The life-long cumulative exposures (exposome) to environmental contaminants (even
low-grade lead, mercury, arsenic etc.) and biological hazards (favoring enteric pathogens
and altered “unhealthy” intestinal microbiota) alone or in combination are now being
increasingly recognized to deleteriously influence the brain's development and potentially
the way the brain copes with aging-related conditions, including neurodegenerative
diseases (Costa et al., 2004; Senut et al., 2012; Tshala-Katumbay et al., 2015). The
latter may involve sub-optimal development of “cognitive reserve,” which is likely
dependent upon a “healthy” and enriched environment to which one is exposed early
in life. The potential importance of cognitive reserves to protect from aging-related
neurodegeneration is suggested by post-mortem evidence showing that some individuals
are better adapted to Alzheimer's disease (AD) related brain injury than others (Marques
et al., 2016); some patients who show post-mortem beta-amyloid plaques in the brain
had not suffered from AD symptoms during life.
The early life intestinal microbiome is now being acknowledged as a determinant factor
influencing human behavior (Oriá et al., 2016; Lima-Ojeda et al., 2017; Carlson et
al., 2018) and for immune system maturation (Mulder et al., 2011; Olszak et al., 2012;
Nash et al., 2017). If chronically disrupted it may even predispose individuals to
neuropsychiatric diseases later in life (Petra et al., 2015; Lima-Ojeda et al., 2017).
In addition, in the first 2 years of life, a critical period when key processes toward
brain maturation such as myelination and synaptogenesis occur, the intestinal microbiota
have not yet reached full “adulthood” maturation (Fanaro et al., 2003; Olivares et
al., 2018). As these biological processes overlap in this critical period, environmental
challenges early in life, such as enteric diseases, malnutrition and altered maturation
of the intestinal microbiota may jeopardize the cognitive development and cause later
life metabolic dysfunction in these children (DeBoer et al., 2012; Guerrant et al.,
2013). However in spite of the potential implications of early-life microbiota colonization
to life-long health, animal models (such as germ-free and antibiotic treatment) widely
used to study causal relationship with gut microbiota have limitations in how they
affect the brain, and in their ability to model human conditions where environmental
conditions are dynamic and constantly changing. Furthermore, the use of mouse models
to research into microbiome-brain-immune should be taken with caution, since the species
composition of the gut microbiota (and its regulation) can be fairly dissimilar in
distinct mammalian taxa (Ericsson and Franklin, 2015; Laukens et al., 2016), therefore
findings from mouse gut research may not be extrapolated to humans. In addition, reproducibility
of findings from animal models may be affected by the intestinal microbiota composition
that may vary with animal housing, including type of caging, type and frequency of
bedding change and water source. In addition, animals from different vendors may have
distinct intestinal microbiota characteristics (Ericsson and Franklin, 2015).
Animal models to study the brain still rely too much on standard “clean-barrier” or
even “enriched” vivarium environments for reproduction of the data, however such models
may truly miss the mark by not accurately reproducing the “realistic” environment
of early life for most of the world's populations (such as in impoverished settings
of the developing world). As the brain is a biological sensor, changes in the environment
are an essential determinant of structural and functional neural modifications to
optimize species survival; including intergenerational transfer of biological information
as a genetic imprint to the offspring. This is a key factor in order to successfully
pass (genetically or epigenetically) the “environmentally-fit” genes to other generations.
The use of laboratory mice in neuroscience studies (and in basic science in general)
have slowly switched over the past four decades from outbred stocks to almost exclusive
use of inbred strains, with C57BL/6 strains now the predominant rodent model. As a
modern biological research tool three important needs have driven this transition:
(1) the need for genetically-engineered mice to understand the role of genes regulating
biological phenomena by knocking-down or knocking-in (e.g., targeted replacement)
techniques, which have required an inbred background; (2) The need to control for
mouse genetic variability (seen in outbred mice), which could mask the “true” phenotype
determined by the experimental condition in a controlled-manner; and (3) The reproducibility
of the findings by other research groups, which is a fundamental caveat of the scientific
method. This has also driven the housing and care of mouse models toward more controlled
and pristine conditions and the pursuit of laboratory animal housing standardization
and specific pathogen free (SPF), barrier-style vivaria for pre-clinical research.
These facilities were designed to prevent potential effects of widely varying environmental
conditions, microbial status, and chemical contaminants. Technologies rapidly developed
to maintain mice in almost sterile conditions including HEPA filtered ventilated housing
racks, HEPA filtered changing stations, autoclaved cages, autoclaved or irradiated
feed, and drinking water treated in some fashion (reverse osmosis, autoclaving, mechanical
filtration, chlorination, and/or acidification); all with the intent to exclude a
range of microbial agents and other environmental contaminants thought to possibly
“interfere” with animal health and research outcomes.
As the role of the intestinal-brain axis arises as a key factor in regulating brain
responses to the environment, understanding how exposure to contaminated or “unhealthy”
environments (fecal coliforms, enteric pathogens, heavy metals, imbalanced diets etc.)
can affect the intestinal microbiome in the first years of life becomes critical to
define and understand. The intestinal microbiome is considered “plastic” early in
life (first 2 years of post-natal life). Maturation of the intestinal microbiota may
be delayed if the host animal is exposed to dietary limitations or to contaminated
environments (Subramanian et al., 2014). The disrupted intestinal microbiota (with
small intestinal bacterial overgrowth and dysbiosis) early in life due to dietary
limitations or contaminated environments (Donowitz and Petri, 2015; Donowitz et al.,
2016) could have a key role in affecting brain development at the same time when the
brain also has its greatest plasticity (Clarke et al., 2013). The way the environmental-born
intestinal microbiota may drive the immune system and associated cognitive outcomes
can be depicted by findings that weanling germ-free mice have increased serum IgE
levels, which rely on upregulated ThCD4-derived IL-4 rather than the IgE baseline
levels seen in mice under pathogen-free vivaria (Cahenzli et al., 2013). It has been
recognized that meningeal ThCD-4-derived IL-4 has been associated with pro-cognitive
effects in mice (Derecki et al., 2010). Interestingly, germ free mice show improved
motor activity and reduced anxiety, compared with SPF mice with a normal gut microbiota
(Diaz et al., 2011; Neufeld et al., 2011), however, with poor social development (Desbonnet
et al., 2014) and with brain neurochemical changes (Clarke et al., 2013). On the other
hand, intestinal microbial dysbiosis induced by antibiotic treatment has been shown
to impair novel object recognition in mice (Fröhlich et al., 2016), and was associated
with decreased hippocampal transcriptions levels of NPY1R, NPY2R, BDNF, and NMDAR2B
(GRIN2B), well-known modulators of hippocampal glutamatergic transmission, therefore
suggesting an association between gut microbiota alterations and novel object recognition
impairment.
Microbiota dysbiosis and intestinal barrier dysfunction induced by chronic inflammation
may lead to circulating endotoxins, such as LPS, that may cause long-term disruption
in the cognitive reserve relying on newly generated neurons from the hippocampus in
adulthood (Valero et al., 2014). Nevertheless, neonatal peripheral injection of LPS
may lead to priming of brain microglia with a second challenge during adulthood causing
impaired neurogenesis and poor spatial memory (Dinel et al., 2014). Of note, microglia
function may be influenced by microbiota-derived short-chain fatty acids (Erny et
al., 2015). Limited microbiota complexity also resulted in defective microglia (Erny
et al., 2015, 2017). The intestinal microbiota composition may also influence the
blood-brain barrier (BBB) permeability (Braniste et al., 2014; Labzin et al., 2018).
This is an important issue, as microbiota dysbiosis may affect innate immune responses.
Such effects may be exemplified by the known induction of Th17 cells by segmented
filamentous bacteria (Gaboriau-Routhiau et al., 2009) (found in rodent microbiota
but not humans) and Bacteroides fragilis-derived polysaccharide A induction of Treg
cells (Mazmanian et al., 2005). Another intriguing aspect is that the gut microbiota
driven by western diets might favor the production of autoantibodies with implications
for systemic inflammation and neurodegenerative diseases (Petta et al., 2018).
The exposure to early intestinal microbial products may lastingly affect the BBB permeability,
a key factor in regulating the brain milieu and function. It has been shown that germ
free mice have more permeable BBB with decreased occludin and claudin-5 levels than
their pathogen-free raised counterparts (Braniste et al., 2014; Sharon et al., 2016).
Few studies have addressed the BBB following enteric infections and malnutrition in
young mice, although bacterial infections following acute stress have been associated
with memory impairment (Gareau et al., 2011). The brain ultrastructure changes in
germ-free mice also include increased hippocampal neurogenesis (Ogbonnaya et al.,
2015), as well as decreased microglial maturation and ramification (Erny et al., 2015),
and increased prefrontal cortex myelination (Hoban et al., 2016). Another interesting
observation is that laboratory mice do not show effector-differentiated mucosal memory
T-cells and therefore do not fully resemble the immune systems of adult humans (Beura
et al., 2016).
In addition, the concept that the brain is considered an “immune privileged” site,
is now shaped by the exciting discovery of lymphatic vessel in the dura sinus and
the trafficking of immune cells between the brain and the deep cervical lymph nodes,
which may shed light on the potential role of the adaptive immune system surveillance
in response to intestinal microbial population changes in homeostasis and disease
and the signals to the central nervous system (Louveau et al., 2015; Kipnis and Filiano,
2018), perhaps with the engagement of the meningeal immune system (Kipnis, 2016) as
well during the course of a life span.
As new gut-brain research emerges and evolves to unravel the key effects of intestinal
microbiota on brain development, there is a need to revisit animal modeling and housing
that can better simulate the impoverished environments faced by many people around
the world, to identify and address brain-related markers and outcomes. The understanding
of this premise is a critical first step for neuroscientists to rethink laboratory
housing standardization in some research initiatives in modeling brain development
studies. Defining laboratory housing conditions in order to evaluate specific dietary,
enteric pathogen and microbiome effects relevant to “real-life” conditions, including
such problems as “environmental enteropathy” is challenging, with a need to preserve
what has been accomplished by the modern vivarium and environmental standardization,
which favors research quality and reproducibility.
One example of this environmental issue can be depicted by murine model studies of
malnutrition that have been conducted in the standard barrier-protected vivarium,
using inbred mouse models (without environmentally contaminated-intestinal microbiota)
and looked at brain markers. Studies that assume no environmental contamination may
have relevance to populations in rich-resource developed countries with a high quality
of life and good public health systems, where in general the level of environment
contamination is low. However they are much less likely to be relevant to the majority
of undernourished people residing in sub-Saharan Africa, Latin America, and Asia where
poverty is still a huge public health problem and fecally-contaminated environments
prevail exposing children to enteric infections and malnutrition. The condition called
environmental enteropathy has been described in children to define a chronic subclinical
intestinal inflammation, mediated by a T-cell response, a cause or consequence of
malnutrition and enteric infections (even without diarrhea) in the first years of
life (Korpe and Petri, 2012). This malnutrition-enteric infections cycle has been
associated with poor cognitive and metabolic outcomes that may be sustained well into
late childhood or even later life (Guerrant et al., 2013).
Early enteric infections and systemic inflammation may profoundly affect neurodevelopment
in children (Donowitz et al., 2018). Initial work to address these problems of dissecting
diet, microbiome, and pathogen effects in carefully defined murine models is underway.
These include studies of diets, antibiotics and pathogen effects, and demonstrate
the importance of recognizing the complexity of these often quite specific interactions.
Examples include recent metabolic studies of protein and zinc deficient diets (Mayneris-Perxachs
et al., 2016) as well as effects of specific pathogens including Cryptosporidium,
Giardia, enteroaggregative E. coli and others (Coutinho et al., 2008; Barash et al.,
2017; Bartelt et al., 2017). Hence, application of the increasingly available tools
of genetics (including knockdown and targeted transgenic murine models as well as
GWAS, SNPchip and epigenetic methylation studies), microbiology (including deep sequencing
of the microbiota, “germ free,” “humanized” as well as TAC card diagnostics) (Liu
et al., 2013) and especially now metabolomics (Farras et al., 2018), selective cell
sorting and signaling and localization of transcriptomics in the gut and CNS, (Hoban
et al., 2016; Zubcevic et al., 2017) are critical to understand the acute and long-term
impact of widespread enteropathy on cognitive/brain development and maturation; as
well as to designing and assessing the effects of targeted safe and effective interventions
to ameliorate these devastating effects and optimize healthy cognition/brain development
and immune system maturation.
We believe that there is no single and definitive response to assess the role played
by environmental contaminants on gut microbiota and health outcomes in the controlled
vivarium; the solutions may depend on specific research questions and on the environmental
particularities of each animal setting. Recently, efforts have been made to reconstitute
the intestinal microbiota from the laboratory mouse living in standard pathogen-free
conditions with a more “natural” microbiota obtained from wild mice. Such efforts
of microbiota transfer from wild mice have led to reduced inflammation and improved
resistance to infectious pathogens in the laboratory mice (Rosshart et al., 2017).
It would be interesting to know whether such transfer could improve behavior following
malnutrition and enteric infections in that host in the most sensitive times of early
post-natal cognitive development. Another good approach is suggested by Shin et al.
(2018), who compared intestinal microbiome in young and aged mice and utilized a cage
switch protocol to promote exchange of microbiota from these different populations
of mice under standard controlled conditions.
In conclusion, the intestinal microbiota is dynamic in the first years of life, when
its constitution is profoundly influenced by the surrounding environment. Early life
intestinal microbiota is therefore considered immature and more “plastic” until it
approaches adult-like characteristics in later childhood. The brain is also very plastic
in the first 2 years of life, the very same time period when children may be exposed
to changing diets and contaminated environments, and afflicted with clinical and subclinical
enteric infections. Enteric infections in the first 2 years of life may alter the
normal progression of the intestinal microbiota to “adult-like” maturation and may
disrupt optimal brain development, especially in children living in impoverished settings
who likely receive a heavier and prolonged infection load. Neuroscience research has
relied on inbred mouse models and barrier-protected housing conditions where mice
are kept in “clean” standardized environments to study the biological phenomena, and
secure conditions that favor the reproducibility of data. In this opinion paper, we
suggest the need to revisit animal models and housing conditions to study early life
brain development, with greater emphasis on modeling contaminated environments, higher
pathogen load, altered microbiota and environmental enteropathy (EE) and better represent
conditions that afflict many children living in impoverished communities around the
world. Such innovative brain research can elucidate mechanisms of brain metabolism
and cognitive function at both extremes of age and potential innovative interventions
to ameliorate their disruption.
Author contributions
All authors listed have made a substantial, direct and intellectual contribution to
the work, and approved it for publication.
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial
or financial relationships that could be construed as a potential conflict of interest.
The handling Editor declared a shared affiliation, though no other collaboration,
with some of the authors DB, RG.