Early effects of lipopolysaccharide-induced inflammation on foetal brain development in rat

Intellectual and Developmental Disability Research Center, Jane and Terry Semel Institute for Neuroscience and Human Behavior, Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine at the University of California at Los Angeles, 90095, USA.
ASN Neuro (Impact Factor: 4.02). 10/2011; 3(4). DOI: 10.1042/AN20110027
Source: PubMed
ABSTRACT
Studies in humans and animal models link maternal infection and imbalanced levels of inflammatory mediators in the foetal brain to the aetiology of neuropsychiatric disorders. In a number of animal models, it was shown that exposure to viral or bacterial agents during a period that corresponds to the second trimester in human gestation triggers brain and behavioural abnormalities in the offspring. However, little is known about the early cellular and molecular events elicited by inflammation in the foetal brain shortly after maternal infection has occurred. In this study, maternal infection was mimicked by two consecutive intraperitoneal injections of 200 μg of LPS (lipopolysaccharide)/kg to timed-pregnant rats at GD15 (gestational day 15) and GD16. Increased thickness of the CP (cortical plate) and hippocampus together with abnormal distribution of immature neuronal markers and decreased expression of markers for neural progenitors were observed in the LPS-exposed foetal forebrains at GD18. Such effects were accompanied by decreased levels of reelin and the radial glial marker GLAST (glial glutamate transporter), and elevated levels of pro-inflammatory cytokines in maternal serum and foetal forebrains. Foetal inflammation elicited by maternal injections of LPS has discrete detrimental effects on brain development. The early biochemical and morphological changes described in this work begin to explain the sequelae of early events that underlie the neurobehavioural deficits reported in humans and animals exposed to prenatal insults.

Full-text

Available from: Cristina A Ghiani
Early effects of lipopolysaccharide-
induced inflammation on foetal brain
development in rat
Cristina A Ghiani*
1
, Natalia S Mattan*
,{2
, Hiroko Nobuta*
,{
, Jemily S Malvar*
,{3
, Julie Boles
1
, Michael G Ross
1
, James A
Waschek*, Ellen M Carpenter*, Robin S Fisher* and Jean de Vellis*
,{
*Intellectual and Developmental Disability Research Center, Jane and Terry Semel Institute for Neuroscience and Human Behavior, Department of
Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine at the University of California at Los Angeles, Los Angeles, CA 90095, U.S.A.
{
Department of Neurobiology, David Geffen School of Medicine at the University of California at Los Angeles, Los Angeles, CA 90095, U.S.A.
{
Neuroscience Interdepartmental Program, David Geffen School of Medicine, University of California at Los Angeles, CA 90095, U.S.A.
1
Department of Obstetrics and Gynecology, Harbor-UCLA Medical Center, Torrance, CA 90509, U.S.A.
Cite this article as: Ghiani CA, Mattan NS, Nobuta H, Malvar JS, Boles J, Ross MG, Waschek JA, Carpenter EM, Fisher RS, de Vellis J (2011) Early effects
of lipopolysaccharide-induced inflammation on foetal brain development in rat. ASN NEURO 3(4):art:e00068.doi:10.1042/AN20110027
ABSTRACT
Studies in humans and animal models link maternal infec-
tion and imbalanced levels of inflammatory mediators in the
foetal brain to the aetiology of neuropsychiatric disorders.
In a number of animal models, it was shown that exposure to
viral or bacterial agents during a period that corresponds
to the second trimester in human gestation triggers brain
and behavioural abnormalities in the offspring. However,
little is known about the early cellular and molecular events
elicited by inflammation in the foetal brain shortly after
maternal infection has occurred. In this study, maternal
infection was mimicked by two consecutive intraperitoneal
injections of 200 mg of LPS (lipopolysaccharide)/kg to timed-
pregnant rats at GD15 (gestational day 15) and GD16.
Increased thickness of the CP (cortical plate) and hippo-
campus together with abnormal distribution of immature
neuronal markers and decreased expression of markers for
neural progenitors were observed in the LPS-exposed foetal
forebrains at GD18. Such effects were accompanied
by decreased levels of reelin and the radial glial marker
GLAST (glial glutamate transporter), and elevated levels of
pro-inflammatory cytokines in maternal serum and foetal
forebrains. Foetal inflammation elicited by maternal injec-
tions of LPS has discrete detrimental effects on brain
development. The early biochemical and morphological
changes described in this work begin to explain the sequelae
of early events that underlie the neurobehavioural deficits
reported in humans and animals exposed to prenatal insults.
Key words: prenatal inflammation, lipopolysaccharide (LPS),
brain development, cytokine, maternal infection, neurode-
velopmental disorder.
INTRODUCTION
Previous studies suggest that modifications of the in utero
environment due to maternal bacterial or viral infections can
have disturbing effects on foetal brain development resulting
in lifelong intellectual and behavioural disorders, such as
schizophrenia and cerebral palsy (Rees and Harding, 2004;
Hagberg and Mallard, 2005; Rees et al., 2008; Fatemi and
Folsom, 2009; Meyer et al., 2009b; Patterson, 2009; Watanabe
et al., 2010). Animal models have been developed to study the
link(s) between functional deficits and cellular and morpho-
logical changes in the offspring’s brain following prenatal
exposure to agents known to stimulate the immune system,
such as LPS (lipopolysaccharide) or poly(I:C) (polyriboinosinic-
polyribocytidilic acid) (Nawa and Takei, 2006; Meyer et al.,
2009a; Boksa, 2010). These studies support the notion that
some gestational periods (e.g. early versus late pregnancy) offer
a higher risk for developing behavioural dysfunction following
maternal infections (Meyer et al., 2007). The foregoing results
are obviously dependent upon the neural cell types maturing
during the gestational window that would be targeted by the
events elicited during maternal immune activation.
The mechanism(s) that mediates the effects of maternal
infection on the developing brain has not been yet identified.
1
To whom correspondence should be addressed (email cghiani@mednet.ucla.edu).
2
Present address: Children’s Hospital Los Angeles, Saban Research Institute, 4661 W. Sunset Blvd. MS 135, Los Angeles, CA 90027, U.S.A.
3
Present address: Childrens Hospital Los Angeles, 4650 Sunset Boulevard, MS 54, Los Angeles, CA 90027-6016, U.S.A.
Abbreviations: Arc, activity-regulated cytoskeletal-associated protein; CNS, central nervous system; CP, cortical plate; DAPI, 49,6-diamidino-2-phenylindole; GD, gestational
day; GFAP, glial fibrillary acidic protein; GLAST, glial glutamate transporter; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H&E, haematoxylin and eosin; IEG,
immediate early gene; IL, interleukin; IZ, intermediate zone; LPS, lipopolysaccharide; MgZ, marginal zone; PFA, paraformaldehyde; poly(I:C), polyriboinosinic-polyribocytidilic
acid; qRT–PCR, quantitative real-time PCR; SVZ/VZ, subventricular zone/ventricular zone; TNFa, tumour necrosis factor a.
E 2011 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licence (http://
creativecommons.org/licenses/by-nc/2.5/) which permits unrestricted non-commercial use, distribution and reproduction in any mediu m, provided the original work is
properly cited.
RESEARCH ARTICLE
ASN NEURO 3(4):art:e00068.doi:10.1042/AN20110027
asnneuro.org / Volume 3 (4) / art:e00068 233
Page 1
Data from different groups have linked elevated cytokine
levels triggered by maternal infection to altered gene
expression and function in the maternal/foetal environments,
including the foetal brain, suggesting that inflammation
and its mediators interfere with normal development. In
fact, within hours (1–24 h) of LPS or poly(I:C) administration
to pregnant rodents, elevated expression levels of pro-
inflammatory cytokines were reported in the placenta and
the amniotic fluid as well as in the foetal plasma, liver
and brain (Urakubo et al., 2001; Bell et al., 2004; Ashdown
et al., 2006; Jonakait, 2007; Boksa, 2010). Cytokines have a
wide range of roles in the innate and adaptive immune
system and influence various neurodevelopmental processes,
including cell differentiation, maturation and survival (Zhao
and Schwartz, 1998; Deverman and Patterson, 2009;
Watanabe et al., 2010). Hence, fluctuations in their maternal
and foetal levels, due for instance to a maternal infection,
signify a disturbance that can impede the ongoing
neurodevelopmental processes, and subsequently affect
proper neural cell maturation (Jonakait, 2007; Meyer et al.,
2009a, 2009b). In support of a role for pro-inflammatory
cytokines in the brain, increased gliosis and apoptosis, and a
loss of pyramidal cells in the hippocampus were also trig-
gered by direct injection of the pro-inflammatory cytokine
IL-6 (interleukin-6) to the mother (Smith et al., 2007). Overall
these findings support the contention that immune activa-
tion at specific gestational times may have diverse effects on
the development of brain regions and further support the
hypothesis that some of the disturbances on brain develop-
ment are mediated by an increase in cytokines.
Notwithstanding major advances made on the long-term
effects of prenatal inflammation on postnatal brain functions
(Boksa, 2010), very few reports have investigated changes
occurring at prenatal stages of brain development shortly
after a maternal challenge (Meyer et al., 2008a; Cui et al.,
2009). Since these changes are more proximal than those
observed postnatally, studies of foetal brains exposed in utero
to maternal infection should be suited to identify the
upstream molecular events of brain pathology and may
eventually help to determine the underlying cause(s) of brain
malfunction later in young adults. The present study was
designed to investigate effects on the development of im-
mature neurons as well as neural progenitors associated with
foetal inflammation, and occurring shortly after maternal im-
mune system stimulation at mid–late gestation. Maternal
infection was mimicked by two consecutive intraperitoneal
injections of LPS (200 mg/kg) to timed-pregnant rats at
GD15 (gestational day 15) and GD16, such that the foetuses
were exposed to increased concentrations of inflammatory
mediators soon after the peak of neurogenesis (Sauvageot and
Stiles, 2002). We show that maternal injections of LPS
at mid–late gestation induced an increase in cytokines in the
foetal brain as well as changes in neural cell maturation
and patterning. These effects may be relevant to defects in
intellectual and behavioural functions described in adult
animals following prenatal exposure to inflammation.
MATERIALS AND METHODS
Animals and treatment
Timed pregnant Sprague–Dawley rats were purchased from
Charles River and housed in AALAC-approved clean animal
facilities with a 12 h light/12 h dark regime. The animals were
divided into two groups: control (saline-treated) and LPS-
treated. The dams were injected intraperitoneally with either
saline or 200 mg of LPS/kg from Escherichia coli 055:B5 (List
Biological Laboratories) at GD15 and GD16. The dams were
observed daily to detect signs of distress and killed at dif-
ferent times after the first or second injection of LPS, or allowed
to give birth. The studies were performed in accordance with
the NIH guidelines for the Care and Use of Laboratory Animals,
and approved by the UCLA Chancellor’s Animal Research
Committee.
ELISA assay in maternal serum
To measure the levels of pro-inflammatory cytokines in
maternal serum, dams were injected intraperitoneally with
either saline or LPS and killed 4 h after the first injection (saline
n55; LPS n59) at GD15, 4 h after the second injection at GD16
(saline n56; LPS n510) or 2 days after the second injection
at GD18 (saline n59 or LPS n59). The amounts of pro-
inflammatory cytokines were assessed as previously reported
(Juarranz et al., 2005) using murine IL-6, IL-1b and TNFa
(tumour necrosis factor a) ELISA Development Kits (Peprotech).
Absorbance was measured at 450 nm on a microplate reader
(SPECTRA max M2; Molecular Devices).
Antibodies
The following mouse monoclonal antibodies were purchased
from the vendors indicated in parentheses: against Nestin and
Arc (activity-regulated cytoskeletal-associated protein; BD
Biosciences), against a-internexin (Millipore), against bIII-tubulin
(Covance) and against b-actin (Sigma). A mouse anti-rat
monoclonal against CD68 (clone ED1) was purchased from
Accurate Chemical and Scientific Corporation. The following
rabbit polyclonal antibodies were used: against doublecortin
(Cell Signaling), against a-internexin and reelin (Clones G10
and 142; Millipore), against bIII-tubulin (Covance), against
GFAP (glial fibrillary acidic protein; Dako). A guinea pig poly-
clonal antibody against GLAST (glial glutamate transporter)
was purchased from Millipore. Details are given in Sup-
plementary Table S1 (available at http://www.asnneuro.org/an/
003/an003e068add.htm).
Immunohistochemistry
Pregnant rats were killed at GD18, 2 days after the second
injection of saline or LPS. The foetuses were rapidly dissected
out, perfused with PBS and fixed overnight in 4% (w/v)
C.A. Ghiani and others
234 E 2011 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/)
which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.
Page 2
PFA (paraformaldehyde) at 4
˚
C. Newborn pups [postnatal day
(P)1] were perfused intracardially with 4% PFA. Brains were
dissected out, post-fixed in 4% PFA at 4
˚
C overnight, cryo-
protected in 15% sucrose and then embedded in Tissue Tek OCT
compound. Immunolabelling of frozen sections (20 mm) was
performed as previously described (Mattan et al., 2010). Briefly,
sections were fixed in 4% PFA for 15 min, blocked in carrier
solution (1% BSA and 0.3% Triton X-100) containing 20%
normal goat serum for 1 h and incubated overnight at 4
˚
C
with primary antibodies diluted in carrier solution containing
5% normal goat serum. Sections were incubated with the
appropriate secondary antibodies conjugated to Cy3 (Jackson
ImmunoResearch Laboratories) or Alexa FluorH 488 (Molecular
Probes), and mounted in Vectashield mounting medium with
DAPI (49,6-diamidino-2-phenylindole; Vector Laboratories).
Immunostained sections were visualized using a Zeiss Axio
Imager 2 with an AxioCam MRm and the ApoTome imaging
system or an Olympus IX81 microscope equipped with a
Hamamatsu ORCA-ER CCD camera.
H&E (haematoxylin and eosin) staining and
measurements
GD18 and P1 brain structures were stained with H&E as
previously reported (Mattan et al., 2010) and inspected using
the Axiovision software on a Zeiss Axioskop with an Axiocam.
Measurements were performed on the third sagittal brain
slice, typically 1.5 mm from the midline, of three or four
consecutive slides from three to six animals. Neocortical
measurements were obtained from presumptive motor areas
slightly caudal to the anterior commissure, and near the level
of the posterior genu of the corpus callosum. Total neocor-
tical and laminar thickness were measured along a line (eyepiece
micrometer) orthogonal to the superficial pial and callosal
surfaces, and averaged across cases for comparisons between
groups. Hippocampal measurements were obtained in a com-
parable fashion from presumptive CA1 area, as evidenced
by its distinct pyramidal cell layer, immediately rostral and
superior to its transition from the dorsal subiculum.
Western-blot analysis
Rat cerebral cortices from GD18 foetuses were rapidly dis-
sected, and the two halves were frozen separately and used
for Western-blot analysis or qRT–PCR (quantitative real-time
PCR). Cortices were homogenized in lysis buffer containing 50
mM Tris/HCl, 0.25% (w/v) DOC (sodium deoxycholate), 150 mM
NaCl, 1 mM EDTA, 1% (w/v) Triton X-100, 0.1% (w/v) SDS,
1mMNa
3
VO
4
(sodium orthovanadate), 1 mM AEBSF [4-(2-
aminoethyl)benzenesulfonyl fluoride], 10 mg/ml aprotinin, 10
mg/ml leupeptin, 10 mg/ml pepstatin and 4 mM sodium fluoride.
Western blottings were performed as previously described
(Ghiani and Gallo, 2001; Ghiani et al., 2010). Then 25–35 mgof
total proteins were loaded on to a 4–20% Tris-glycine gel
(Invitrogen). Protein bands were detected by chemilumin-
escence using the Amersham ECL kit (GE Healthcare) with HRP
(horseradish peroxidase)-conjugated secondary antibodies
(Cell Signaling). Relative intensities of the protein bands were
quantified by scanning densitometry using the NIH Image
Software (Image J, http://rsb.info.nih.gov/ij/). Equal protein
loading was verified by Ponceau S solution (Sigma) reversible
staining of the blots, and each extract was also analysed for
relative protein levels of b-actin. For the comparison of relative
protein levels in GD18 cerebral cortices of foetuses from
saline- and LPS-injected dams, each background-corrected
value was normalized according to the relative b-actin level of
the sample, and then referred to the average of the saline
values calculated from the same immunoblot image.
Real-time RT–PCR
Total RNA was extracted using TRIzolH (Invitrogen), following
the manufacturer’s protocol. Samples were further purified by
treatment with TURBO DNA-free
TM
(Ambion), followed by a
second extraction with phenol/chloroform. RNA was reverse
transcribed using iScript cDNA Synthesis Kit (Bio-Rad
Laboratories) then analysed for various transcript expressions
(see Supplementary Table S2 available at http://www.asnneuro.
org/an/003/an003e068add.htm). The primers for Egr-1 were
part of the Qiagen QuantiTectH Primer Assay (Qiagen). qRT–
PCR was set up using iQ SYBRH Green Supermix (Bio-Rad
Laboratories) and performed in triplicate as previously
described (Ghiani et al., 2006; Mattan et al., 2010) on an
iCycler MyiQ Real Time PCR machine (Bio-Rad Laboratories).
Negative controls (samples in which reverse transcriptase was
omitted) were amplified individually using the same primer
sets (Supplementary Table S2) to ensure the absence of geno-
mic DNA contamination. PCR amplification resulted in the
generation of single bands. Amplification specificity was
assessed by melting curve and standard curves made from
serial dilutions of control RNA were used for quantifica-
tion. Data were normalized to the internal control GAPDH
(glyceraldehyde-3-phosphate dehydrogenase).
Statistical analysis
Statistical analysis was performed using GraphPad Prism 4.01
(GraphPad Software) by Student’s t test or one-way ANOVA
followed by Bonferroni’s multiple comparison test when three
or more experimental groups were compared.
RESULTS
Maternal injections of LPS elicited an
inflammatory response in the foetal brain
Maternal bacterial infection was modelled in timed-pregnant
rats at GD15 and GD16 by administering two consecutive
injections of LPS (200 mg/kg intraperitoneally) to study the
Early effects of maternal LPS on brain development
E 2011 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/)
which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.
235
Page 3
impact of the maternal and foetal inflammatory response on
brain development. The dose was chosen based on previous
reports showing a minimal rate of foetal re-absorption (Bell
and Hallenbeck, 2002). Such a model is known to induce
cellular and behavioural deficits in adult rodents, although
little is known about the cellular and molecular events that
take place in the foetal brain shortly after activation of the
maternal immune system has occurred (Jonakait, 2007; Meyer
et al., 2008b, 2009a; Boksa, 2010).
In order to determine if administration of LPS elicited a
response in the dams, the levels of three pro-inflammatory
cytokines (IL-6, IL-1b and TNFa), previously associated with
the maternal and foetal response triggered by activators
of the immune system, were analysed by ELISA in the maternal
serum. The levels of these cytokines were significantly in-
creased in the serum of LPS-injected dams (Figure 1A) 4 h after
the first LPS-injection at GD15 as compared with saline-
injected dams. Similar changes in cytokine gene expression
Figure 1 Maternal injections of LPS elicited an inflammatory response in the foetal forebrain
(A) Cytokine levels were significantly increased in maternal serum 4 h after the first injection of LPS (200 mg/kg, intraperitoneally)
Dams were injected at GD15 and GD16 and killed 4 h after the first (4 h) or the second LPS-injection (28 h). Results were plotted as
the means¡S.E.M. for five or six saline-in jected dams and nine or ten LPS-injected dams per time point. *P,0.05 versus respective
control (saline-injected dams), One-way ANOVA followed by Bonferroni’s multiple comparison test. (B) Pro-inflammatory cytokine
expression levels were increased in the foetal forebrain at different time-points after maternal injections of LPS. Cytokine expression
levels were measured in the foetal cerebral cortex by qRT–PCR at GD15, 4 h after the first (4 h) maternal injection of LPS or saline; at
GD16, 4 h after the second injecti on (28 h), and at GD18, 48 h after the second maternal injection of LPS or saline (72 h). At least
three foetuses/dam/group/time-point were analysed. Levels were normalized to GAPDH. Histograms represent the means¡S.E.M. of
9–30 foetuses from five or six saline-injected dams and eight to ten LPS-injected dams per time point. *P,0.05, **P,0.01 versus
respective saline-exposed foetuses, One-way ANOVA followed by Bonferroni’s multiple comparison test. (CF) Dams received two
consecutive injections of LPS (200 mg/kg, intraperitoneally) at GD15 and GD16 and were killed at GD18. (C) The microglial marker
CD68 was strongly expressed in the LPS-exposed foetal forebrain at GD18. LV, lateral ventricle. Scale bar550 mm. (D) A significant
increase in GFAP protein levels was found at GD18 in LPS-exposed foetal cerebral cortex. Values derived from the densitometric
analysis were corrected for the background, normalized to b-actin and are shown as a percentage of the value for saline-exposed
foetuses. The histogram shows represents the means ¡ S.E.M. for 11–12 foetuses from seven saline-injected and seven LPS-injected
dams. *P,0.05 versus saline-exposed foetuses, Student’s t test. (E) GFAP immunoreactivity was mainly localized in the IZ in both
LPS- and saline-exposed foetuses. Scale bar550 mm. (F) Higher magnification of the insets in (B) shows that GFAP-positive cells
display a reactive phenotype in the IZ of LPS-exposed foetuses (arrow). Nuclei were identified by DAPI staining. Scale bar520 mm.
C.A. Ghiani and others
236 E 2011 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/)
which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.
Page 4
were observed in the foetal forebrains at corresponding times
by qRT–PCR (Figure 1B), indicating that a foetal inflam-
matory response was taking place in response to maternal
injections of LPS. In particular, IL-6 gene expression dis-
played a time-dependent increase. Its levels were significantly
increased not only 4 h after the first and the second maternal
injections (28 h), but were still greatly elevated 72 h after
the first injection, i.e. on GD18, which suggested that high
levels of this inflammatory mediator persisted in the foetal
forebrain (Figure 1B), albeit no changes in the maternal
serum IL-6 were seen at this time (results not shown). Gene
expression levels of IL-1b and TNFa in the foetal cerebral
cortex were significantly increased 4 h after the second
maternal injection of LPS (28 h; Figure 1B), but not 4 h after
the first LPS injection. TNFa levels in experimental animals
reverted to saline levels by 72 h (GD18), whereas IL-1b gene
expression levels were still elevated, although this increase
was not statistically significant (Figure 1B). In agreement with
an up-regulation of the expression levels of inflammatory-
related genes in the forebrain of foetuses from LPS-injected
dams, increased immunoreactivity for CD68, a marker for
microglia/macrophages, was seen 72 h after maternal
injections of LPS (GD18; Figure 1C). These data appear to
indicate that an extended foetal response was induced, as
maternal levels of reactive cytokines decreased rapidly, while
foetal levels remained elevated well after exposure to LPS.
A hallmark of the response of the CNS (central nervous
system) to injury or inflammation is the presence of reac-
tive astrocytes, characterized by increased expression of the
specific marker GFAP. Western-blot analysis of GD18 saline-
and LPS-exposed foetal cerebral cortices at 72 h revealed a
40% increase in GFAP protein levels in the latter group
(Figure 1D). Furthermore, even though GFAP immunoreacti-
vity could be seen in both the forebrains of LPS-exposed
and control groups, and was mainly localized in the IZ
(intermediate zone), a stronger signal was present in the LPS-
exposed forebrains (Figure 1E), where GFAP-positive cells
displayed the characteristic morphology of reactive astrocytes,
which appear as hypertrophic process-bearing cells (Figure 1F,
arrow).
Figure 2 Maternal injections of LPS hindered the development of brain structures
(A) H&E staining of GD18 foetal forebrain showed a significant enlargement of the CP in the forming cerebral cortex of LPS-exposed
foetuses compared with saline-exposed foetuses. No significant differences were found in the thickness of other laminae of the
cerebral cortex or in the total thickness of the cortex. (B) The hippocampus of LPS-exposed foetuses was significantly larger than in
control animals. (C) Quantifications of the differences in the thickness of the forebrain, CP and hippocampus. Histograms represent
the means¡S.E.M. for six rat foetuses from six saline-injected and six LPS-injected dams. *P,0.05 versus saline-exposed foetuses,
Student’s t test. MgZ, marginal zone; IZ, intermediate zone; VZ/SVZ, ventricular zone/subventricular zone.
Early effects of maternal LPS on brain development
E 2011 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/)
which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.
237
Page 5
Maternal injections of LPS impaired brain
morphogenesis
To determine whether activation of the maternal immune
system triggered by LPS affected the cellular and laminar
organization of the neocortex, coronal sections from LPS-
exposed and control foetal forebrains collected at GD18 were
analysed using H&E staining. Despite the fact that the total
thickness of the forebrains, measured at the level of the
parietal neocortex, was not significantly different between
LPS-treated and control foetuses, analysis of the various
laminae of the foetal cerebral cortex revealed a significant
enlargement (24%) of the CP (cortical plate) in the neocortex
of LPS-exposed foetuses compared with saline-injected
animals (Figures 2A and 2C). No significant changes were
seen in the MgZ (marginal zone), IZ or SVZ/VZ (subventricular
zone/ventricular zone). Additionally, LPS-exposed foetuses
displayed significantly thickened hippocampi (15%) as
compared with controls animals (Figures 2B and 2C). These
data suggest that activation of the maternal immune res-
ponse affected development of foetal brain cytoarchitecture.
Maternal injections of LPS altered the expression
pattern of immature neuronal markers
In rats, cortical neurogenesis begins around GD11 and GD12,
peaks at GD14, and declines through the remainder of
gestation into the postnatal period (Bayer and Altman, 1991;
Sauvageot and Stiles, 2002). At the time of the LPS injections,
GD15 and GD16, neurons destined to settle in the infra-
granular layers VI and V are being generated followed by
neurons destined to settle in the supragranular layers IV–II at
later stages (GD17–21) (Bayer and Altman, 1991). We surmised
that foetal inflammation could affect those neurons whose
generation and migration coincided with the time of the in-
jections or soon after and were ordained to settle in the
supragranular layers. To address this possibility, the expression
of doublecortin, a microtubule-associated protein expressed in
immature neurons and involved in the regulation of neuronal
migration, was investigated. In control foetuses, doublecortin
positive cells were detected in both the IZ and CP (Figure 3A).
In contrast, in the foetal forebrains from LPS-injected dams,
doublecortin-positive cells were predominantly found in the IZ
and were nearly absent in the CP (Figure 3A). Likewise
abnormal distributions of cells that expressed the immature
neuronal markers a-internexin and bIII-tubulin were seen in
the neocortex (Figures 3B and 3C, respectively). In fact, similar
to the expression pattern of doublecortin (Figure 3A), a-
internexin- and bIII-tubulin-positive cells (Figures 3B and 3C,
respectively) populated both the CP and the IZ in the forming
the neocortex of foetuses from saline-injected dams. In the
forebrains of LPS-exposed foetuses, cells positive for these
markers were mainly concentrated in the IZ with notably fewer
immunoreactive cells detected in the CP (Figures 3B and 3C).
Nonetheless, as shown in Figure 3(D), the total protein levels of
a-internexin and bIII-tubulin were not significantly changed,
suggesting that foetal inflammation predominantly affected
the distribution pattern of neuronal cells.
The cellular and molecular machinery involved in
neuronal migration is perturbed in LPS-exposed
foetal forebrains
Proper migration of immature neurons to their final des-
tination in the CP is essential for the development of a
functioning CNS. Neuronal migration is a complex process
that occurs with a defined temporal pattern and is regulated
by soluble cues as well as interactions between migratory
neurons and other cell types (Gupta et al., 2002; Nadarajah
and Parnavelas, 2002). During this period in brain develop-
ment, the majority of the neurons, destined to form the
different layers of the CP migrate along specialized cells
named radial glia. At GD9 and GD10 radial glial processes
span the cortical wall from the VZ to the pial surface. Radial
glial cells serve as both progenitor cells and a primitive
migratory scaffold for post-mitotic neurons (Campbell and
Gotz, 2002). To further ascertain the cellular changes
occurring in GD18 LPS-exposed foetal forebrains that might
underlie the defect in neuronal patterning, we examined
the expression levels and distribution of GLAST, a marker for
radial glia. In the developing neocortex of control foetuses,
cell fibres positive for GLAST extended throughout the IZ
and the CP, up to the pial surface (Figure 4A). GLAST
immunoreactivity displayed a marked decrease in the IZ of
LPS-exposed foetuses and was practically absent in the CP,
where fewer positive processes could be seen (Figure 4A,
arrows in left lower panel). Furthermore, GLAST protein levels
showed a 30% decrease in the forebrain of GD18 foetuses
from LPS-injected dams as compared with saline-injected
foetuses (Figure 4B), potentially suggesting a weakening of
the migratory scaffold formed by radial glia.
Among the factors involved in the regulation of radial glia
development, maintenance of the radial glia scaffold as
well as proper orientation of the radial glia processes is the
glycoprotein reelin (Hartfuss et al., 2003; Forster et al., 2010).
In the mammalian brain, reelin has different roles depending
upon the developmental stage. Prenatally, it is secreted by
the Cajal-Retzius cells in the MgZ and plays major roles
in the regulation of neuronal migration and cortical layer
formation; whereas, in the adult brain, reelin is secreted by
GABAergic interneurons and participates in synaptic plasticity
and memory formation (Fatemi, 2005; Forster et al., 2010).
In addition to its proposed role as a susceptibility gene
for neuropsychiatric disorders, such as schizophrenia and
autism, its levels were reported to be decreased in adult mice
following exposure to antenatal insults such as maternal viral
infection (Fatemi, 2005). Reelin expression was analysed in
foetuses from LPS- and saline-injected dams at GD18 by both
immunohistochemistry and Western-blot analysis. Reelin
expression was present in the Cajal-Retzius cells of the MgZ
in both animal groups, even though its immunoreactivity was
lower in the LPS-exposed foetuses compared with controls
C.A. Ghiani and others
238 E 2011 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/)
which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.
Page 6
(Figure 4C). These results were further confirmed by Western-
blot analysis. Reelin exists in various isoforms, which are
generated by cleavage of the 400 kDa isoform, considered to
be the full form and the others proteolytic products (Ignatova
et al., 2004; Jossin et al., 2004, 2007). Three main isoforms
(400, 330 and 180 kDa) were revealed by Western blotting in
the cerebral cortex of LPS-exposed foetuses and controls. A
significant decrease (30%) in the protein levels of the 180
kDa form was found in the LPS-exposed foetuses (Figure 4D)
as compared with controls, whilst the other bands displayed
no significant changes, suggesting that foetal inflammation
may interfere with cleavage of reelin.
The expression of neural progenitor cell markers
is altered by inflammation
Brain development from GD17 to GD20 is characterized
by the presence of actively proliferating precursor cell
populations, which will give rise to late-born neurons and
cells of the glial lineage. Maturation of neural precursors is
tightly regulated by both cell specific, intrinsic and extrin-
sic factors (Berger-Sweeney and Hohmann, 1997; Cameron
et al., 1998; Pomeroy and Kim, 2000; Nguyen et al., 2001;
Sauvageot and Stiles, 2002). Because of the documented role
of radial glial cells as neural progenitors (Campbell and Gotz,
2002; Pinto and Gotz, 2007), and the decrease in GLAST
levels found in LPS-exposed foetuses (Figures 4A and 4B),
we reasoned that activation of the foetal inflammatory
response might also affect progenitor cell development.
Hence, the expression of markers for neural progenitors was
analysed.
The effects of foetal brain inflammation on neural pro-
genitor cells were investigated by examining the distribution
and protein levels of nestin, an intermediate neurofilament
typically found in neural progenitors. As shown in Figure 5(A),
nestin immunoreactivity was decreased in the neocortex of
Figure 3 Abnormal distribution of markers for immature neurons in the foetal forebrain after maternal injections of LPS
(A) Doublecortin immunoreactivity was seen in both the CP and IZ in GD18 foetuses from saline-injected dams. Conversely,
doublecortin-positive cells were mostly detected in the IZ of age-matched foetuses exposed to LPS, while the CP displayed lower
immunoreactivity. Lower panels are higher magnifications of the area marked by the two arrows showing the atypical distribution of
doublecortin positive cells in the CP and IZ of LPS-exposed animals. (B) Expression of the immature neuronal marker a-internexin
could be seen in the IZ of GD18 LPS-exposed foetuses and was almost absent in the CP as compared with age-matched control
(Saline). (C) Expression of bIII-tubulin could be observed throughout the cerebral cortex in GD18 saline-exposed foetuses. In the
cerebral cortex of LPS-exposed foetuses, immunoreactivity for this marker was mainly found in the IZ, while it was almost absent in
the CP. Lower panels are higher magnifications of the area marked by the two arrows. Scale bar550 mm. LV, lateral ventricle. ( D)No
differences were found in the protein levels of a-internexin and bIII-tubulin measured in whole tissue lysates prepared from cerebral
cortices of GD18 saline- and LPS-exposed foetuses. Values derived from the densitometric analysis were corrected for the back-
ground, normalized to b-actin and are shown as a percentage of the value for saline-exposed animals. Histograms are the
means¡S.E.M. for 17 foetuses from seven dams injected with saline and 18 foetuses from eight LPS-injected dams.
Early effects of maternal LPS on brain development
E 2011 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/)
which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.
239
Page 7
LPS-exposed foetuses compared with controls. In agreement
with this finding, decreased protein levels (25%) for nestin
were found in forebrains of LPS-exposed animals (Figure 5B).
Finally, expression of Sox2, a transcription factor expressed in
neural precursors, was measured by qRT–PCR and found to be
significantly decreased (15%) in the forebrain of LPS-exposed
animals compared with foetuses from saline-injected dams
(Figure 5C).
Figure 4 Altered expression levels of GLAST and Reelin in the foetal forebrain after maternal immune system activation with LPS
(A) Processes immunopositive for GLAST were observed throughout the IZ and the CP of GD18 saline-exposed foetuses, whereas they
were evidently decreased in the corresponding areas of GD18 LPS-exposed animals. Nuclei were identified by DAPI staining. (B) The total
protein levels of GLAST were significantly decreased in the cerebral cortex of GD18 LPS-exposed foetuses compared with control. Values
derived from the densitometric analysis were corrected for the background, normalized to b-actin, and are shown as a percentage of the
value for saline-exposed animals. Histograms are the means¡S.E.M. for 12 foetuses from seven dams injected with saline and 15
foetuses from eight LPS-injected dams. *P,0.05 versus saline-exposed foetuses, Student’s t test. (C) Immunoreactivity of the
glycoprotein protein reelin was decreased in the Cajal-Retzius cells in the MgZ of forebrains from GD18 LPS-exposed foetuses compared
with saline. Scale bars550 mm. (D) Representative immunoblots showing that the protein levels of the 180 kDa isoform of reelin were
significantly decreased in whole tissue lysates from the cerebral cortex of GD18 LPS-exposed foetuses. Values are shown as a
percentage of the value for saline-exposed animals. Histograms are the means¡S.E.M. for five foetuses from three dams injected with
saline and five foetuses from three LPS-injected dams. *P,0.05 versus saline-exposed foetuses, Student’s t test.
C.A. Ghiani and others
240 E 2011 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/)
which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.
Page 8
The expression levels of the immediate early gene
Arc are influenced by maternal injections of LPS
IEGs (immediate early genes) are among the most highly
expressed genes during CNS development and their activation
in response to neuronal activity is fundamental for con-
trolling downstream genes involved in specification and
maturation of neural progenitors in the neocortex as well as
in other brain areas (Herdegen and Leah, 1998; Kaufmann
and Worley, 1999). We sought to determine whether the
anomalous neuronal patterning seen in GD18 foetal fore-
brains after maternal injections of LPS at GD15 and GD16 was
accompanied by abnormal expression levels of IEGs. The
activity-regulated IEGs Ania-3, cfos, and Egr-1 showed a
trend towards decrease, although such changes were not
significant. Conversely, Arc displayed a significant decrease in
gene expression levels (20%) in the neocortex of LPS-exposed
foetuses as compared with controls (Figure 6A). Furthermore,
a significant decrease in the total protein levels of Arc was
found (27%) in forebrains of LPS-exposed foetuses when
compared with control (Figure 6B).
Postnatal effects of maternal injections of LPS at
GD15 and GD16
In order to investigate if the changes in brain structures and
neural cell development identified at GD18 were still present
postnatally and to assess whether foetal inflammation
elicited long-lasting effects, the brains of offspring from
saline- and LPS-injected dams were analysed at P1. H&E
staining of P1 brains revealed lack of differences in the size of
the CP between pups born to dams injected with LPS or saline
at GD15 and GD16 (Figures 7A, 7B and 7G). However, the
total thickness of the neocortex was significantly enlarged
(17%; Figure 7G) when compared with control pups and the
cells appeared to be more closely aggregated than in control
(Figures 7C and 7D). The mean value of the hippocampal
areas, measured at the transition between CA1 and CA2 areas,
were increased in the LPS-exposed pups, although this dif-
ference was not significant (Figures 7E–7G). Furthermore, the
expression pattern of bIII-tubulin (Supplementary Figures S1A
and S1B available at http://www.asnneuro.org/an/003/an003
e068add.htm) and doublecortin (Supplementary Figures S1C
and S1D) was still altered in P1 cerebral cortex of pups born to
LPS-injected dams compared with control pups.
DISCUSSION
Among many proposed environmental factors, epidemiologi-
cal data support an association between maternal infections
Figure 5 Expression of neural progenitor cells markers in the foetal forebrain is disturbed after maternal injections of LPS
(A) Immunoreactivity for nestin, a marker for neural progenitors, was decreased at GD18 in the foetal cerebral cortex of LPS-exposed
foetuses compared with foetuses from saline-injected dams. Upper panels: double immunostaining for nestin in green and GLAST in
red; lower panels: single immunostaining for nestin. Scale bar550 mm. (B) Representative immunoblots showing that nestin protein
levels were significantly decreased in whole tissue lysates from the cerebral cortex of GD18 LPS-exposed foetuses. Values derived
from densitometric analysis were corrected for the background, normalized to b-actin, and are shown as a percentage of the value
for saline-exposed animals. Histograms are the means ¡ S.E.M. for ten foetuses from five dams injected with saline and ten foetuses
from five LPS-injected dams. *P,0.05 versus saline-exposed foetuses, Student’s t test. (C) The gene expression levels of the
transcription factor Sox2 were decreased in the foetal cerebral cortex after maternal injections of LPS. Sox2 expression was
measured by qRT–PCR in the cerebral cortex of GD18 foetuses from saline and LPS-injected dams. The levels were normalized to
GAPDH. Histograms represent the means¡S.E.M. for 15 foetuses from seven saline-injected dams and 15 foetuses from seven LPS-
injected dams. *P,0.05 versus saline-exposed foetuses, Student’s t test.
Early effects of maternal LPS on brain development
E 2011 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/)
which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.
241
Page 9
and neuropsychiatric disorders (Mednick et al., 1988; Brown,
2006; Ellman and Susser, 2009). Here, we present evidence
that maternal infection, mimicked by injections of LPS, and
subsequent foetal inflammation during mid–late gestation in
rats (equivalent to mid-second trimester in human) con-
siderably perturbed brain development, shortly after the
inflammatory response was triggered in the foetus, by
interfering with signalling pathways involved in neuronal cell
distribution pattern. Enhanced microglia activation, reactive
astroglia and increased expression of pro-inflammatory
cytokines were detected in the foetal brain after maternal
exposure to LPS, suggesting that the observed effects might, at
least in part, be generated by these immune mediators.
Previous studies have reported increased levels of pro-
inflammatory cytokines in the maternal serum as well as at
the maternal/foetal interface (amniotic fluid, placenta) after
stimulation of the maternal immune system with different
agents [LPS, poly(I:C), etc.] (Boksa, 2010). Altogether these
findings advocate for a role of these immune mediators in the
morphological and neurobehavioural changes in offspring
exposed to prenatal inflammation.
Studies in humans as well as in accepted animal models of
maternal infection have described a series of abnormalities in
adult brain cytoarchitecture, including decreased dendritic
arborization and aberrant neuronal migration (Fatemi and
Folsom, 2009; Deutsch et al., 2010), pointing at a neurodeve-
lopmental origin of severe psychiatric disorders and implying
that certain defects are present before the onset of the
disorder. Alterations in normal cortical development caused by
small disturbances of neurogenesis and neuronal migration
may elicit maldevelopment of these areas, affecting the
formation of neuronal networks and resulting in the neuro-
pathological defects described in the post-mortem brain of
persons affected by schizophrenia and other neurodevelop-
mental disorders. Perhaps some pathways active in the adult
brain are already dysregulated during foetal life by maternal
conditions leading to the formation of malfunctioning
neuronal circuits in the adolescent and adult brain. In the
present study, an enlarged CP and abnormal expression of
markers for immature neurons were observed in the neocor-
tex of LPS-exposed foetuses at GD18, i.e. 2 days after LPS
exposure, in comparison with the brains of age-matched
control foetuses. In addition, a number of important molecules,
including reelin, GLAST and Arc, were decreased in foetal brains
following LPS exposure. Finally, at P1, the cerebral cortex of
LPS-exposed animals was significantly larger than in age-
matched control offspring and the cells appeared to be more
compact, resulting in a reduction of the space these cells have
available to extend processes compared with control.
Our results suggest an effect on the cleavage of reelin
triggered by inflammation and the consequent cascade of
events. In the developing mammalian brain, reelin has a
pivotal role in cortical layer formation by regulating neuronal
migration (Fatemi, 2005; Forster et al., 2010). In adult brain,
this glycoprotein participates in synaptic plasticity and
memory formation and is considered a susceptibility gene
for neuropsychiatric disorders, such as schizophrenia and
autism (Fatemi, 2005; Forster et al., 2010). Proteolytic cleavage
of reelin produces five fragments, among them the central
fragment was shown to mimic reelin functions in vitro. The
function(s) of the full-length and shorter fragments of reelin
has hitherto not been completely elucidated, albeit it was
reported that inhibition of reelin processing in vivo prevents
signalling and hampers development in cortical embryonic
slices (Jossin et al., 2004, 2007). Cleavage appears to be
required for reelin to be released in the intercellular space and
to bind to its receptors on receptive cells. In reeler Orleans
(relnorl/orl) mice, lack of reelin signalling is due to abnormal
protein processing and expression of a truncated, non-
releasable reelin fragment (de Bergeyck et al., 1997; Derer
et al., 2001). In contrast with the C-terminus fragments, both
the N-terminus and the central fragments have been detected
in the CP and are considered important for reelin function.
Moreover, Jossin et al. (2007) showed that, while the larger
fragments as well as the full length could be only detected in
Figure 6 Gene and protein expression levels of IEG in the foetal forebrain
following maternal immune system activation with LPS
(A) Only the gene expression levels of Arc were significantly decreased in the
cerebral cortex of GD18 foetuses from LPS-treated dams compared with
control. Changes in gene expression levels of Arc, cfos, Ania-3 and Egr-1 were
measured by qRT–PCR in the cerebral cortex of foetuses from saline and LPS-
injected dams. The levels were normalized to GAPDH. Histograms represent
the means¡S.E.M. for 16 foetuses from five saline-injected dams and 14 from
five LPS-injected dams. *P,0.05 versus saline-exposed foetuses, Student’s
t test. (B) The total protein levels of Arc were decreased in whole tissue lysates
from GD18 foetal cerebral cortex after maternal injections of LPS. Values
derived from densitometric analysis were corrected for the background,
normalized to b-actin, and are shown as a percentage of the value for saline-
exposed animals. Histograms are the means¡S.E.M. for ten foetuses from five
dams injected with saline and 11 from five LPS-injected dams. *P,0.05 versus
saline-exposed foetuses, Student’s t test.
C.A. Ghiani and others
242 E 2011 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/)
which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.
Page 10
the proximity of the MgZ, antibodies, raised against the N-
terminal region or the central fragment, detected reelin among
the cells of the CP. Hence, reelin processing seems important
for his diffusion, which might be influenced by the fragments’
size (ranging from 100 to 330 kDa), i.e. the smaller N-terminal
and central fragments may diffuse farther into the CP and
influence late born neurons which are at a greater distance
from the MgZ. These differences would create a gradient of
reelin throughout the developing cortex (Zhao and Frotscher,
2010). The decreased levels of the 180 kDa isoform reported
in this study suggest an impairment of reelin processing,
which could, at least in part, explain the increased presence of
doublecortin and bIII-tubulin positive cells in the IZ in the LPS-
exposed foetuses compared with controls.
During brain development, proper synaptic activity must
activate a cascade of genes involved in transforming immature
neuronal connections into functional circuits. IEGs are highly
expressed during CNS development and have important
roles in the adult brain. Their expression is developmentally
regulated and influenced by exogenous signals such as neuro-
transmitters and second messenger signalling pathways. IEG
activation in response to neural activity is fundamental for
controlling expression of downstream genes and their
products, which are involved in specification and maturation
of neural progenitors in the cerebral cortex as well as in other
brain areas (Herdegen and Leah, 1998; Kaufmann and Worley,
1999). Interference with the early expression and activity of
IEGs, such as Arc, likely plays a role in the brain maldevelopment
Figure 7 Changes in the offspring forebrain after prenatal foetal and maternal immune activation with LPS
Dams received two consecutive injections of LPS (200 mg/kg, intraperitoneally) at GD15 and GD16 and the offspring were killed at
postnatal day (P)1. (A, B) H&E staining revealed lack of significant differences in the thickness of the CP in P1 rats born to dams
injected with saline or LPS; conversely, a significant enlargement of the cerebral cortex was found in P1 rats born to LPS-injected
dams. (C, D) Higher magnification images showing increased cell density in the CP of P1 rats prenatally exposed to the effects of
maternal injections of LPS. (E, F) No significant differences were found in the thickness of the hippocampus measured at the
transition between the CA1 and CA2 areas. White bars indicate where the measurements were performed. LV, lateral ventricle.
(G) Quantifications of the differences in the thickness of the cerebral cortex, CP and hippocampus. Histograms represent the
means¡S.E.M. for three P1 control (saline) and three P1 rats prenatally exposed to LPS. *P,0.05 versus saline-exposed P1 rats,
Student’s t test.
Early effects of maternal LPS on brain development
E 2011 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/)
which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.
243
Page 11
found in individuals with schizophrenia. As a consequence,
synaptic transmission and plasticity are affected in young
adulthood, when refinement of synaptic connections requires
higher activity potentially leading to a loss of synaptic plasticity
(Fatemi and Folsom, 2009).
These findings portray an intricate process by which foetal
inflammation perturbs neuronal patterning and cortical
development contributing to cognitive and/or psychotic
manifestations later in adulthood. Such a process acts upon
a number of different pathways, a number of which then have
additional roles in mediating some of the experience-
dependent plasticity in the adult brain. Based on these results,
we surmise that the formation of neuronal networks in
offspring from LPS-injected dams is altered, and such abnor-
malities may represent a major underlying pathophysiology of
psychiatric disorders with a neurodevelopmental origin.
The LPS model used in this study does not fully recapitulate
the events triggered by bacterial pathogens and their toxins
in the foetal brain, and reproduces only part of the
inflammation-mediated effects. Nonetheless, our study has
set the stage to unravel the sequelae of events that underlie
the neurobehavioural deficits reported in animals exposed to
an antenatal insult.
ACKNOWLEDGEMENTS
We thank Donna Crandall for her help with the preparation
of the Figures, Catalina Abad for insightful discussions and
help with the ELISA assays, Elvira Khialeeva and Diane
Anthony for their help with the H&E staining, Justin Lam and
Linda L. Kalamkeryan for technical help. We are grateful to
Christopher S. Colwell and David E. Krantz for critically
reading the manuscript before submission.
FUNDING
N.S.M. was supported by an NINDS-Rita L. Kirschstein NRSA
Fellowship. This work was supported by a Pilot and Feasibility
grant from the UCLA Center for Neurobiology of Stress and a
Stein-Oppenheimer Award to J.d.V and C.A.G., a Semel Young
Investigator Award to C.A.G., and grants from the National
Institutes of Health [P01-HD06576 and HD04612] to J.d.V.
REFERENCES
Ashdown H, Dumont Y, Ng M, Poole S, Boksa P, Luheshi GN (2006) The role
of cytokines in mediating effects of prenatal infection on the fetus:
implications for schizophrenia. Mol Psychiatry 11:47–55.
Bayer SA, Altman J (1991) Neocortical Development: Raven Press.
Bell MJ, Hallenbeck JM (2002) Effects of intrauterine inflammation on
developing rat brain. J Neurosci Res 70:570–579.
Bell MJ, Hallenbeck JM, Gallo V (2004) Determining the fetal inflammatory
response in an experimental model of intrauterine inflammation in rats.
Pediatr Res 56:541–546.
Berger-Sweeney J, Hohmann CF (1997) Behavioral consequences of abnormal
cortical development: insights into developmental disabilities. Behav
Brain Res 86:121–142.
Boksa P (2010) Effects of prenatal infection on brain development and
behavior: a review of findings from animal models. Brain Behav Immun
24:881–897.
Brown AS (2006) Prenatal infection as a risk factor for schizophrenia.
Schizophr Bull 32:20 0–202.
Cameron HA, Hazel TG, McKay RD (1998) Regulation of neurogenesis by
growth factors and neurotransmitters. J Neurobiol 36:287–306.
Campbell K, Gotz M (2002) Radial glia: multi-purpose cells for vertebrate
brain development. Trends Neurosci 25:235–238.
Cui K, Ashdown H, Luheshi GN, Boksa P (2009) Effects of prenatal immune
activation on hippocampal neurogenesis in the rat. Schizophr Res
113:288–297.
de Bergeyck V, Nakajima K, Lambert de Rouvroit C, Naerhuyzen B, Goffinet
AM, Miyata T, Ogawa M, Mikoshiba K (1997) A trun cated Reelin protein is
produced but not secreted in the ‘Orleans’ reeler mutation (Reln[rl-Orl]).
Brain Res Mol Brain Res 50:85–90.
Derer P, Derer M, Goffinet A (2001) Axonal secretion of Reelin by Cajal-
Retzius cells: evidence from comparison of normal and Reln(Orl) mutant
mice. J Comp Neurol 440:136–143.
Deutsch SI, Burket JA, Katz E (2010) Does subtle disturbance of neuronal
migration contribute to schizophrenia and other neurodevelopmental
disorders? Potentia l genetic mechanisms with possible treatment
implications. Eur Neuropsychopharmacol 20:281–287.
Deverman BE, Patterson PH (2009) Cytokines and CNS development. Neuron
64:61–78.
Ellman LM, Susser ES (2009) The promise of epidemiologic studies: neuroimmune
mechanisms in the etiologies of brain disorders. Neuron 64:25–27.
Fatemi SH (2005) Reelin glycoprotein: structure, biology and roles in health
and disease. Mol Psychiatry 10:251–257.
Fatemi SH, Folsom TD (2009) The neurodevelopmental hypothesis of
schizophrenia, revisited. Schizophr Bull 35:528–548.
Forster E, Bock HH, Herz J, Chai X, Frotscher M, Zhao S (2010) Emerging
topics in Reelin function. Eur J Neurosci 31:1511–1518.
Ghiani CA, Gallo V (2001) Inhibition of cyclin E-cyclin-dependent kinase 2
complex formation and activity is associated with cell cycle arrest and
withdrawal in oligodendrocyte progenitor cells. J Neurosci 21:1274–1282.
Ghiani CA, Lelievre V, Beltran-Parrazal L, Sforza DM, Malvar J, Smith DJ,
Charles AC, Ferchmin PA, de Vellis J (2006) Gene expression is
differentially regulated by neurotransmit ters in embryonic neuronal
cortical culture. J Neurochem 97(Suppl 1):35–43.
Ghiani CA, Starcevic M, Rodriguez-Fernandez IA, Nazarian R, Cheli VT,
Chan LN, Malvar JS, de Vellis J, Sabatti C, Dell’Angelica EC (2010)
The dysbindin-containing complex (BLOC-1) in brain: developmental
regulation, interaction with SNARE proteins and role in neurite outgrowth.
Mol Psychiatry 15:115, 204–115.
Gupta A, Tsai LH, Wynshaw-Boris A (2002) Life is a journey: a genetic look at
neocortical development. Nat Rev Genet 3:342–355.
Hagberg H, Mallard C (2005) Effect of inflammation on central nervous
system development and vulnerability. Curr Opin Neurol 18:117–123.
Hartfuss E, Forster E, Bock HH, Hack MA, Leprince P, Luque JM, Herz J,
Frotscher M, Gotz M (2003) Reelin signaling directly affects radial glia
morphology and biochemical maturation. Development 130:4597–4609.
Herdegen T, Leah JD (1998) Inducible and constitutive transcription factors in
the mammalian nervous system: control of gene expressi on by Jun, Fos
and Krox, and CREB/ATF proteins. Brain Res Brain Res Rev 28:370–490.
Ignatova N, Sindic CJ, Goffinet AM (2004) Characterization of the various
forms of the Reelin protein in the cerebrospinal fluid of normal subjects
and in neurological diseases. Neurobiol Dis 15:326–330.
Jonakait GM (2007) The effects of maternal inflammation on neuronal
development: possible mechanisms. Int J Dev Neurosci 25:415–425.
Jossin Y, Ignatova N, Hiesberger T, Herz J, Lambert de Rouvroit C, Goffinet AM
(2004) The central fragment of Reelin, generated by proteolytic
processing in vivo, is critical to its function during cortical plate
development. J Neurosci 24:514–521.
Jossin Y, Gui L, Goffinet AM (2007) Processing of Reelin by embryonic
neurons is important for function in tissue but not in dissociated cultured
neurons. J Neurosci 27:4243–4252.
Juarranz Y, Abad C, Martinez C, Arranz A, Gutierrez-Canas I, Rosignoli F,
Gomariz RP, Leceta J (2005) Protective effect of vasoactive intestinal
peptide on bone destruction in the collagen-induced arthritis model of
rheumatoid arthritis. Arthritis Res Ther 7:R1034–R1045.
Kaufmann WE, Worley PF (1999) Neural activity and immediate early gene
expression in the cerebral cortex. Ment Retard Dev Disabil Res Rev 5:41–50.
Mattan NS, Ghiani CA, Lloyd M, Matalon R, Bok D, Casaccia P, de Vellis J
(2010) Aspartoacylase deficiency affects early postnatal development of
oligodendrocytes and myelination. Neurobiol Dis 40:432–443.
C.A. Ghiani and others
244 E 2011 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/)
which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.
Page 12
Mednick SA, Machon RA, Huttunen MO, Bonett D (1988) Adult schizophrenia
following prenatal exposure to an influenza epidemic. Arch Gen
Psychiatry 45:189–192.
Meyer U, Yee BK, Feldon J (2007) The neurodevelopmental impact of prenatal
infections at different times of pregnancy: the earlier the worse?
Neuroscientist 13:241–256.
Meyer U, Engler A, Weber L, Schedlowski M, Feldon J (2008a) Preliminary
evidence for a modulation of fetal dopaminergic development by
maternal immune activation during pregnancy. Neuroscience 154:701–709.
Meyer U, Nyffeler M, Schwendener S, Knuesel I, Yee BK, Feldon J (2008b)
Relative prenatal and postnatal maternal contributions to schizophrenia-
related neurochemical dysfunction after in utero immune challenge.
Neuropsychopharmacology 33:441–456.
Meyer U, Feldon J, Fatemi SH (2009a) In-vivo rodent models for the
experimental investigation of prenatal immune activation effects in
neurodevelopmental brain disorders. Neurosci Biobehav Rev 33:1061–
1079.
Meyer U, Feldon J, Yee BK (2009b) A review of the fetal brain cytokine
imbalance hypothesis of schizophrenia. Schizophr Bull 35:959–972.
Nadarajah B, Parnavelas JG (2002) Modes of neuronal migration in the
developing cerebral cortex. Nat Rev Neurosci 3:423 –432.
Nawa H, Takei N (2006) Recent progress in animal modeling of immune
inflammatory processes in schizophrenia: implication of specific
cytokines. Neurosci Res 56:2–13.
Nguyen L, Rigo JM, Rocher V, Belachew S, Malgrange B, Rogister B, Leprince
P, Moonen G (2001) Neurotransmitters as early signals for central
nervous system development. Cell Tissue Res 305:187–202.
Patterson PH (2009) Immune involvement in schizophrenia and autism:
etiology, pathology and animal models. Behav Brain Res 204:313–321.
Pinto L, Gotz M (2007) Radial glial cell heterogeneity the source of diverse
progeny in the CNS. Prog Neurobiol 83:2–23.
Pomeroy SL, Kim JY (2000) Biology and pa thobiology of neuronal
development. Ment Retard Dev Disabil Res Rev 6:41–46.
Rees S, Harding R (2004) Brain development during fetal life: influences of
the intra-uterine environment. Neurosci Lett 361:111–114.
Rees S, Harding R, Walker D (2008) An adverse intrauterine environment:
implications for injury and altered development of the brain. Int J Dev
Neurosci 26:3–11.
Sauvageot CM, Stiles CD (2002) Molecular mechanisms controlling cortical
gliogenesis. Curr Opin Neurobiol 12:244–249.
Smith SE, Li J, Garbett K, Mirnics K, Patterson PH (2007) Maternal immune
activation alters fetal brain development through interleukin-6.
J Neurosci 27:10695–10702.
Urakubo A, Jarskog LF, Lieberman JA, Gilmore JH (2001) Prenatal exposure to
maternal infection alters cytokine expression in the placenta, amniotic
fluid, and fetal brain. Schizophr Res 47:27–36.
Watanabe Y, Someya T, Nawa H (2010) Cytokine hypothesis of schizophrenia
pathogenesis: evidence from human stud ies and animal models .
Psychiatry Clin Neurosci 64:217–230.
Zhao B, Schwartz JP (1998) Involvement of cytokines in normal CNS
development and neurological diseases: recent progress and perspectives.
J Neurosci Res 52:7–16.
Zhao S, Frotscher M (2010) Go or stop? Divergent roles of Reelin in radial
neuronal migration. Neuroscientist 16:421–434.
Received 31 August 2011/12 October 2011; accepted 14 October 2011
Published as Immediate Publication 18 October 2011, doi 10.1042/AN20110027
Early effects of maternal LPS on brain development
E 2011 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/)
which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.
245
Page 13
  • Source
    • "A recent review pointed out that premature babies are more vulnerable to infections and inflammation that can lead to neurodevelopmental problems and higher risk for ASD [12]. In an animal study, maternal infections caused by multiple intraperitoneal injections of lipopolysaccharide damaged the layer formation of the fetal brain, possibly linked to neuropsychiatric disorders, such as schizophrenia and autism [37]. A clinical study with 1.2 million pregnancies showed that the risk of autism in the children of women with the highest levels of C-reactive protein, a wellknown marker of inflammation, was 43 % higher than women with the lowest levels [38] . "
    [Show abstract] [Hide abstract] ABSTRACT: Background Autism spectrum disorder (ASD) affects many children and juveniles. The pathogenesis of ASD is not well understood. Environmental factors may play important roles in the development of ASD. We examined a possible relationship of inflammatory pain in neonates and the development of ASD in juveniles. Methods Acute inflammation pain was induced by 5 % formalin (5 μl/day) subcutaneous injection into two hindpaws of postnatal day 3 to 5 (P3–P5) rat pups. Western blot, immunohistochemical, and behavioral examinations were performed at different time points after the insult. Results Formalin injection caused acute and chronic inflammatory responses including transient local edema, increased levels of inflammatory cytokines, TNF-α, and IL-1β in the blood as well as in the brain, and increased microglia in the brain. One day after the pain insult, there was significant cell death in the cortex and hippocampus. Two weeks later, although the hindpaw local reaction subsided, impaired axonal growth and demyelization were seen in the brain of P21 juvenile rats. The number of bromodeoxyuridine (BrdU) and doublecortin (DCX) double-positive cells in the hippocampal dentate gyrus of P21 rats was significantly lower than that in controls, indicating reduced neurogenesis. In the P21 rat’s brain of the formalin group, the expression of autism-related gene neurexin 1 (NRXN1), fragile X mental retardation 1 (FMR1), and oxytocin was significantly downregulated, consistent with the gene alteration in ASD. Juvenile rats in the formalin group showed hyperalgesia, repetitive behaviors, abnormal locomotion, sleep disorder, and distinct deficits in social memory and social activities. These alterations in neuroinflammatory reactions, gene expression, and behaviors were more evident in male than in female rats. Importantly, an anti-inflammation treatment using indomethacin (10 mg/kg, i.p.) at the time of formalin injections suppressed inflammatory responses and neuronal cell death and prevented alterations in ASD-related genes and the development of abnormal behaviors. Conclusions These novel observations indicate that severe inflammatory pain in neonates and persistent inflammatory reactions may predispose premature infants to development delays and psychiatric disorders including ASD. The prevention of pain stimuli and prompt treatments of inflammation during development appear vitally important in disrupting possible evolution of ASD syndromes. Electronic supplementary material The online version of this article (doi:10.1186/s12974-016-0575-x) contains supplementary material, which is available to authorized users.
    Full-text · Article · Dec 2016 · Journal of Neuroinflammation
  • Source
    • "Activated microglia and oxidative damage to both DNA and lipids significantly increased in our mouse model compared with the control mice. These findings were consistent with the results of a previous report using rats [45]. In addition, elevation of the fetal serum IL-6 level, which is part of the definition for FIRS [7], was also confirmed. "
    [Show abstract] [Hide abstract] ABSTRACT: Exposure to inflammation in utero is related to perinatal brain injury, which is itself associated with high rates of long-term morbidity and mortality in children. Novel therapeutic interventions during the perinatal period are required to prevent inflammation, but its pathogenesis is incompletely understood. Activated microglia are known to play a central role in brain injury by producing a variety of pro-inflammatory cytokines and releasing oxidative products. The study is aimed to investigate the preventative potential of molecular hydrogen (H2), which is an antioxidant and anti-inflammatory agent without mutagenicity. Pregnant ICR mice were injected with lipopolysaccharide (LPS) intraperitoneally on embryonic day 17 to create a model of perinatal brain injury caused by prenatal inflammation. In this model, the effect of maternal administration of hydrogen water (HW) on pups was also evaluated. The levels of pro-inflammatory cytokines, oxidative damage and activation of microglia were determined in the fetal brains. H2 reduced the LPS-induced expression of pro-inflammatory cytokines, oxidative damage and microglial activation in the fetal brains. Next, we investigated how H2 contributes to neuroprotection, focusing on microglia, using primary cultured microglia and neurons. H2 prevented LPS- or cytokine-induced generation of reactive oxidative species by microglia and reduced LPS-induced microglial neurotoxicity. Finally, we identified several molecules influenced by H2, involved in the process of activating microglia. These results suggested that H2 holds promise for the prevention of inflammation related to perinatal brain injury.
    Full-text · Article · Feb 2016 · Free Radical Biology and Medicine
  • Source
    • "Normally, LPS does not cross the placental barrier, but it induces the release of proinflammatory cytokines (Ashdown et al., 2006). Much evidence has linked elevated cytokine levels triggered by maternal infection to various neurodevelopmental processes, including cell differentiation , maturation, and survival (Deverman and Patterson, 2009; Ghiani et al., 2011; Paris et al., 2011; Zhao and Schwartz, 1998). Hence, fluctuations in the levels of maternal and fetal immune mediators induced by maternal infection signify disturbances that can impede the ongoing of neurodevelopmental processes and subsequently affect proper neural cell maturation (Meyer et al., 2009). "
    [Show abstract] [Hide abstract] ABSTRACT: Objective: The present study analyzed the effects of lipopolysaccharide (LPS) on maternal behavior during lactation and possible correlations with changes in emotional and immune responses in offspring. Methods: Lactating rats received 100 μg/kg LPS, and the control group received saline solution on lactation day (LD) 3. Maternal general activity and maternal behavior were observed on LD5 (i.e. the day that the peak of fever occurred). In male pups, hematological parameters and ultrasonic vocalizations (USVs) were assessed on LD5. At weaning, an additional dose of LPS (50 µg/kg, i.p.) was administered in male pups, and open-field behavior, oxidative burst and phagocytosis were evaluated. Results: A reduction in the time in which dams retrieved the pups was observed, whereas no effects on maternal aggressive behavior were found. On LD5, a reduction of the frequency of USVs was observed in pups, but no signs of inflammation were found. At weaning, an increase in immune system activity was observed, but no differences in open-field behavior were found. Conclusion: These results indicate that inflammation in lactating mothers disrupted mother/pup interactions and may have produced short- and long-term effects on pup behavior as well as biological pathways that modulate inflammatory responses to bacterial endotoxin challenge in pups.
    Full-text · Article · Aug 2014 · NeuroImmunoModulation
Show more