcus,

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Composition of the gut microbiota during fetal life

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Until recently, it was thought that the fetus and intrauterine environment were sterile

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and that the gut microbiota would develop only at birth after passage through the vaginal

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canal or, in cases of cesarean section, after contact with environmental microbes.20,

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However, recently collected data suggest that maternal-fetal exchange of commensal

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bacteria may occur before birth via placental colonization. Together with the demonstration

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of bacteria in amniotic fluid,22 umbilical cord blood,23 meconium,24 and fetal membranes,25

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research has shown the existence of a unique placental microbiota niche composed of

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non-pathogenic commensal microbiota from the Firmicutes and Fusobacteria phyla.26

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Existence of placental microbiota and early colonization of the fetus through the placenta

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was recently confirmed by Collado et al., who reported that the placenta and the amniotic

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fluid harbor a distinct microbiota characterized by low richness, low diversity and the

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predominance of Proteobacteria.27 Moreover, they found that the placenta and meconium

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microbiota composition were similar, suggesting microbial transfer at the feto-maternal

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interface.27

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The composition of placental and fetal gut microbiota can significantly vary,

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according to several maternal conditions. Antibiotic therapy, malnutrition or over-nutrition,

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obesity, diabetes, and eczema are all factors that can condition a significant modification

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of the gut microbiota in pregnant woman with a predominance of potentially negative

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bacteria such as Enterobacteriaceae and Pseudomonadaceae and a poor presence of

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protective microbiota such as the Lactobacillus genus.28 Similar findings have been

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reported in studies that have evaluated pregnant women with stress. Animal studies have

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shown that a single 2-hour exposure to a social stressor can greatly reduce the relative

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proportions of two genera and one family of highly abundant, probably protective, intestinal

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bacteria, including the species of the genus Lactobacillus.29

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the first 110 days after birth in a healthy cohort of 56 vaginally born infants.30 They

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reported that maternal prenatal stress was strongly and persistently associated with the

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infants' gut microbiota composition. Infants of mothers with high stress during pregnancy

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had significantly higher relative abundances of Escherichia, Serratia, and Enterobacter

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and lower relative abundances of lactic acid bacteria (i.e., Lactobacillus, Lactococcus,

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Aerococcus) and Bifidobacteria. Moreover, a study by Jasarevic et al. showed that

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maternal stress altered proteins related to vaginal immunity and abundance of species of

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the genus Lactobacillus, the prominent taxa in the maternal vagina.31 Loss of maternal

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vaginal Lactobacillus resulted in decreased transmission of this bacterium to the offspring.

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Postnatal period

Colonization after birth is strictly dependent on the duration of gestation, mode of

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delivery, use of antibiotics and type of feeding. In term infants born to healthy mothers with

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vaginal delivery, breastfeeding, and no antibiotic use, the gut microbiota is characterized

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by low diversity and a relative dominance of Proteobacteria and Actinotobacteria.32

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Bacteria found in the feces of these neonates are similar to those normally detectable in

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the maternal vagina.33 With time, the gut microbiota becomes diverse with the emergence

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of Firmicutes and Bacteroidetes. During the first few months, these changes are mainly

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due to breast milk, which contains great amounts of streptococci and staphylococci.

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Moreover, it includes many complex oligosaccharides that stimulate the growth of

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Staphylococci and Bifidobacteria.34, 35 Further differentiation occurs after the introduction of

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solid foods with enhanced introduction of butyrate producers, including Bacteroides and

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Clostridium species.36 At the end of the third year, when the final composition of gut

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microbiota is achieved, members of the Firmicutes and Bacteroidetes phyla are the most

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common bacteria, followed by Actinobacteria, which is mainly represented by the genus

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Bifidobacterium. Once the final composition is achieved, the gut microbiota tends to

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remain stable.19 Infants born through cesarean section are not exposed to maternal gut

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microorganisms and are colonized mainly by microbiota derived by their mother’s skin.37

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Staphylococcus, Corynebacterium, and Propionibacterium species are the most common

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colonizers, together with certain bacteria that come from environmental sources such as

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health-care workers and the hospital environment. Colonization with Clostridium difficile is

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more common than in vaginally delivered infants, whereas that with Bacteroides and

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Bifidobaterium species occurs later.37 Modifications of the gut microbiota induced by

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cesarean section persist for a long time. Longitudinal studies have shown that the delayed

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and altered colonization pattern in cesarean-section-delivered infants persists at least until

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the age of one year,38 and even after 7 years of age, minor differences can be detected.39

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The gut microbiota of the formula-fed infant differs from that of the breastfed infant

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because it is characterized by a wider microbiota spectrum. In particular, the counts and

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incidences of Clostridium (C. paraputrificum, C. perfringens, C. clostridiiforme, C. difficile,

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and C. tertium) and Streptococcus (S. bovis, S. faecalis, and S. faecium) species, Bacillus

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subtilis, Bacteroides vulgatus, Veillonella parvula, Lactobacillus acidophilus, Escherichia

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coli, and Pseudomonas aeruginosa in bottle-fed infants are significantly higher than those

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in breastfed infants. By contrast, Lactobacillus rhamnosus and Staphylococci prevails in

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breast-fed infants, whereas Staphylococcus epidermidis is almost absent in samples from

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the feces of formula-fed infants.40

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Premature infants have a markedly different gut microbiota from term infants, with a

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bacilli-dominated phase, delayed or missed acquisition of Bifidobacteria, and an earlier

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acquisition of Firmicutes.27,

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the gut microbiota, with differences that are mainly related to the spectrum of activity of the

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Furthermore, the use of antibiotics can significantly modify

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administered drugs.6 However, an increase in Gram-negative rods with a negative

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metabolic influence has been frequently reported.33

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GUT MICROBIOTA AND THE CENTRAL NERVOUS SYSTEM (CNS)

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Importance of the normal microbiota for normal brain development

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The gut microbiota is essential for normal CNS development. Generally, gut

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microbiota absence is associated with several CNS developmental problems. Diaz Heiitz

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et al. reported that compared with conventionally raised mice, germ-free (GF) animals had

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an increased expression of PSD-95 and synaptophysin in the striate nucleus.42-44 Because

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these proteins are, respectively, a stimulator of excitatory synapse maturation and a

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hallmark of synaptic vesicle maturation, these findings seem to indicate that when the

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conventional microbiota is lacking, synaptic maturation is accelerated. This might affect

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motor control and anxiety-like behavior in later life, as shown in GF animals.45 Hoban et al.

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investigated changes in the homeostatic regulation of the neuronal transcription in GF

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mice within the prefrontal cortex.46 They found a marked, concerted upregulation of genes

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linked to myelination and myelin plasticity, leading to hypermyelinated axons.

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Colonization of experimental animals by a conventional microbiota reversed the

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myelin changes, confirming the necessity of intestinal flora for normal CNS development.

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Absence of microbiota during early life increases activity-related transcriptional pathways

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in the amygdala. Stilling et al. exploited unbiased genome-wide transcriptional profiling to

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determine gene expression in the amygdala of GF and GF mice that were colonized after

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weaning.47 Using RNA-sequencing and a comprehensive downstream analysis pipeline,

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these authors studied the amygdala transcriptome and found significant differences at the

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levels of differential gene expression, exon usage and RNA-editing. Upregulation of

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several immediate early response genes such as Fos, Fosb, Egr2 or Nr4a1 in association

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with increased CREB signaling in GF mice was evidenced. In addition, the differential

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expression and recoding of several genes implicated in brain physiology processes such

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as

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demonstrated.

neurotransmission,

neuronal

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metabolism

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morphology

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Microbes also control microglia maturation and function. GF mice display global

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defects in microglia, with altered cell proportions and an immature phenotype.48 Because

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microglia are formed by immune cells, this leads to impaired innate immune responses. In

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animals without risk factors for intestinal flora modifications, temporal gut microbiota

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eradication or limited microbiota complexity severely change the microglia properties.48

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Moreover, colonization with a complex microbiota partially restores microglia features,

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highlighting the role of gut microbiota in conditioning normal CNS development.

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Mechanisms by which the gut microbiota can interfere with central nervous system

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(CNS) development

Interaction between the CNS and the gut microbiota involves the gut microbes, the

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brain, the endocrine system, the immune system, and the autonomic nervous system

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(Table 2). Signals from the brain can influence the motor, sensory, and secretory

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modalities of the gastrointestinal tract, and conversely, visceral messages from the

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gastrointestinal tract can influence brain function.49 Despite the exact mechanisms by

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which the gut and brain reciprocally influence structure and function not being precisely

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defined, several data indicate that the passage of certain bacterial components through

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the intestinal wall plays a fundamental role in this regard. The most important bacterial

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components are lipopolysaccharides in the cell wall of some Gram-negative bacteria (such

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as Enterobacteriaceae and Pseudomonadaceae); the lipopolysaccharides may translocate

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from the intestinal mucosa to the systemic circulation, where they can affect immune

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regulation and CNS function.50 Lipopolysaccharides act upon toll-like receptor 4 to activate

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the systemic inflammation response with the production of pro-inflammatory cytokines

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such as tumor necrosis factor-α (TNF-α), interleukin(IL)-6, and IL-1β.51 These cytokines, in

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turn, critically affect the CNS.52 By contrast, other bacteria, such as those included in the

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genus Lactobacillus, seem to exert a protective effect, probably because they secrete

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chemicals that limit colonization by inflammatory microorganisms.53 Microbial metabolites, mainly gut-derived tryptophan metabolites, can have a critical

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importance. These metabolites are capable of modulating the immune system by various

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mechanisms, including the regulation of T cells, notably Th17 lymphocytes.54, 55 Moreover,

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high levels of tryptophan metabolites increase kynurenine concentration, which can also

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engage aryl hydrocarbon receptors. This induces the differentiation of regulatory T cells

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and increases the levels of indoleamine 2,3-dioxygenase, an enzyme that further induces

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the catabolism of tryptophan and an increase in the amount of circulating metabolites of

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this amino acid.56 In mice, it has been demonstrated that even when no transfer of the

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microbiota to the fetus from the mother has occurred, tryptophan metabolites that cross

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the placenta are active on the aryl hydrocarbon receptors and can significantly influence

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CNS development of the fetus.57 Finally, the level of systemic tryptophan metabolites

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influences the concentration of serotonin and γ- aminobutyric acid the CNS and the

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production of neurotoxic molecules in astrocytes and microglia.58, 59

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A further modulation of CNS function is provided by short chain fatty acids (SCFAs)

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produced by bacterial fermentation of complex polysaccharides (starches and fibers) in the

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colon.60 Particularly, Clostridia and Bacteroides are important SCFA producers. Major

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effects of these compounds are the alteration of mitochondrial function via the citric acid

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cycle and carnitine metabolism, or the epigenetic modulation of genes controlling brain

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function.

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neuroinflammatory, metabolic, and epigenetic changes. MacFabe et al. have shown that

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when propionic acid or other short-chain fatty acids are injected into the cerebral ventricles

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All

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reversible

behavioral,

electrographic,

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of rats, the rats show biologic, chemical, and pathologic changes that are characteristic of

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autism.61

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It

has

also

been

suggested

that

intestinal

bacteria

can

affect

fetal

neurodevelopment by influencing 5-hydroxytryptamine (5-HT) serum levels.62 Indigenous

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spore-forming bacteria from the microbiota promote 5-HT biosynthesis from colonic

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enterochromaffin cells (ECs), which supply 5-HT to the mucosa, lumen, and circulating

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platelets. Yano et al. identified select fecal metabolites that are increased by spore-forming

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bacteria and that elevate 5-HT in chromaffin cell cultures, suggesting direct metabolic

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signaling of gut microbes to ECs.62 Furthermore, elevating luminal concentrations of

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particular microbial metabolites increases colonic and blood 5-HT in GF mice and,

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consequently, fetal neuronal cell division and differentiation, physiologically regulated by

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this compound.63 Depletions of 5-HT during development were reported to have effects on

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the maturation of cortical neurons64, 65 and to alter barrel cortex development.66. 67

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A further mechanism with which gut microbiota can influence CNS is through the

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endocrine pathway. Prenatal stress is accompanied by increased cortisol levels, mainly via

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the systemic and peripheral release of corticotropin-releasing factor (CRF) from the

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amygdala, which is important in the control of emotional and autonomic responses to

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stress and contains CRF nerve terminals, CRF cell bodies, and CRF receptors.68

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Hypercortisolemia increases gut permeability and favors bacterial lipopolysaccharide

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leakage across the gut wall.69 Moreover, it is associated with an increased amygdala

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volume70 and this with depression.71

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Modification of the blood-brain barrier (BBB) can play a role in conditioning

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alterations in CNS development. The BBB acts as a gatekeeper to control the passage

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and exchange of molecules and nutrients between the circulatory system and the brain

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parenchyma. The BBB also ensures homeostasis of the CNS. Braniste et al. reported that

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GF mice, beginning with intrauterine life, display increased BBB permeability compared 11

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with non-GF mice with a normal gut microbial composition.72 The increased BBB

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permeability is maintained in GF mice after birth and during adulthood and is associated

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with reduced expression of the tight junction proteins occludin and claudin-5, which are

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known to regulate BBB function in endothelial tissues. Finally, the gut microbiota interacts with intestinal cells to stimulate the production of

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peptides that activate afferent endings of the vagus nerve. The resultant signals are

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transmitted to the CNS, affecting behavior and efferent neural activity. The same function

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appears to be exerted by pro-inflammatory cytokines. Consequently, the brain receives

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information regarding systemic inflammation, contributing to affective symptoms and

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initiating behavioral responses, including depression and other sickness behaviors.73

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Vagal signals from the gut are also thought to be responsible for “gut feelings,” which act

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as signals to the brain that an environment may be threatening or anxiety provoking.74

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Activated efferent fibers of the vagus, in turn, carry anti-inflammatory signals to the

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periphery, reducing the production of proinflammatory cytokines.75

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If the gut microbiota plays an essential role in the modulation of brain structure and

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function, it is clear that when unbalance between “positive” and “negative” bacteria occurs

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and the latter microbes prevail, inflammation arises, chronic immune disease can occur,

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and the CNS structure and function may be altered.50 Moreover, when chronic dysbiosis

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occurs, a systemic inflammatory state tends to persist.

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IMPACT OF DYSBIOSIS ON THE CENTRAL NERVOUS SYSTEM (CNS)

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The potential clinical effects of dysbiosis on the CNS are significantly more

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important during the developmental period. Younger individuals can suffer from relevant

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and permanent modification of CNS structure and function.43 However, even older

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subjects, despite a complete development of the CNS, can have associated psychiatric

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problems or so-called sickness behaviors, including fatigue, insomnia, lack of appetite and 12

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depression.76,

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disease78 and Alzheimer’s disease79 have been associated with gut microbiota

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modifications.

Moreover, also neurodegenerative disorders, such as Parkinson’s

All the conditions that can cause gut dysbiosis have been associated with

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impairment of the CNS structure and function. However, in some instances, the data

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collected in humans have been conflicting. Moreover, in cases exhibiting simultaneous

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conditions, the importance of the various factors is difficult to ascertain.

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Pregnant women with an unhealthy alteration or imbalance in the microbial

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composition frequently have higher circulating levels of pro-inflammatory cytokines such

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TNF-α, IL-6, and IL-1β and can produce sons with behavioral problems.6, 80 Infants born to

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stressed mothers frequently exhibit increased impulsivity, anxiety problems, attention

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deficit hyperactivity disorder (ADHD) and autism spectrum disorders (ASD).81-85

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Studies employing animal models have revealed that delivery by cesarean section

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is frequently associated with alterations of the mesolimbic and mesostriatal dopamine

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pathway, with persistent blunting of stress-induced dopamine release in the right prefrontal

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cortex.86 Moreover, compared with vaginally delivered animals, those delivered by

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cesarean section suffer more frequently from behavioral abnormalities that have been

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associated with ASD.87 However, the importance of the cesarean section in conditioning

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negative effects on the CNS is debated. A meta-analysis by Curran et al., who reviewed

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the literature published up to 28 February 2014, concluded that children born through

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cesarean section had a 23% increased risk of developing ASD, compared with those born

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by vaginal delivery.88 However, more recently, the same authors reported contrary

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conclusions in two different studies. In the first study, they enrolled sibling pairs discordant

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with respect to ASD status and found that subjects born via elective or emergency

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cesarean section were approximately 20% more likely to be diagnosed as having ASD

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compared with those with unassisted vaginal delivery.89 However, in the sibling control

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implying that this association was likely due to familial confounding factors (genetic and/or

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environmental) rather than the mode of delivery. In the second study, Curran et al.

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evaluated a nationally representative UK cohort of 13,141 children and did not find any

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association between cesarean section and ASD or ADHD.90 However, more favorable for

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a possible negative impact of cesarean section are the data collected by Adler and Wong-

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Kee-You.91 They explored whether differences in spatial attention would occur in infants

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delivered by cesarean or vaginal section. Three-month-old infants performed either a

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spatial cueing task or a visual expectation task. The cesarean-section-delivered infants'

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stimulus-driven, reflexive attention was slower than that of the vaginally delivered infants,

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whereas their cognitively driven, voluntary attention was unaffected. Based on the authors’

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conclusions, these findings suggest that the types of birth experience influence at least

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one form of infant attention, and possibly any cognitive process that relies on spatial

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attention.

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Conflicting results were also reported in studies regarding breastfeeding and

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cognitive development. In the USA, a national cohort study of 5,475 children of normal

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birth weight, having ever being breastfed, was associated with a significant higher child

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intelligence quotient (IQ).92 However, the positive effect of breastfeeding was no longer

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evident after an adjustment for confounders, mainly maternal intelligence. On the contrary,

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in UK, even after adjusting for confounders, a significant difference in the mean score of

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cognitive development between children who were breastfed and children who were never

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breastfed was evidenced. The differences were greater in children with long-term

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breastfeeding and in premature infants.93 Similarly, in Asia, Cai et al. demonstrated that

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higher breastfeeding exposure was associated with better memory at 6 months,94 as

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demonstrated by increased preferential gazing toward correctly matched items during the

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early portions of a relational memory task. At 24 months of age, breastfed children were

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more likely to display sequential memory during a deferred imitation memory task, and

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toddlers with more exposure to breastfeeding scored higher in receptive language as well

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as expressive language. Further data suggesting a positive impact of breastfeeding on infants’ neurological

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development have been collected by Krol et al.95 These authors examined whether and

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how the duration of exclusive breastfeeding affected the neural processing of emotional

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signals by measuring electro-cortical responses to body expressions in 8-month-old

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infants. The analyses revealed that infants with high-exclusive breastfeeding showed a

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significantly greater neural sensitivity to happy body expressions than did those with low-

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exclusive breastfeeding. Moreover, regression analyses revealed that the neural bias

347

toward happiness or fearfulness differed as a function of the duration of exclusive

348

breastfeeding. Specifically, a longer duration was associated with a happy bias, whereas a

349

shorter breastfeeding duration was associated with a fearful bias.

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ASD is a neurologic condition for which a strict relationship with gut dysbiosis has

351

been repeatedly hypothesized because, in children with this condition, particularly in those

352

with associated gastrointestinal dysfunctions (i.e., altered bowel habits, chronic abdominal

353

pain, reflux and vomiting), several studies have shown a frequent significant modification

354

of the gut microflora.96, 97 Finegold et al. compared the gut microflora of children exhibiting

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ASD of various severities with the gut microflora of healthy controls and reported that at

356

the phylum level, Bacteroidetes were found at higher concentrations in the severely

357

autistic group while Firmicutes were more predominant in the control group.98 Smaller but

358

significant differences also occurred in the Actinobacterium and Proteobacterium phyla.

359

Desulfovibrio species and Bacteroides vulgatus were detected more commonly in the

360

stools of severely autistic children than in controls. However, in several cases (those with

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late-onset ASD), 10-fold higher levels of Clostridium species, included in the Firmicutes

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phylum, were observed.99

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Recent

studies

have

evidenced

a

possible

relationship

between

some

neurodegenerative disorders that usually occur in adults or in old people such as

365

Parkinson's and Alzheimer’s diseases and gut dysbiosis. Interestingly, it has been reported

366

that in patients with Parkinson's disease the abundance of Prevotellaceae in feces was

367

reduced by 77.6% as compared with controls.100 The relative abundance of

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Enterobacteriaceae was positively associated with the severity of postural instability and

369

gait difficulty suggesting not only that the intestinal microbiota is altered in Parkinson’s

370

disease but that it is related to motor phenotype.100

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Regarding Alzheimer’s disease, it has been demonstrated that in germ-free mouse

372

models a drastic reduction of cerebral amyloid pathology could be demonstrated when

373

these animals were compared to control Alzheimer’s disease animals with intestinal

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microbiota. Sequencing bacterial 16S rRNA from fecal samples revealed a remarkable

375

shift in the gut microbiota of conventionally raised Alzheimer’s disease mice as compared

376

to healthy, wild-type mice.101 Colonization of germ-free Alzheimer mice with harvested

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microbiota from conventionally-raised Alzheimer mice dramatically increased cerebral Aβ

378

pathology. In contrast, colonization with microbiota from control wild-type mice was

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ineffective in increasing cerebral amyloid level, suggesting a microbial involvement in the

380

development of Alzheimer’s disease pathology.101

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THERAPEUTIC MANIPULATION OF THE GUT MICROBIOTA

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The evidence that gut dysbiosis can be associated with significant alterations of

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CNS structure and function has led to the supposition that gut microbiota manipulation

386

might be a rational approach to limit neurologic clinical problems.

387

Attempts to normalize the gut microbiota by treating children with ASD and

388

gastrointestinal problems with specific antibiotics active against Clostridium species have 16

ACCEPTED MANUSCRIPT been suggested.102 A significant reduction in these bacteria, which have been

390

demonstrated to have a negative effect on the CNS and were found in higher

391

concentrations in children with ASD, was proposed as an option. Oral vancomycin was

392

considered the best solution because this antibiotic is minimally absorbed by the intestinal

393

tract. Unfortunately, this attempt had only partial success. Administration of the antibiotic to

394

children with regressive-onset ASD was followed by a significant improvement of

395

neurological conditions, but only during the period of drug use.84 Neurological conditions

396

returned to baseline shortly after treatment suspension.

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A second approach was the administration of probiotics and/or prebiotics. Probiotics

398

are live microorganisms that when administered in adequate amounts, confer a health

399

benefit to the host.103 Studies have revealed that many of these bacteria can modify the

400

bacterial gut composition, with a reduction of negative strains and an increase in those

401

considered protective. Moreover, probiotics can reduce the concentration of certain

402

bacterial products that once they have crossed the intestinal wall, cause inflammation,

403

immune system alterations, modulation of gene expression and modification of the CNS

404

structure and function.1044 However, the beneficial effects are divergent and are dependent

405

on the strain. Bifidobacterium breve NCIMB 702258 and Bifidobacterium breve DPC 6330

406

were found to have contrasting effects on the gut microbiota. Compositional sequencing of

407

the gut microbiota showed a tendency for greater proportions of Clostridiaceae (25%,

408

12%, and 18%; p=0.08) and lower proportions of Eubacteriaceae (3%, 12%, and 13%;

409

p=0.06) in mice supplemented with Bifidobacterium breve DPC 6330 than in mice

410

supplemented with the second microorganism and unsupplemented controls.105

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However, studies in the experimental animals have shown that the administration of

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selected probiotics to animals may be effective in reducing neurologic signs and symptoms

413

associated with gut dysbiosis. Chronic administration of Bifidobacterium infantis to rats

414

deprived of maternal contact at an early age and having stress-related gastrointestinal and 17

ACCEPTED MANUSCRIPT mood disorders results in normalization of the immune response, reversal of behavioral

416

deficits, and restoration of basal noradrenaline concentrations in the brainstem.106

417

Moreover, Bifidobacterium longum NCC3001 normalizes anxiety-like behavior and

418

hippocampal brain derived neurotrophic factor (BDNF) in mice with infectious colitis. 107 In

419

rats, the administration of Lactobacillus helveticus and Lactobacillus rhamnosus can

420

prevent

421

demonstrated with Lactobacillus farciminis.109 Hsiao et al. demonstrated gastrointestinal

422

barrier defects and microbiota alterations in the maternal immune activation (MIA) mouse

423

model, which is known to display features of ASD.110 Oral treatment of the offspring with

424

the human commensal Bacteroides fragilis corrected the gut permeability, altered the

425

microbial composition, and ameliorated the defects in communicative, stereotypic, anxiety-

426

like and sensorimotor behaviors. MIA offspring displayed an altered serum metabolomic

427

profile, and Bacteroides fragilis modulated the levels of several metabolites. One

428

metabolite of particular interest was 4-ethylphenylsulfate (4EPS). 4EPS is thought to be a

429

uremic toxin, as is p-cresol (4-methylphenol), a chemically related metabolite reported to

430

be a possible urinary biomarker for ASD.111 This study showed a 46-fold increase in 4EPS

431

in the MIA model and normalization upon Bacteroides fragilis treatment. Moreover, the

432

treatment of mice with 4EPS potassium salt was sufficient to induce anxiety (but not

433

autism)-like behaviors similar to that of the MIA offspring.

intestinal

abnormalities.108 The

same

effect

was

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chronic-stress-induced

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Unfortunately, studies in humans are scarce and have not always yielded

435

satisfactory results, highlighting the difficulties in extrapolating experimental findings to

436

human physiology. Daily consumption of a fermented milk product with probiotics

437

(Bifidobacterium animalis subspecies lactis, Streptococcus thermophiles, Lactobacillus

438

bulgaricus, and Lactococcus lactis subspecies lactis) for 4 weeks was associated with a

439

significant modification of the activity of CNS regions that control emotion and

440

sensation.112 Moreover, it was shown that probiotic supplementation early in life might 18

ACCEPTED MANUSCRIPT reduce the risk of neuropsychiatric disorder development later in childhood.113 Seventy-five

442

infants who were randomized to receive Lactobacillus rhamnosus GG or placebo during

443

the first 6 months of life were followed for 13 years. At this age, ADHD was diagnosed in

444

6/35 (17.1%) children in the placebo group and none in the probiotic group (p=0.008). The

445

mean (standard deviation) numbers of Bifidobacterium species bacteria in feces during the

446

first 6 months of life were significantly lower in affected children [8.26 (1.24) log cells/g]

447

than in healthy children [9.12 (0.64) log cells/g; p=0.03)]. A positive effect on CNS function

448

was also demonstrated by Romeo et al., who examined the administration of Lactobacillus

449

reuteri or Lactobacillus rhamnosus to preterm infants for 6 weeks or until discharge.114 The

450

neurological structured assessment performed at 1 year of age revealed a significantly

451

lower incidence of neurological abnormalities in children receiving probiotics than in

452

untreated controls. By contrast, the administration of a mixture of Lactobacillus acidophilus

453

and Bifidobacteria infantis to preterm very-low-birth-weight infants until discharge from the

454

hospital (approximately 45 days) did not modify the psychometric parameters measured at

455

3 years corrected age in comparison with control subjects not receiving probiotic

456

supplementation.115

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Very few studies have evaluated the effects of probiotics on ASD clinical features in

458

humans. Parracho et al. investigated the potential of Lactobacillus plantarum WCSF1 to

459

modulate the gut microbiota of autistic subjects through a double-blind, placebo-controlled,

460

crossover-designed feeding study.116 The fecal microbiota, gut function and behavior

461

scores of subjects were examined throughout the 12-week study. Probiotic administration

462

significantly increased the Lactobacilli and Enterococci group and significantly reduced

463

Clostridium cluster XIVa compared with placebo. In addition, probiotic feeding caused

464

significant differences in behavior scores compared to baseline. Another study reported

465

that probiotic administration can induce significant metabolic modifications considered

466

indicative of the disruption of gut dysbiosis but not marginal behavioral changes such as

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improvements in the ability to concentrate and carry out orders.1107 Considering the need

468

for further studies able to definitively establish whether probiotics can reduce ASD

469

symptoms, a very well-designed protocol for a large, definitive, randomized controlled trial

470

has recently been proposed.118 Prebiotic oligosaccharides are indigestible nutritional constituents that are purported

472

to have antimicrobial, immunomodulatory and anti-inflammatory properties.119 These

473

effects are attributable to a direct effect on the gut microbiota because they promote the

474

growth of Bifidobacterium species and consequently reduce (through competition)

475

colonization by pathogenic flora. Moreover, they can direct interact with gut cells, assuring

476

the integrity of the intestinal barrier.120 In rodents, in which the absence of gut bacteria is

477

associated with decreased central expression of brain derived neurotropic factor (BDNF)

478

and N-methyl-d-aspartate receptor subunits, oral probiotics increase brain BDNF and

479

impart associated anxiolytic effects.121 Moreover, prebiotic administration normalizes

480

lipopolysaccharide-induced anxiety and the cortical 5-HT2A receptor and IL1-β levels.122

481

Finally, neonatal prebiotic supplementation seems to modify neurotransmission with a

482

long-duration effect persisting until the adult age.123 As with probiotics, prebiotic studies

483

regarding the impact of prebiotic use in humans with neurological problems are few and

484

not conclusive. In adult volunteers, the administration of galactooligosaccharides was

485

associated with a reduction of the waking cortisol response and significant modification of

486

emotional bias, suggesting a potential direct effect on brain function. However, in the same

487

studies, a different prebiotic had no effect, suggesting that as with probiotics, the choice of

488

the right compound is critical to obtain satisfactory modification of the CNS activity.124

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489

Finally, dietary modification has been considered, and animal studies have been

490

conducted. In most of these instances, certain effects on the gut microbiota were shown,

491

with favorable modifications of CNS function. For example, in rats, ingestion of a diet with

492

a high beef content was associated with the exclusive presence in the gut of certain 20

ACCEPTED MANUSCRIPT 493

bacterial genera, including Proteus, Serratia, Sarcina, and Staphylococcus. These animals

494

displayed increased learning and memory behaviors as well as decreased levels of

495

anxiety-like behavior.125 However, no relevant data have been collected in humans.

496

CONCLUSIONS

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Data seem to indicate that the gut microbiota influences CNS development and

499

function and that gut dysbiosis is associated with significant neurological problems.

500

However, most of these results have been collected in experimental animals and cannot

501

be transferred to humans. The human gut-brain axis fundamentally differs from the rodent

502

axis primarily because of the great expansion of the prefrontal cortex and the frontoinsular

503

regions, which play a major role in human emotional regulation. Moreover, it is not

504

definitively established whether neurologic diseases depend on a generic modification of

505

the gut microbiota or whether a single bacterial phylum or species plays a specific role for

506

any single condition. Furthermore, limited information exists regarding protective bacteria.

507

The evidence that both probiotics and prebiotics can have different impacts according to

508

the microbial species or oligosaccharides that are administered indicates the difficulty of

509

evaluating the relationship between bacteria and the CNS, as well as the best therapeutic

510

approach. In humans, particularly in children, several factors may be important in

511

conditioning gut microbiota modifications; unfortunately, most of these factors act

512

simultaneously and can induce different gut microbiota alterations. More efforts are

513

required to fully define the array of complex behaviors that are influenced by the gut

514

microbiota at the CNS level, as well as the mechanisms involved. This seems particularly

515

important for younger children, who are at greater risk of significant damage to CNS

516

structure and function.

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ACKNOWLEDGMENTS

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This review was partially supported by a grant from the Italian Ministry of Health

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(Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico Ricerca Corrente 2016

523

850/01).

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Table 1. Factors influencing gut microbiota during development. Factor Antibiotic therapy, malnutrition or overnutrition, obesity, diabetes, eczema, stress during pregnancy Duration of gestation, mode of delivery, use of antibiotics, type of feeding

RI PT

Development phase Fetal life

Postnatal period 873

SC

874 875

M AN U

876 877 878 879

883 884 885 886 887 888

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889 890 891 892 37

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Table 2. Mechanisms of interaction between gut microbiota and central nervous system. Mechanism Passage of lipopolysaccharides in the cell wall of some Gram-negative bacteria from the intestinal mucosa to the systemic circulation Short chain fatty acid production by bacteria fermentation 5-hydroxytryptamine biosynthesis from gut bacteria Stress-related increase in the cortisol level

Result Production cytokines

of

pro-inflammatory

RI PT

895 896 897

Alteration of mitochondrial function

M AN U

SC

Increase in neuronal cell division and differentiation Increased gut permeability and increased volume of the amygdala Modification of the blood-brain barrier Reduced expression of the tight junction proteins occludin and claudin5 Production of peptides that activate afferent Influence on behavior and neural endings of the vagus nerve activity 898

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38

ACCEPTED MANUSCRIPT HIGHLIGHTS •

Gut dysbiosis has been associated with alterations of central nervous system (CNS).

•

The effects of dysbiosis on the CNS are significantly more important during the

RI PT

developmental period. •

Both probiotics and prebiotics can have different impacts on CNS.

•

In humans, several factors may be important in conditioning gut microbiota

EP

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microbiota at the CNS level.

M AN U

Efforts are required to clarify the mechanisms that are influenced by the gut

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•

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modifications.